( 12 ) United States Patent ( 10 ) Patent No.: US 10,610,125 B2 Dang Et Al

US010610125B2

( 12 ) United States Patent ( 10 ) Patent No.: US 10,610,125 B2 Dang et al. (45 ) Date of Patent : * Apr. 7, 2020

(54 ) METHODS AND COMPOSITIONS FOR C12Q 1/6886 (2018.01 ) CELL - PROLIFERATION - RELATED G06F 19/00 ( 2018.01) DISORDERS (52 ) U.S. CI. ??? A61B 5/055 ( 2013.01 ) ; A61K 31/41 (71 ) Applicant: Agios Pharmaceuticals , Inc. , (2013.01 ) ; A61K 31/426 (2013.01 ) ; A61K Cambridge , MA ( US ) 45/06 (2013.01 ) ; C12N 15/1137 ( 2013.01) ; (72 ) Inventors : Lenny Dang , Boston , MA ( US ); C12Q 1/32 (2013.01 ) ; C12Q 1/6886 Valeria Fantin , Burlingame, CA (US ) ; (2013.01 ); C12Y 101/01042 ( 2013.01) ; GOIN Stefan Gross, Brookline , MA (US ) ; 33/574 (2013.01 ) ; GO6F 19/328 (2013.01 ); Hyun Gyung Jang , Waltham , MA C12N 2310/14 ( 2013.01 ) ; YO2A 90/24 ( US ) ; Shengfang Jin , Newton , MA ( 2018.01) ; YO2A 90/26 ( 2018.01) ( US ) ; Francesco G. Salituro , (58 ) Field of Classification Search Marlborough , MA ( US ); Jeffrey 0 . CPC GOIN 2800/52 ; GOIN 2800/7028 ; A61P Saunders, Lincoln , MA (US ) ; Shin -San 43/00 , A61P 35/00 Michael Su , Boston , MA (US ); See application file for complete search history . Katharine Yen , Wellesley, MA (US ) ( 73 ) Assignee : Agios Pharmaceuticals, Inc. , (56 ) References Cited Cambridge , MA (US ) U.S. PATENT DOCUMENTS ( * ) Notice : Subject to any disclaimer, the term of this 2,390,529 A 12/1945 Friedheim patent is extended or adjusted under 35 3,755,322 A 8/1973 Winter et al. 3,867,383 A 2/1975 Winter U.S.C. 154 (b ) by 101 days . 4,084,053 A 4/1978 Desai et al . This patent is subject to a terminal dis 5,021,421 A 6/1991 Hino et al. 5,489,591 A 2/1996 Kobayashi et al. claimer . 5,807,876 A 9/1998 Armistead et al . 5,834,485 A 11/1998 Dyke et al. ( 21) Appl. No .: 15 /589,615 5,965,559 A 10/1999 Faull et al. 5,965,569 A 10/1999 Camps Garcia et al. ( 22 ) Filed : May 8 , 2017 5,984,882 A 11/1999 Rosenschein et al. 6,262,113 B1 7/2001 Widdowson et al . Prior Publication Data 6,274,620 B1 8/2001 Labrecque et al . (65 ) 6,313,127 B1 11/2001 Waterson et al. US 2017/0325708 A1 Nov. 16 , 2017 6,399,358 B1 6/2002 Williams et al . 6,723,730 B2 4/2004 Bakthavatchalam et al. 6,979,675 B2 12/2005 Tidmarsh 7,173,025 B1 2/2007 Stocker et al . Related U.S. Application Data 7,858,782 B2 12/2010 Tao et al . (63 ) Continuation of application No. 13/ 939,519, filed on 8,133,900 B2 3/2012 Hood et al. Jul . 11, 2013 , now abandoned , which is a continuation (Continued ) of application No. 13 /256,396 , filed as application No. PCT /US2010 /027253 on Mar. 12 , 2010 , now FOREIGN PATENT DOCUMENTS abandoned . CN 101296909 A 10/2008 CN (60 ) Provisional application No.61 / 266,929 , filed on Dec. 101575408 A 11/2009 4 , 2009, provisional application No.61 / 253,820 , filed ( Continued ) on Oct. 21 , 2009, provisional application No. 61/ 229,689, filed on Jul. 29, 2009, provisional OTHER PUBLICATIONS application No. 61/ 227,649 , filed on Jul. 22 , 2009 , provisional application No. 61/ 220,543 , filed on Jun . Dykxhoorn , D. M. et al . “ The silent treatment: siRNAs as small 25 , 2009 , provisional application No. 61/ 180,609 , molecular drugs. ” Gene Therapy ( 2006 ) 13 541-552 . ( Year: 2006 ). * filed on May 22, 2009 , provisional application No. (Continued ) 61/ 173,518 , filed on Apr. 28 , 2009 , provisional application No. 61/ 160,664, filed on Mar. 16 , 2009 , Primary Examiner Christopher Adam Hixson provisional application No. 61 / 160,253 , filed on Mar. (74 ) Attorney, Agent, or Firm — Goodwin Procter LLP 13 , 2009. ( 51 ) Int . Cl. (57 ) ABSTRACT GOIN 33/574 ( 2006.01 ) A61B 5/055 ( 2006.01 ) Methods of treating and evaluating subjects having neoac A61K 31/41 (2006.01 ) tive mutants are described herei A61K 31/426 ( 2006.01) A61K 45/06 ( 2006.01 ) C12N 15/113 ( 2010.01 ) 12 Claims, 49 Drawing Sheets C12Q 1/32 ( 2006.01 ) Specification includes a Sequence Listing . US 10,610,125 B2 Page 2

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(55 ) 55 60 w 110 120 130 140 164 IDHI( CDS ) ( 1 ) ANGTCCAAAAAAATCA.GIGGOGGIICICTGGTAGAGATGCAAGGAGATGAANTGACACGAATCATTIGEGAATIGATTIAACAGAAACICATTTTTCCCTACCIGGAATT PETALJIDHI (55 ) ATGTCCAALALAATCAGIGGCGGTICIGIGGTAGAGATGCAAGGAGATGAANTGACACGARICATTIGGGAATIGITTAAAGAGARACTCATTITTCCCTACGIGGAATT Consensus (55 ) ANGICCAAAAAAATCAGIGGCGGIICIGIGGTAGAGATGCAAGGAGATGAAATGACACGAATCATIIGGCAATIGATTAAAGAGARACTCATTITICCCTACGIGGAATT (163 ) 163 180 220 250 260 272 IDHI (CDS ) ( 109 ) TIGGAICIACATAGCTATGATITAGGCATAGAGAATOGIGATGCCACCAACGACCAAGICACCAAGGANGCIGCAGAAGCIATA AAGAAGCATAATGTIGGCGICAAAIG PET4 IaIDHI ( 163) TIRGGATCTACATAGCTATGATITAGGCATAGAGANICGTGAIGCCACCAACGACCAACTCACCAAGGA TGCICCAGAAGCTATAAAGAAGCATAAIGTIGGCGICAANIG Consensus (163 ) TIGGATCTACATAGCTATGATITAGGCATAGACAATCGTGATECCACCAACGACCAAGICACCAAGGA TGCIGCAGAAGCTATAAAGAAGCATAATGTIGGCGTCAMATG 271 , 271 370 380 IDHI ( CDS ) (217 ) IGIGCCACTATCACICCIGATGAGAAGAGGOTTGAGGAGTTCAAGTIGAAACAAAIGIGGAAATCACCAARTGGCACCATACGAAATATICTGGGTGCCACGGICTICAO perdlaIDH1 (271 ) TCTGCCACTATCACICCIGATGAGAAGAGGOTICAGGAGIICAAGTIGAAACAAAICIGGAAITCACCAMATGGCACCATACGALATATTCIGGGICCCACGGICTICAG Consensus ( 271 ) IGIGCCACTAICACICCIGATGAGAAGAGGOTIGAGGAGTICAAGTIGAAACAAAIGIGGAAATCACCAMAIGGCACCATACCAAATATTCTGGGIGGCACGGICTICAG 379 379 390 IDHI (CDS ) (325 ) AGAGAAGCCATTATCIGCAALAATATCCCCCGGCTIGIGAGIGGAIGGGIAAAACCTATCATCATAGGICGICATOCHTAIGGGGATCAATACAGAGCAACIGATYTIGT PET4 laid 1 ( 379 ) AGAGAAGCCATIATCIGCAAAAATATCCCCCGGCTTGTGAGIGGATGGGTAAAACCIATCATCATAGGICGICAPGCITATGGGGATCAATACAGAGCAACTGATTTIGT Consensus (679 ) AGAGAAGCCATTAICIGCAALLATATCCCCCGCCTIGIGAGICCATEGGIALACCIATCAICATAGGICGICOTCC TIANGGGGATCAATACAGACCAACTGATTTIGT ( 487 ) 487 510 550 560 584 596 IDHI ( CDS ) (433 ) GYIGITCCTGGGCCTGGAAAAGTAGAGATAACCIACACACCAAGIGACGGAACCCAALAGGIGACATACCTGCTACATAACTTIGAAGAAGGTGGTGGIGTTGCCATGGG perdlaIDHI (487 ) GITGIICCIGGGCCTGGAAAAGTAGAGATAACCTACACACCAACIGACGGAACCCAAAAGGIGACATACCIGCTACATAACITIGAAGAAGGIGGICCIGITGCCATGGG Consensus ( 487 ) GTIGTICCIGGGCCIGGAAAAGTAGAGATAACCIACACACCAAGIGACGGAACCCAAMAGGIGACATACCIGGIACATAACTTIGAAGAAGGIGGIGGIGITCCCAIGGG (595 ) 595 600 650 660 690 IDHI ( CDS ) (541 ) GGGATGTATAATCAAGATAAGICAATIGAAGATTTIGCACACAGTICCTICCAAATGGCTCIGICTAAGGGTIGGCCTTIGIATCTGAGCACCAAAAACACTATIC TGAA PET4laidh1 (595 ) GGGATGTATAATCAAGATAAGICAATIGAAGATITIGCACACAGTICCTICCAAANGGCTCIGICTAAGCGTIGGCCTTTGTATCIGAGCACCAAAAACACTATTCIGAA Consensus ( 595 ) GEGAIGTATAATTAAGATAAGICAATIGAAGATITIGCACACACTICCTICCAPATGGCICIGICTAAGGGTICGCCITTGIATCTCAGCACCALADACACTATICTGAA (703 ) 703 710 120 812 IDHI ( CDS ) (649 ) AAGALATATGATGGGCGTTTTAAAGACATCIFICAGGAGATATAIGACAAGCAGIACLAGICCCAGITTGAAGCTCAALAGATCIGGIATGAGCATACGCICATCGACGA PET4laIDHI (703 ) AAGAAATATGATGGGCGITTTAAAGACATCIIICAGGAGATATATGACAAGCACIACAAGICCCAGITIGAAGCTCAAAAGATCIGGIATGAGCATAGOCICATCGAUGA Consensus ( 703 ) AAGAASTAIGANGGGCGITTTAAAGACAICITICAGGAGATATANGACAAGCAGIACLAGICCCAGTITGAAGCICAALAGAIC OGGIAIGACCAPACGCICATCGACGA (B11 811 920 IDH1 (CDS ) ( 757 ) GACANGGIGGCCCAAGCTATGAAAICAGAGGGAGGCTICATCIGGGCCIGTAAAAACTATGATGGIGACGIGCAGIC GGACTCTCTCGCCCAAGGGTATGGCICICTOGG PET4 IaIDH1 (811 ) GACANGGIGGCCCAAGCTAIGAAATCACACGGAGGCTICATCIGGGCCIGTAAAAACTAIGAIGGIGACCIGCAGTCGGACICTGTGGCCCAAGGGIATGGCTCTCTCGG Consensus ( 011 ) GACANGGIGGCCCAAGCTATGAAATCAGAGGGAGGCTICATCTGGGOCIGTAAAAACTATGAIGGTGACGIGCAGICGGACICIGIGGOCCAAGGGTAIGGCICICICGG ( 919 ) 919 990 1028 IDHI ( CDS ) (665 ) GGCATGATGACCAGCGIGCIGGITIGTCCAGAIGGCAAGACAGTAGAAGCAGAGGCIGCCCACGGGACTGIAACCCGICACTACCGCAIGTACCAGAAAGGACAGGAGAC PET41aIDH1 (919 ) GGCAIGATGACCAGCGIGCTGGITIGACCAGANGGCAAGACAGTAGAAGCAGAGGCIGCCCACGGGACIGIAACCOGICACTACCGCAIGTACCAGAAAGGACAGGAGAO Consensus (919 ) GGCANGATGACCAGOCIACIGATINGTCCAGAIGGCAAGACAGTAGAAGCAGAGGCIGCCCACGGGACIGTAACCCGICACIACCGCAIGTACCAGAHAGGACAGGAGAC (1027 1027 1040 1060 1080 1120 1136 IDF1 (CDS ) ( 973 ) ACGTCCACCIATOCCAFIGCITCCATITTIGCCTGGACCAGAGGGTTAGCOCACAGAGCAAAGCTTGATAACAATAMAGAGCTIGCCTICHTIGCAAAIGCTTIGGAAGA PET41aIDH1 ( 1027 ) ACGICCACCAATOCCATTGCTIC CATITTTGCCTGGACCAGAGGGTTACCCCACAGAGCAAAGCTIGATAACAATAAAGACCTTGCCTICTTTGCAAAIGCTTTGGAAGA Consensus ( 1027 ) ACGTCCACCADICCCATIGCTICCATTITIGCCIGGACCAGAGGGITAGCCCACAGAGCARAGCIIGATAACAITRAAGAGCIICCCTICITIGCAAAIGCTTIGGAAGA ( 1135 1135 1140 1150 1160 1170 1180 1190 1200 1210 1230 1244 IDH1 ( CDS ) ( 1081) GAAGTCTCTATTGAGACAATTGAGGC'NGGCTICAIGACCAAGGACTIGGCTGCTIGCATTAMAGGITIACCCAATGTGCAACGTTCTGACTACTIGAATACATITGAGIT PET41aIDHI( 1135 ) GAACICICTATTGAGACAATTGAGGCIGGCTICATGACCAAGGACTIGGCIGCTIGCATTAALGCTTTACCCAATCTGCAACCIICIGACTACITGAATACATTIGACIT Consensus ( 1135 ) GAAGICICTATIGAGACAATIGAGGCTEGCITCAIGACCAACGACTIGGCTACTIGCATTAAAGGTTTACCCAATGTGCAACCHICOGACTACTIGAATACATTIGAGIT ( 1242 1242 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1351 IDHI ( CDS ) ( 1188 ) GHICATGGATAAACTIGGAGASAAC TIGAAGATCARACTAGCICAGGCCAMACTTTAA - + . . *** ORDCOM PET41a1DH1 ( 1242 ) GTICATGGATAAACINGGACARACTIGAAGATCAAACTAGCTCAGGCCAAACTTICACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAATACCTAGGCIG Consensus ( 1242 ) GIICATGGATAMACTTGGAGALACTIGAAGAICARACTAGCTCAGGCCARACTIT A Fig . 1 U.S. Patent Apr. 7 , 2020 Sheet 2 of 49 US 10,610,125 B2

(55 ) 55 60 80 110 130 IDH1 ( CDS ) ( 1 ) ATGICCAAAAAAATCAGTGGCGGI ICIGIGGIAGAGAIGCAAGGAGAIGAAMIGACACGAATCATTTGGGAATTGATTAAAGAGAAACICATIITICCCIACGIGGAAM PET41aIDH1 (55 ) ATGICCAAAAAAA GIGG GGTAGAGATGCAAGGAGATGAAATGACACGAATCATTTGGGAATTGATTTIGAAMIGACACGAATICATIIGGGAATIGATTAAAGAGAAACICATTITICCCTACGIGGA Consensus ( 55 ) AIGICCALARAJATCAGICG IGIGGTAGAGATGCAAGGAGATGAMAIGACACG.LTCATTTGGGAATIGATIALAGAGAAACICATIITICCCTACGIGGAATI ( 163 ) 163 210 220 250 260 272 IDH1 ( CDS ) ( 109 ) TIGGAICTACATAGCTATGATTTAGGCATAGAGAATCGTGAIGCCACCAACGACCAAGTCACCAAGGATGCIGCAGAAGCTATAAAGAAGCATAAIGTIGGCGICAMATG pôr41a1dH1 ( 163 ) TIGGATCTACATAGCIAIGATITAGGCATAGAGAATCGIGATGCCACCAACGACCAAGICACCAACGAIGCIGCAGAA AAIGTIGGCGICAAAR Consensus ( 163) TIGCAICTACATAGCTAIGATTIACCCATAGAGAATOGIGA'TGCCACCAACGACCAAGICACCAAGGAIGCIGCAGHAGCTATAAACAACCATAATGTIGGOGICALATO ( 271 ) 271 280 290 310 IDH1( CDS ) (217 ) ICTGCCACTAICACICCIGATGAGAAGAGGGTIGAGGAGTICAAGTIGALACAAN6395A ! mut?tionAICARAGUA CAANIGGCACCHIHCGAAATATICI -gives - Atg.137 PET4làidhi (271 ) TGTGCCACTATC ACTCCTGATGAGRAGAGGGTTGAGGAGTICALGTIGAC394A1mutation clives aro 132 Consensus (271 ) TGIGCCACTATCACTCCTGAIGAGAAGAGGGTTGAGGAGTTCAAGTIGALACANANGTTGGAAATCACCARATGGCACCHACGARATA ITUGTO TCTICAG (379 ) 379 488 IDH1 ( CDS ) (325 ) AGAGAAGCCATTATCIGCAAAAATATCCCC GIGGANGGGTAAAACCIATCAXCATAGETCG CATCCTTANGGGGAICAATACAGAGCHACUGATTINGT PET41a1DH1 ( 379 ) AGAGAAGCCATTATCIGCAAAAATATCCCCCGG GAIGGGTAAAACCIATCATCATAGGICGICATGCTIATGGGGATCAATACAGAGCAACTGATTOIGT Consensus (379 ) AGAGAAGCCATTATCIGCAAAAATATCCCCCGGCTIGTGAGIGGA TCGGTAAAACCIATCATCATAGGICGICATGCTIATGGGGATCAATACAGAGCAACIGATITIGT 520 550 560 570 580 596 IDH1 ( CDS ) ( 433 ) CITCTICCIGGGCCTCGAAAAGTAGAGATAATAAOCTACACACCAAGIGACGGAACCCAA GIGACATACCIGGTACATAACTTTGAAGAAGGT UIVIT000 PET41aIDH1 ( 487) CITGIICCIGGGCCTGG CACACCAAGIGACGI CCCAAAAG GACATACCAGGIACATAACTTIGAAGAA M Consensus ( 487) GPTGTICCIGGGCCAGGAAR TAGAGATAACCTACACACCAAGIGACGGAACCCAAMAGGIGACATACCIGGTACATAACTINGAAGAAGGTCGIGGIGITCCCANGGG (595 ) 595 600 690 IDHI( CDS ) ( 541 ) GGGATGTATAATCAAGATAAGTCAATIGAAGATTTIGCACACAGTICCTICCAANIGGCTCIGICTAAGGGTIGGCCTTIGTAICTGAGCACCAMARACACIAPICIGAM P?T41a1DH1 (595 ) GGGATGIATAATCAAGATAAGICAATIGAAGATITIGCACACAGTICCTICCAAATGGCTCTGICTAAGGGTIGGCCTIIGIATCIGAGCACCAAAAACACTATICIGAA Consensus (595 ) GGGAIGTATAATCAAGATAAGICAAITGAAGATITIGCACACAGTICCTICCALATCGCTCIGICTAAGGGIICGCCTTIGTAICICAGCACCARADACACTATICICAA (703 ) 703 710 730 750 780 790 300 312 IDH1( CDS ) 1649 ) AAGAAATA'TGATGGGCGITTTAAAGACATCTITCAGGAGATATATGACAAGCAGTACAAG'ICCCAGITIGAAGCICAAAAGATCIGGIATGAGCATAGGCTCATCGACGA PET41PIDH1 (703 ) AAGAAATATGATGCGCGTITTAAACACATCTCGTTTTAANGACATCTINCAGGAGATATATGACAAGCAGYACAACICCCAGITIGAAGCICAAAAGAICIGGIATGAGCATAGGCICATCGACGA Consensus ( 703 ) AAGAAATATGATGGGCGTITIAAAGACAICTITCAGGAGATATATGACAACCAGTACAAGIOCCAGTTIGAAGCICAAAAGAICIGGIATGACCATAGGCICATCGACGA (811 ) 811 820 830 960 910 920 IDH1( CDS ) (757 ) GACATGCIGGCCCAAGCTATGARATCAGAGGGAGGCTICATCIGGGCCIGTAAAAACTATGATGGIGACGIGCAGICGGACIC IGIGGCCCAAGGGTATGGCTCICIOGG PET41a1DH1 ( 811 ) CACATGGIGCCCCAAGCTATGAAATCAGAGCGAGGCITCAICTGGGOCIGTAAAAACTATGATGGIGACGTCCAGTCGGACIC TGTGCCCCAAGGGTAIGGCTCTCTCGG Consensus ( 811 ) GACATGGIGGCCCAAGCTATGADATCAGAGGGAGGCTICARCIGGGCCTGTAAAADCTATGAIGGIGACGIGCAGTCGCACTCIGICGCCCAAGGGTATGGCTCICICGG ( 919 ) 919 970 980 990 1010 1028 IDA1( CDS ) ( 865 ) GGCAIGATGACCAGCGIGCIGGITIGICCAGATGGCARGACAGTAGAAGCAGAGGCIGCCCACGGCACIGTAACCCGTCACTACCGCAIGTACCAGAAAGGACAGGAGAC pwT41aID 1 (919 ) GGCATGAIGACCAGCGIGCIGGITIGTCCAGAIGGCAAGACAGTAGAAGCAGAGGCIGCCCACGGGACIGPAACCCGICACTACCGCAIGIACCAGAAAGGACAGGAGAC Consensus ( 919 ) GGCATGAIGACCAGCGIGCTGGITIGTCCAGAIGGCAAGACAGTAGAAGCAGAGGCIGCCCACGGGACIGTAACCCGICACTACCCCATGTACCAGAAAGGACAGGAGAC ( 1027 ) 1027 1050 1060 1070 1080 1100 1110 1120 1136 IDHI( CDS ) (973 ) ACGICCACCAATOCCATIGCTICCATTTTICOCIGGACCAGAGGGTTAGCCCACAGAGCAMAGCIIGATAACAATAMAGAGCTIGCCTICITIGCAAATGCTTIGGAAGA PET41a1DH1( 1027) ACGICCACCAATCCCATIGCTTCCATITITGCCIGGACCAGAGGGTTAGCCCACAGAGCAMAGCTIGATAACAATAMAGAGCTTGCCTICINIGCAAAIGCTTIGGAAGA Consensus ( 1027 ) ACGICCACCAATCCCATIGCTICCATTTTIGCCIGGACCAGAGGGTTAGCCCACACAGCAAAGCTIGATAACAATAAAGAGCIIGCCTTCTTIGCARATGOTYNGGAAGA (1135 ) 1135 1140 1160 1190 1200 1210 1220 1230 IDHI ( CDS ) ( 1081 ) GAAGICICTATTGAGACAATIGAGGCIGGCTICATGACCAAGGACTIGGCIGCTIGCATTAMAGGITTACCCALIGIGCAACGTICIGAC TACTIGAATACATTIGACTI PET41a1DH1( 1135 ) GAAGTCICTATTGAGACAATTGAGGCTGGCTICATGACCAAGGACTTGGCTGCTACCATTAMAGGITTACCCAATGTGCAACGTICIGACTACTIGA ATACATTIGAGTT Consensus ( 1135 ) GAAGICICTATTGAGACAATIGAGGCIGGCTICATGACCAAGGACTIGGCTGCTIGCATTAAAGGITTACCCAATGIGCALACGTICTGACTACTICAATACATTIGACII ( 1242 ) 1242 1250 1260 1270 1280 1290 1300 1310 1320 1340 1351 IDH1 ( CDS ) ( 1188 ) CITCATCGATA ACTIGGAGAAAACTIGAAGATCAAACTACCICAGGCCARACTITA - WOODWOOD PET41 aIDF1( 1242 ) GITCAIGGATAAACTICGAGAMAACTIGAAGATCAPACTAGCTCAGGCCAPACITICACTCGAGCACCACCACCACCACCACCACCACTAATIGATTAATACCIAGGCIG Consensus ( 1242 ) GIICATGGATAACHIGGAGAMAACTTGAAGATCAPACTAGCTCACGCCAAACTII A Fig . 2 U.S. Patent Apr. 7, 2020 Sheet 3 of 49 US 10,610,125 B2

Collect 1000 # 17 # 27 ColumnColum : 6 mlNi -Sepharose column Sample : 30ml Bacteria Extract 800 Buffer A : 20 mM Tris , pH7.4 500MM Nao 600 5 mM b -mercaptoethanol 10 % glycerol 400 Buffer B : Buffer A 400mM Imidazole 200 1 protease inhibitor tablet /200 ml Elution : Liner gradient ofBuffer B 3579114 12 60123 261291321 Waste ( 10-100 % ) in 20CV 150 200 250 ml Fig . 3

M T S L 17 18 19 20 21 M 22 23 24 25 26 27

Fig . 4 U.S. Patent Apr. 7, 2020 Sheet 4 of 49 US 10,610,125 B2

MAU 200 Column: Sephacryl S - 200 (320ml ) Sample : 10mlIDHwt sample from Ni Sepharose column 1501 Buffer C : 50 mM Tris pH 7.4 ; 500 MM NaCl; 5 mm B -mercaptoethanol 10 % glycerol 50 Loading 0 1023111911141ten 7505350 50 100 150 200 ml Fig . 5A

IDHwt

Fig . 5B U.S. Patent Apr. 7, 2020 Sheet 5 of 49 US 10,610,125 B2

MAU # 3-7 7 31

+ Column : 5 mlNi -Sepharose 1500 Sample : 30 mlBacteria Extract Buffer A : 50 mM Tris , pH 7.4 ; 500 MM NaCl; 5 mM 1000 B -mercaptoethanol 10 % glycerol Buffer B : Buffer A : 400mm Imidazole 500 9%0L+V06 Elution : liner gradient of Buffer B Loading elute ( 10--100 % ) in 20 CV kabázel Waste 250 300 350 mi Fig . 6

Fermentas x x x x x x x x x x x x x x x x Protein Marker So in 4 5 7 116.0 kDa

66.2 kDa w :: 45.0 kDa 35.0 kDa 25,0 kDa 18,4 kDa 14,4 kDa $ ??? ??? ?? poo po po pod pog poo ooo ooo ooo ooo ooo ooo Fig . 7 U.S. Patent Apr. 7, 2020 Sheet 6 of 49 US 10,610,125 B2

MAU # 13-22 500 Column: S - 200 XK2660 400 Sample 10 cd sample from Ni- Sepharose column Buffer C : 50 mM Tris, pH7.4 300 500ml NaCl, 5 mM B mercaptoethanol 10 % glycerol 200 100 Loading

20 40 60 80 100 120 140 ml Fig . 8A

M DHR132S

Fig . 8B U.S. Patent Apr. 7, 2020 Sheet 7 of 49 US 10,610,125 B2

MAU # 5 # 10 . that 3 $ 1 :1 Column: 5 mlNi - Sepharose 2000 w Sample : 30 mlBacteria Extract Buffer A : 50 mM Tris ,pH 7.4 ; 500 mm NaC ); 5 MM 6 -mercaptoethanol : 10 % glycerol; Protease Inhibitor Cocktail (Roche ) 1 tablet/ 200 ml Buffer B : Buffer A plus 400 mM Imidazole 500 Elution ; liner gradient of Buffer B ( 10-100 % ) in 20 CV 0 230km Waste 100 U120 140 160 180 200 220 ml Fig . 9

3 Fermentas So in Ft M5 6 7 9 10 E Ni Protein Markering 116.0 kDa 66.2 kDa 45.0 kDa 35.0 kDa .* 25.0 kDa *** 18.4 kDa 14.4 KDa co *** a * x x x x x x x x x x Fig . 10 U.S. Patent Apr. 7, 2020 Sheet 8 of 49 US 10,610,125 B2

# 6- # 9 MAU Column: S - 200 XK26'60 600 Sample : 9 ml sample from Ni- Sepharose column Buffer C : 50 mM Tris ,pH7.4 400 500 mM NaC1,5 mMB mercaptoethanol 10 % glycerol 200 Loading

0 20 40 60 80 100120 140 ml Fig . 11A

M DHR132H

Fig . 11B U.S. Patent Apr. 7 , 2020 Sheet 9 of 49 US 10,610,125 B2

ICDH1wild - type forward reaction

30 EXTY 25 20 Vmax = 30,5 Rate(umol/minmg) Km = 56.8 15 - 10 en

0 --- 200 400 600 800 1000 1200 DL- Isocitrate (UM ) Fig . 12A IDH1 R132H 0.5 0.4

Rate(umolmin/mg) 0.3 Vmax = 0.6052 Km = 171.7 n = 0.6001 0.1

0.0 ???? 0 200 400 600 800 1000 1200 Isocitrate (UM ) Fig . 12B ICDH1 R132S 3.5

2.5 mgmV/sec)(umolmin 2.0 Vmax 93.5 Km - 1.125e - 1-6 n = 0.4794 0.5 0.0 0 200 400 600 800 1000 1200 Isocitrate (UM ) Fig . 120 U.S. Patent Apr. 7 , 2020 Sheet 10 of 49 US 10,610,125 B2

a - KG inhibition of ICDH1 wt 0.35 0.30 0.25 1/Rate(umolminmg) 0.20 * 1 = 1167 who 0.15 1 = 3500 0.10 0.05-2 .02 -0.01 0.00 0.01 0.02 0.03 0.04 1 / [isocitrate ] (UM ) Fig . 13A

a - KG inhibition of ICHDI R132H

1/Rate(umolminmg) 6

w 4 Ol = 30 nom 85.3 2 mom 256 -0.005 0.000 0.005 0.010 0.015 0.020 1 / [ isocitrate ] ( UM ) Fig . 13B a - KG inhibition of ICHDI R132S

8 1/Rate(umolmin/mg) 4 1 - 30 . 85.3 2 : 1 256 ww -0.005 0.000 0.005 0.010 0.015 0.020 1 / [isocitrate ] ( UM ) Fig . 130 U.S. Patent Apr. 7, 2020 Sheet 11 of 49 US 10,610,125 B2

Reverse Reaction a - KG to isocitrate

or or

au CELL DUTTO

LETTEOW DEUD OD340 0.8 0.6 FONNUDO IT ANNOR NORZNA NG NON ICDH1 wt 0.4 ICDH1 R132HWOLLE OOTDTT NU AM NOMY ICDH1 R132S 0.2

200 400 600 800 1000 time (sec ) Fig . 14 U.S. Patent Apr. 7, 2020 Sheet 12 of 49 US 10,610,125 B2

ICDH R1324 Reverse Reaction

1.2 1.0 Vmax = 1.3 Rate(umol/minmg) 0.8 Km = 0.9657 0,6 n = 1.8 0.4 0.2 un 10 15 20 25 [a - ketoglutarate ] (MM ) Fig . 15A

ICDH R132S 2.5 2.0 Rate(umol/minmg) Vmax = 2.7 Km = 0.1813 n = 24.6 0.5

0.0 0.04 20 25 (a -ketoglutarate ] (MM ) Fig . 15B U.S. Patent Apr. 7, 2020 Sheet 13 of 49 US 10,610,125 B2

NADH in reverse reaction

tw IDHIWt 30 ug /ml 1.4 IDH15 30 ug /ml --3-- IDH1h 30 ug /mi Absorbance(OD340) 1 www no enzyme 0.8 Starriage 0.6 0.4 0.2

200 600 Time ( s ) Fig . 16 U.S. Patent Apr. 7, 2020 Sheet 14 of 49 US 10,610,125 B2

Oxalomalate Inhibition of IDH1 wt 2.0 Vmax 31.2 Km = 55.5 Ki - 955.4 1Rate/(umolminmg) mu non { 12390 mom 37170

-0.01 0.00 0.01 0.02 0.03 1 / [ isocitrate ] (UM ) Fig . 17A

Oxalomalate Inhibition of IDH R132H

35 Vmax = 0.5353 km = 84.3 Ki = 950.8 25 1/Rateumol(minmg) 20 *** 805 ... : 2414 10 7242 5

-0.02 -0.01 0.00 0.01 0.02 0.03 0.04 0,05 1 / [ isocitrate ] (UM ) Fig . 17B U.S. Patent Apr. 7, 2020 Sheet 15 of 49 US 10,610,125 B2

Oxalomalate inhibition of IDH R132S

Vmax 3.2 Km = 211.3

6 mm 1/Rate(umolminmg) 246 www 737 * ] 2211

-0.01 0.00 0.02 0.03 0.04 0.05 1 / [ isocitrate ) (UM ) Fig . 170 U.S. Patent Apr. 7, 2020 Sheet 16 of 49 US 10,610,125 B2

11.50 Max6100.0cps. 11.03 10.44 tukipindikuonyeshapesardeteruportesplastici 11.0 10.0 9.71 10.27 9.549.26.

7.43 7.998.77 7.26 6.07.08.0 Detale 6.176.606,74 Time,min LC-MS/analysisofthecontrolreaction.ThepresenceaKGisindicatedbypeakat7.26 18A.Fig OXICof-MRM(3pairs):144.9/100.9DalDakofromSample1negativeneat20090417F.wiffTurboSpray 5.535.83 5.17 4.40 4.33 13.813.07 minutes(blue)whilenoisocitrateisobservedred. 2.652.743.44 3.02.0 2.15

0.58 6000 5500 5000 40001 3500 3000 2500 2000 1000 500 0 U.S. Patent Apr. 7, 2020 Sheet 17 of 49 US 10,610,125 B2

11.48 Max1613.3cps. 10.2410.3710.81 11.25 9.439.87 9.191 8.81 9.0 8,128.49 8.05 7.49 7.36 6.81 6.52 LC-MS/analysisofthereactioncontainingenzyme.Noisocitrateisobserved(red),andaKG Time,min 18B.Fig OXICof-MRM(3pairs):144.9/100.9DalDaKGfromSample2R132Hneat20090417F.wiffTurboSpray 6.36 6.04 5.33 6.05.0 5,20 4.64 3.89 3.502.81 3.42 hasbeencompletelyconsumed(blue). 1.68 2.17 wem 1.55, 2.01.0 0.57 10.88 0.52 1600 1500 1400 1300 1200 1000 800 500 400 300 200 1000 0 U.S. Patent Apr. 7, 2020 Sheet 18 of 49 US 10,610,125 B2

Max1533,3cps. 11.1510.02 10.71111.35 ?????).???????? ??

9.0 0.379.5110.07.211,15(9.218.578.357.914 18.51 7.91

6.07.05.0 Time,min finalconcentrationofisocitrate.WhentheterminatedR132H-containingreactionwasspikedtoaconcentration 18C.Fig OXICO-MRM(3pairs):144,9/100.9DalDaKGfromSample41mMISCCof20090417FwiftTurboSpray 1mMisocitrate,itwasreadilyobserved(red);essentiallycompleteconsumptionofa-KGconfirmedblue. empreunaofrecemosSe LC-MS/analysisofthespikedcontrolreaction.Theinstrumentasconfiguredcanreadilydetectpresumptive

2.03.0

0.57 5.084 4,6e4 4.4e4 4.284 4.0e4 3.8e4 3.664 3.4e4 3.2e4 3.024 2.8e4 2.664 2.4e4 2.2e4 2.0e4 1.6e4 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.00 0.0 U.S. Patent Apr. 7, 2020 Sheet 19 of 49 US 10,610,125 B2

Max3.2e4cps. 11.19 10.640.319.46

9.20 28.01 8.58 7.75 7.14 6.426,75 5.82 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5 Time,min identifiedthe147.1/128.7MRMtransitionasapeakretainedat7.14minutes.The0.52isaninstrumentartifact 19.Fig OXICof-MRM(5pairs):147.1/128.7DaIDDahydroxyglutaricacidfromSample150ugmlDAHG20090422A.wiffTurboSpray TT LC-MS/analysisofalphahydroxyglutarate.Theinstrumentwasoptimizedforthedetection2hydroxyglutarateand 4,535.254.714.17 ,3.69

2.642.90 diversionvalve.switchingofaninlinebycaused 0.52 3.2e47 3.0e4, 2.8e4 2.6e4. 2.4e4 2.2e4. 2.0e4 1.824 1.6e4. 1.4e4 1.2e4 1.0e4 8000.0 6000.0 4000.0 2000.00 0.0 U.S. Patent Apr. 7, 2020 Sheet 20 of 49 US 10,610,125 B2

Max8820.0cps. 11.38 9.99 9.870.33.03 8.458.859.44

8.17 28 6.907 *************www.primaparte

.92 - ****** 0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5 Time,min 20.Fig 4.90 OXICof-MRM(5pairs):147.1/128.7DalDDahydroxyglutaricacidfromSample216Apro9Negative200904228.wiffTurboSpray

4.03 21784.154.62 3.11 1.64 1.101.23 5.0e4 4.8e4. 4.6e4 4.4e4 4.2e41 4.0e4 3.8241 3.6e4 3.4e4 3.2e4 3.084 2.8e4 2.6e4 2.4e4 2.2e4 2.004 1.8e41 1.6041 1.4e4 1.2e4 1.0e4 6000.010.51 4000.0 2000.0 Audio0.0 U.S. Patent Apr. 7, 2020 Sheet 21 of 49 US 10,610,125 B2

1 mskkisggsy venggdemtr iiwelikekl ifpyveldlh sydlgienrd atndqvtkda 61 aeaikkhnvg vkcatitpde krveefklkq mwkspngtir nilggtvfre aiickniprl 121 vsgwvkpiii grhaygdqyr atdfvvpgpg kveitytpsd gtqkvtylvh nfeeggqvam 181 gmyngdksie dfahssfqma Iskgwplyls tkntilkkyd grfkdifgei ydkqyksqfe 241 agkiwyehrl iddmvaqank seggfiwack nydgdvgsds vaqgygslgm mtsvlvcpdg 301 ktveaeaahg tvtrhyrmyg kggetstnpi asifawtrgl ahrakldnnk elaffanale 361 evsietieag fntkdlaaci kolpnvqrsd ylntfefmdk igenlkikla qakl Fig . 21 U.S. Patent Apr. 7, 2020 Sheet 22 of 49 US 10,610,125 B2

1 atgtccaaaa aaatcagtgg cggttctgtg gtagagatge aaggagatga aatgacacga 61 atcatttggg aattgattaa agagaaactc atttttccct acgtggaatt ggatctacat 121 agctatgatt taggcataga gaatcgtgat gccaccaacg accaagtcac caaggatgct 181 gcagaagcta taaagaagca taatgttggc gtcaaatgtg ccactatcac tcctgatgag 241 aagagggttg aggagttcaa gttgaaacaa atgtggaaat caccaaatgg caccatacga 301 aatattctgg gtggcacggt cttcagagaa gocattatct gcaaaaatat cccccggett 361 gtgagtggat gggtaaaacc tatcatcata ggtcgtcatg cttatgggga tcaatacaga 421 gcaactgatt ttgttgttcc tgggcctgga aaagtagaga taacctacac accaagtgac 481 ggaacccaaa aggtgacata cctggtacat aactttgaag aaggtggtgg tgttaccato 541 gggatgtata atcaagataa gtcaattgaa gattttgcac acagttcctt ccaaatgget 601 ctgtctaagg gttggccttt gtatctgage accaaaaaca ctattctgaa gaaatatgat 661 gggcgtttta aagacatott tcaggagata tatgacaagc agtacaagtc ccagtttgaa 721 gotcaaaaga tctggtatga gcataggctc atcgacgaca tggtggocca agctatgaaa 781 tcagagggag gettcatctg ggcctgtaaa aactatgatg gtgacgtgca gtcggactct 841 gtggcccaag ggtatggotc totcggcatg atgaccagcg tgctggtttg tocagatggo 901 aagacagtag aagcagaggc tgcccacggg actgtaaccc gtcactaccg catgtaccag 961 aaaggacagg agacgtccac caatcccatt gottccattt ttgcctggac cagagggtta 1021 gcccacagag caaagcttga taacaataaa gagcttgcct tctttgcaaa tgctttggaa 1081 gaagtctcta ttgagacaat tgaggctggc ttcatgacca aggacttggc tgottgcatt 1141 aaaggtttac ccaatgtgca acgttctgac tacttgaata catttgagtt catggataaa 1201 cttggagaaa acttgaagat caaactaget caggccaaac tttaa Fig . 21A U.S. Patent Apr. 7 , 2020 Sheet 23 of 49 US 10,610,125 B2

1 cctgtggtoc cgggtttctg cagagtctac ttcagaagcg gaggcactgg gagtccggtt 61 tgggattgcc aggctgtggt tgtgagtctg agcttgtgag cggctgtggc gccccaactc 121 ttcgccagca tatcatcccg gcaggcgata aactacattc agttgagtct gcaagactgg 181 gaggaactgg ggtgataaga aatctattca ctgtcaaggt ttattgaagt caaaatgtcc 241 aaaaaaatca gtggcggttc tgtggtagag atgcaaggag atgaaatgac acgaatcatt 301 tgggaattga ttaaagagaa actcattttt ccctacgtgg aattggatct acatagctat 361 gatttaggca tagagaatcg tgatgccacc aacgaccaag tcaccaagga tgctgcagaa 421 gctataaaga agcataatgt tggcgtcaaa tgtgccacta tcactcctga tgagaagagg 481 gttgaggagt tcaagttgaa acaaatgtgg aaatcaccaa atggcaccat acgaaatatt 541 ctgggtggca cggtcttcag agaagccatt atctgcaaaa atatcoccog gottgtgagt 601 ggatgggtaa aacctatcat cataggtcgt catgottatg gggatcaata cagagcaact 661 gattttgttg ttcctgggcc tggaaaagta gagataacct acacaccaag tgacggaacc 721 caaaaggtga catacctggt acataacttt gaagaaggtg gtggtgttgc catggggatg 781 tataatcaag ataagtcaat tgaagatttt gcacacagtt ccttccaaat ggctctgtct 841 aagggttggc ctttgtatct gagcaccaaa aacactatto tgaagaaata tgatgggcgt 901 tttaaagaca tctttcagga gatatatgac aagcagtaca agtoccagtt tgaagctcaa 961 aagatctggt atgagcatag gotcatcgac gacatggtgg cccaagctat gaaatcagag 1021 ggaggcttca tctgggcctg taaaaactat gatggtgacg tgcagtcgga ctctgtggcc 1081 caagggtatg gctctctcgg catgatgacc agcgtgctgg tttgtccaga tggcaagaca 1141 gtagaagcag aggctgccca cgggactgta acccgtcact accgcatgta ccagaaagga 1201 caggagacgt ccaccaatcc cattgcttcc atttttgcet ggaccagagg gttagcccac 1261 agagcaaagc ttgataacaa taaagagctt gccttctttg caaatgottt ggaagaagtc 1321 totattgaga caattgaggc tggettcatg accaaggact tggctgottg cattaaaggt 1381 ttacccaatg tgcaacgttc tgactacttg aatacatttg agttcatgga taaacttaga 1441 gaaaacttga agatcaaact agctcaggcc aaactttaag ttcatacctg agctaagaag 1501 gataattgtc ttttggtaac taggtctaca ggtttacatt tttctgtgtt acactcaagg 1561 ataaaggcaa aatcaatttt gtaatttgtt tagaagccag agtttatott ttctataagt 1621 ttacagcctt tttcttatat atacagttat toccaccttt gtgaacatgg caagggactt 1681 ttttacaatt tttattttat tttctagtac cagcctagga attcggttag tactcatttg 1741 tattcactgt cactttttct catgttctaa ttataaatga ccaaaatcaa gattgctcaa 1801 aagggtaaat gatagccaca gtattgctcc ctaaaatatg cataaagtag aaattcactg 1861 ccttcccctc ctgtccatga ccttgggcac agggaagttc tggtgtcata gatatcccgt 1921 tttgtgaggt agagctgtgc attaaacttg cacatgactg gaacgaagta tgagtgcaac 1981 tcaaatgtgt tgaagatact gcagtcattt ttgtaaagac cttgctgaat gtttccaata 2041 gactaaatac tgtttaggcc gcaggagagt ttggaatccg gaataaatac tacctggagg 2101 tttgtcctct ccatttttct ctttctcctc ctggcctggc ctgaatatta tactactcta 2161 aatagcatat ttcatccaag tocaataatg taagctgaat cttttttgga cttctgctgg 2221 cctgttttat ttcttttata taaatgtgat ttctcagaaa ttgatattaa acactatctt 2281 atcttctcct gaactgttga ttttaattaa aattaagtgc taattaccaa aaaaaaaaa Fig . 21B U.S. Patent Apr. 7, 2020 Sheet 24 of 49 US 10,610,125 B2

MAGYLRVVRSLCRASGSRPAWAPAALTAPTSQEQPRRHYADKRIKVAKPV VEMDGDEMTRIIWQFIKEKLILPHVDIQLKYFDLGLPNRDOTDDQVTIDS ALATOKYSVAVKCATITPDEARVBEFKLKKMWKSPNGTIRNILGGTVFRE PIICKNIPRLVPGWTKPITIGRHAHGDOYKATDFVADRAGTFKMVFTPKD GSGVKEWEVYNFPAGGVGMGMYNTDESISGFAHSCFQYAIQKKWPLYMST KNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKIWYEHRLIDDMVAQVLKS SGGFVWACKNYDGDVOSDILAQGFGSLGLMISVLVCPDGKTIEAEAAHGT VTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNODLIRFAQMLEK VCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALG RO Fig . 22 U.S. Patent Apr. 7, 2020 Sheet 25 of 49 US 10,610,125 B2

1 atggccggct acctgcgggt cgtgcgctcg ctctgcagag cctcaggctc gcggccggcc 61 tgggcgccgg cggccctgac agcccccacc tcgcaagagc agccgcggcg ccactatgcc 121 gacaaaagga tcaaggtggc gaagcccgtg gtggagatgg atggtgatga gatgacccgt 181 attatctggc agttcatcaa ggagaagctc atcctgcccc acgtggacat ccagctaaag 241 tattttgacc tcgggctccc aaaccgtgac cagactgatg accaggtcac cattgactct 301 gcactggcca cccagaagta cagtgtggct gtcaagtgtg ccaccatcac ccctgatgag 361 gcccgtgtgg aagagttcaa gctgaagaag atgtggaaaa gtcccaatgg aactatccgg 421 aacatcctgg gggggactgt cttecgggag cccatcatct gcaaaaacat cccacgccta 481 gtccctggct ggaccaagcc catcaccatt ggcaggcacg cccatggcga ccagtacaag 541 gocacagact ttgtggcaga ccgggccggo actttcaaaa tggtcttcac cccaaaagat 601 ggcagtggtg tcaaggagtg ggaagtgtac aacttccccg caggcggcgt gggcatgggc 661 atgtacaaca ccgacgagtc catctcaggt tttgcgcaca gctgcttcca gtatgccato 721 cagaagaaat ggccgctgta catgagcacc aagaacacca tactgaaagc ctacgatggg 781 cgtttcaagg acatcttcca ggagatcttt gacaagcact ataagacega cttcgacaag 841 aataagatct ggtatgagca ccggctcatt gatgacatgg tggctcaggt cctcaagtct 901 tcgggtggct ttgtgtgggc ctgcaagaac tatgacggag atgtgcagtc agacatcctg 961 goccagggct ttggctccct tggcctgatg acgtccgtcc tggtctgccc tgatgggaag 1021 acgattgagg ctgaggccgc tcatgggacc gtcacccgcc actatcggga gcaccagaag 1081 ggccggccca ccagcaccaa ccccatcgcc agcatctttg cctggacacg tggcctggag 1141 caccggggga agctggatgg gaaccaagac ctcatcaggt ttgcccagat gctggagaag 1201 gtgtgcgtgg agacggtgga gagtggagcc atgaccaagg acctggcggg ctgcattcac 1261 ggcctcagca atgtgaagct gaacgagcac ttcotgaaca ccacggactt cctcgacaco 1321 atcaagagca acctggacag agccctgggc aggcagtag Fig . 22A U.S. Patent Apr. 7, 2020 Sheet 26 of 49 US 10,610,125 B2

1 ccagogttag cccgoggcca ggcagccggg aggagoggcg cgcgctcgga cctctcccgc 61 cctgctcgtt cgctctccag cttgggatgg ccggctacct gcgggtcgtg cgctcgctct 121 gcagagcctc aggctcgcgg ccggcctggg cgccggcggc cctgacagcc cccacctcgc 181 aagagcagec goggcgccac tatgccgaca aaaggatcaa ggtggcgaag cccgtggtgg 241 agatggatgg tgatgagatg acccgtatta tctggcagtt catcaaggag aagctcatcc 301 tgccccacgt ggacatccag ctaaagtatt ttgacctcgg gctcccaaac cgtgaccaga 361 ctgatgacca ggtcaccatt gactctgcac tggccaccca gaagtacagt gtggctgtca 421 agtgtgccac catcacccct gatgaggccc gtgtggaaga gttcaagctg aagaagatgt 481 ggaaaagtcc caatggaact atccggaaca tcctgggggg gactgtcttc cgggagccca 541 tcatctgcaa aaacatccca cgcctagtcc ctggctggac caagcccatc accattggca 601 ggcacgccca tggcgaccag tacaaggcca cagactttgt ggcagaccgg gccggcactt 661 tcaaaatggt cttcacccca aaagatggca gtggtgtcaa ggagtgggaa gtgtacaact 721 tccccgcagg cggcgtgggc atgggcatgt acaacaccga cgagtccatc tcaggttttg 781 cgcacagctg cttccagtat gccatccaga cgccctggcc gctgtacatg agcaccaaga 841 acaccatact gaaagcctac gatgggcgtt tcaaggacat cttccaggag atctttgaca 901 agcactataa gaccgacttc gacaagaata agatctggta tgagcaccgg ctcattgatg 961 acatggtggc tcaggtcctc aagtcttcgg gtggctttgt gtgggcctgc aagaactatg 1021 acggagatgt gcagtcagac atcctggccc agggctttgg ctcccttggc ctgatgacgt 1081 ccgtcctggt ctgccctcat gggaagacga ttgaggctga ggccgctcat gggaccgtca 1141 cccgccacta tcgggagcac cagaagggcc ggcccaccag caccaacccc atcgccagca 1201 tctttgcctg gacacgtggo ctggagcacc gggggaagct ggatgggaac caagacctca 1261 tcaggtttgc ccagatgctg gagaaggtgt gcgtggagac ggtggagagt ggagccatga 1321 ccaaggacct ggcgggctgc attcacggcctcagcaatgt gaagctgaac gagcacttcc 1381 tgaacaccac ggacttcctc gacaccatca agagcaacct ggacagagcc ctgggcaggo 1441 agtaggggga ggcgccaccc atggctgcag tggaggggcc agggctgagc cggcgggtcc 1501 tcctgagcgc ggcagagggt gagcctcaca gcccctctct ggaggccttt ctaggggatg 1561 tttttttata agccagatgt ttttaaaagc atatgtgtgt ttcccctcat ggtgacgtga 1621 ggcaggagca gtgcgtttta cctcagccag t?agtatgtt ttgcatactg taatttatat 1681 tgcccttgga acacatggtg ccatatttag ctactaaaaa gctcttcaca aaaaaaaaaa Fig . 22B U.S. Patent Apr. 7, 2020 Sheet 27 of 49 US 10,610,125 B2

ICDH1s Forward Reaction 0.4 0.35 0.3 0.25 0.2 0.15 . 0.1 0.05 ^^^0 500 1000 1500 2000 2500 3000 3500 4000

ICDH1 R132H Forward Reaction 0.2 0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0 500 1500 2000 2500 3000 3500 4000 Fig . 23 U.S. Patent Apr. 7, 2020 Sheet 28 of 49 US 10,610,125 B2

OXIC OF -MRM ( 1 pair ): 362.9 / 146.6 Da ID : DATAN -2HG from Sample 1 (874 -DHAG ) of 20090617K ... Max 9033.3 cps. 9000 8.02 8500 10.12 8000 Area : 1.575e + 5 -8.11 Area : 1.6510 + 5 7500 7000 -10.26 6500 AG1-874 Racemate Alone 6000 5500 5000 Intensity,cps 4500 4000 3500 10.47 3000 7.91 2500 2000 8.37 1500 -10.61 1000 8.58 10.75 500 speciaticaciliatibha nos Apie kennis ????? ?? saith indian capthasahasranatana pani ? ?? 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 Time, min Fig . 24A U.S. Patent Apr. 7, 2020 Sheet 29 of 49 US 10,610,125 B2

OXIC of -MRM (1 pair) : 362.9 / 146.6 Da ID : DATAN -2HG from Sample 2 ( R -2HG - DHAG ) of 200906 ... Max 4680.0 cps . 4680 9.78 4600 4400 4200 9.85 4000 3800 3600 3400 3200 3000 R - 2HG Standard 2800 2600 Intensity,cps 2400 2200 2000 1800 1600 -10.08 1400 1200 1000 800 600 -10.27 400 2004 Aynanagh 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 Time, min Fig . 24B U.S. Patent Apr. 7, 2020 Sheet 30 of 49 US 10,610,125 B2

OXIC of -MRM ( 1 pair) : 362.9 /146.6 Da ID : DATAN -2HG from Sample 3 (874 + RSTD ) of 20090618 ... No peak detection if number of points exceeds 1040 Max 3890.0 cps. 3900 3800 3700 3600 3500 3400 3300 3200 ) 3100 3000 Racemate + R - Standard 2900 2800 2700 2600 2500 2400 2300 2200 2100 Intensity,cps 2000 1900 1800 1700 1600 1500 1400 1300 1200 1000 900 800 700 600 400 300 200N 100 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 Time, min Fig . 240 U.S. Patent Apr. 7, 2020 Sheet 31 of 49 US 10,610,125 B2

OXIC OF -MRM ( 1 pair) : 362.9 / 146.6 Da ID : DATAN -2HG from Sample 1 (Derivitized Enzyme Rxn 1" ... Max 4126.7 cps. 4127 10.03 4000 3800 3600 3400 3200 3000 2800 Enzyme Rxn Alone 2600 2400 Intensity,cps 2200 2000

1600 1400 1200 1000 800 600 400 4.08 9.80 200N

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 Time, min Fig . 24D U.S. Patent Apr. 7, 2020 Sheet 32 of 49 US 10,610,125 B2

OXIC of -MRM ( 1 pair ) : 362.9 / 146.6 Da ID : DATAN -2HG from Sample 2 (Rxn + RSTD ) of 20090618 ... No peak detection if number of points exceeds 1040 Max 4726.7 cps . 4727 4600 4400 4200 4000 3800 3600 3400 Enzyme Rxn + 2 - R -HG Standard 3200 3000 2800 2600 Intensity,cps 2400 2200 Su 2000 1800 1600 1400 1200 1000 800 600 400 2004 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 Time, min Fig . 24E U.S. Patent Apr. 7, 2020 Sheet 33 of 49 US 10,610,125 B2

OXIC OF -MRM ( 1 pair ) : 362.9 / 146.6 Da ID : DATAN -2HG from Sample 5 (Rxn +874 ) of 200906188 ... 10.14 Max 2413.3 cps. 24001 2300 22001 Racemate + Enzyme Rxn 2100 2000 1900 1800 1700 1600 1500 8.28 1300 w 1200

1000 900 800 700 600 8,42 500 400 300 10.45 11.90 200 5.40 6.77 8.67 10.56 13.43 100 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13,0 14,0 15,0 16,0 17.0 18.0 Time, min Fig . 24F U.S. Patent Apr. 7, 2020 Sheet 34 of 49 US 10,610,125 B2

Stereo - Specific inhibition of ICHD1 wt by hydroxyglutaric acid 1.2

1.0

0.8 INOA 0.6 IC50 = 17 UM e reduction of aKG to aHG by ICDH1 R132H OR -alpha - hydroxyglutaric acid standard 0.2

0.0 1e - 10 1e - 7 le - 6 1e - 3 ( alpha- hydroxyglutaric acid ] M Fig . 25 U.S. Patent Apr. 7, 2020 Sheet 35 of 49 US 10,610,125 B2

GBM - 2610 (LN - 18 ) 90 80 2 -HG 60 Concentrations(UM) 50

hand 40 30 20 10 p = 0.007 ( W : R )

Parental WT R132 Fig . 26A

HTB - 14 ( U - 87 )

700 600

Concentrations(UM) 500 2 - HG

300 200 p = 0.001 (W :R ) 100

Parental R132 Fig . 26B U.S. Patent Apr. 7 , 2020 Sheet 36 of 49 US 10,610,125 B2 gcataatgagctctatatgccatcactgcagttgtaggttataactatccatttgtctgaaaaactttgcttc taatttttctctttcaagCTATGATTTAGGCATAGAGAATCGTGATGCCACCAACGACCAAGTCACCAAGGAT GCTGCAGAAGCTATAAAGAAGCATAATGTTGGCGTCAAATGTGCCACTATCACTCCTGATGAGAAGAGGGTTG AGGAGTTCAAGTTGAAACAAATGTGGAAATCACCAAATGGCACCATACGAAATATTCTGGGTGGCJGTCTTCA GAGAAGCCATTATCTGCAAAAATATCCCCCGGCTTGTGAGTGGATGGGTAAAACCTATCATCATAGGTCGTCA TGCTTATGGGGATCAAgtaagtcatgttggcaataatgtgattttgcatgbtggcccagaaatttccaacttg tatgtgttttattcttatcttttogtatctacacccattaagcaaggta 27.Fig U.S. Patent Apr. 7, 2020 Sheet 37 of 49 US 10,610,125 B2

2HG akG malate fumarate succinate isocitrate 100.00

MetaboliteConcentrationumole(/g) 10.00

0.10 mutant mutant***** mutantu?unud . mutant wt mutant mutant Fig . 28 U.S. Patent Apr. 7, 2020 Sheet 38 of 49 US 10,610,125 B2

San CLOSED CLOSED CLOSED OPEN SED VS. VS. (R132H ) (R132H ) Fig . 29A U.S. Patent Apr. 7, 2020 Sheet 39 of 49 US 10,610,125 B2

Thr77 NADP+ Ser94 Thr214

Asn95 139Tyr 212Lys 252Asp 100Arg 109Arg 275Asp 132Arg 29B.Fig Thr77

Ser94 4: Thr214 NADPH

95Asn

Tyr139 Xxx Lys212 252Asp 100Arg 275Asp 23+ +++++00 109Arg R132H His132 U.S. Patent Apr. 7, 2020 Sheet 40 of 49 US 10,610,125 B2

" NADP + NADPH " 1000 wt 800 NADPH(umol) 600 I

200 R1324 SYVYÅUNAYOYOXSAYOYNOYAYSYNOYAUNAY.YAY.YAYNYALNY NOYNOYSYY AYGUNDY 10 20 25 30 Time (min ) Fig . 30A

" NADP + NADPH "

www wt NADPHConsumed(umol) -500 BR** RAWA **** PARK *** DRUR *** RARTPAHARREBRPRA *** BRER **** DABR *** DAH *** ADR **** ADRE -1000 I - 1500 R1324 5 10 20 25 30 Time (min ) Fig . 30B U.S. Patent Apr. 7, 2020 Sheet 41 of 49 US 10,610,125 B2

0.44 965 1031.0x

*a/n n/a a/n

*n/a=Nomeasurableenzymaticactivity Reductive(?NADP+) )UM(NADPH,KM )UM(QKG,KM ")iskcat 30C.Fig R132H 84 370 10085 24 37.5 49 65 29 1871 105x4.4

Oxidative(?NADPH) )UM(+NADP.KM )UMisocitrate(,KM )?M(??????, )UM(KaKG 57)(Kear U.S. Patent Apr. 7 , 2020 Sheet 42 of 49 US 10,610,125 B2

100 80 Holo 60 2 -HG control Reductive Rxn LC/MSsignal(normalized) Product 20 Startet Para

5 HPLC Elution Time (min ) Fig . 31A

150 S ( + ) - 2HG R ( -) - 2HG 100 HoHO Rxn + Racemate LC/MSsignal(normalized) 2HG Racemate Rxn + R ) 2HG

R (- ) - 2HGdopoppopoo std .,

Reductive rån???? 70 20 HPLC Elution Time (min ) Fig . 31B U.S. Patent Apr. 7 , 2020 Sheet 43 of 49 US 10,610,125 B2

250 150 100 75

25 20 .

15 : SDS PAGE Western blot Fig . 32A

MAU 74.92 300

200

100

20 40 80 100 mi Fig . 32B U.S. Patent Apr. 7 , 2020 Sheet 44 of 49 US 10,610,125 B2

Fig . 33

Fig . 34 U.S. Patent Apr. 7, 2020 Sheet 45 of 49 US 10,610,125 B2

IDH2 R172K mutant has elevated NADPH reductive catalysis compared to WT enzyme. 0.4 0.35 0.3 0.25 SpecificActivity(umol/min mg) 0.2 0.15 0.1 0.05 DH2wt-1 DH1-Wt IDH2R172K-2 * IDH1-R132H IDH2wt2 w

Comparison of NADPH reduction between IDH2 -R172K , IDH2- wt , IDH1- R132H , and IDH1- Wt enzymes. Final reactant concentrations for each reaction was as follow 20mM Tris 7.5 , 150 mM Naci, 2 mM MnCl2 , 10 % Glycerol, 0.03 % BSA , varied enzyme ( 1-120 ug /mL ) , 1 mM NADPH , 5 mM akG . Fig . 35 U.S. Patent Apr. 7 , 2020 Sheet 46 of 49 US 10,610,125 B2

1000001

10000

1000 ! 2x106cells)(ng/2-HG 100

IDH1 Mutant Mutant Fig . 36ACultures

1000

2-HG(ng/ml) *****

IDH1 Mutant Fig . 36B 7 IDH1 wt 6 IDHI R132 mutant PH## On M 5 OD OD 4 careBath Hoop SM ***** E H* Che WanWar Mr 3 Relativecellnumber tha se * Water 2 KOSONOA MO 1 e sono moool +0000 5 20 Time (days ) Fig . 36C U.S. Patent Apr. 7, 2020 Sheet 47 of 49 US 10,610,125 B2

IDH1mutant A IDH2 mutant 10000.00 O IDH1/ 2 wt

obwoo o Su 1000.00

O (ng/2x106cells) yon ***** *****volt Metaboliteconcentration 100.00 mlo000

10.00 O - KG Succinate Malate Fumarate IDH1/ 2 mutant AML cells do not display altered levels of central carbon metabolites. Fig . 37 U.S. Patent Apr. 7, 2020 Sheet 48 of 49 US 10,610,125 B2

2HG MRM SOC MRM bbgu8 5 in

234 Retentiontime(min) 3 Retentiontime(min)

1 1 IDH2R172K Product IDH1R172K Product whermocon 0 washawishi 5 4

2354

Retentiontime(min) 3 Retentiontime(min) 38.Fig 2

} } IDH1R1320 Product 0 IDHIR1320 Product JOUXANXwwwwwwwwwwwwwwww 0

5 10

10000OOUOOOOO 4 4

3 Retentiontime(min) 3 Retentiontime(min)

2 2

1 1

cocoloLOOOOOOOnaanuarmOCODE 0 1.042HG Standard 0.8 socitrateStandard 0.8 0.6 0.2 0.0 1.0 0.6 0.4 0.2 0.0

Intensity Signal Intensityw Signal U.S. Patent Apr. 7, 2020 Sheet 49 of 49 US 10,610,125 B2

IDH1 / 2 i OH HO NADP NADPH Fig . 39A

DHI R1320 *** IDH2 R172K OH NADPH NADP iresOH Fig . 39B US 10,610,125 B2 1 2 METHODS AND COMPOSITIONS FOR treatment for the subject , on the basis of neaoctivity geno CELL - PROLIFERATION - RELATED type or phenotype . This evaluation can allow for optimal DISORDERS matching of subject with treatment , e.g., where the selection of subject, treatment, or both , is based on an analysis of CLAIM OF PRIORITY 5 neoactivity genotype or phenotype . E.g., methods describe herein can allow selection of a treatment regimen compris This application is a continuation of U.S. Ser . No. 13/939 , ing administration of a novel compound , e.g. , a novel 519, filed Jul. 11 , 2013 , which is a continuation of U.S. Ser. compound disclosed herein , or a known compound, e.g., a No. 13 / 256,396 , filed Nov. 29 , 2011 , which is a national known compound not previously recommended for a stage application under 35 U.S.C. § 371 of International 10 selected disorder. In embodiments the known compound Application No. PCT/ US2010 /027253 , filed Mar. 12 , 2010 , reduces levels of neoactivity or neoactive product or ame published as International Publication No. WO 2010/105243 liorates an effect of a neoactive product. Methods described on Sep. 16 , 2010 , which claims priority to U.S. Ser. No. herein can guide and provide a basis for selection and 61/ 160,253 , filed Mar. 13 , 2009 ; U.S. Ser . No. 61/ 160,664 , administration of a novel compound or a known compound , filed Mar. 16 , 2009 ; U.S. Ser. No. 61/ 173,518 , filed Apr. 28 , 15 or combination of compounds, not previously recommended 2009 ; U.S. Ser. No. 61 / 180,609 , filed May 22 , 2009 ; U.S. for subjects having a disorder characterized by a somatic Ser. No. 61 / 220,543 , filed Jun . 25 , 2009; U.S. Ser. No. neoactive mutation in a metabolic pathway enzyme. In 61/ 227,649 , filed Jul. 22 , 2009 ; U.S. Ser. No. 61/ 229,689 , embodiments the neoactive genotype or phenotype can act 2009filed ;Jul and. 29 U.S. , 2009 Ser ; . U.S. No. 61Ser/ . 266,929 No. 61, /253,820 filed Dec. , filed 4, 2009Oct., the21, 20 as a biomarker the presence of which indicates that a contents of each of which are incorporated herein by refer compound , either novel, or previously known , should be ence . administered , to treat a disorder characterized by a somatic neoactive mutation in a metabolic pathway enzyme. Neo FIELD OF THE INVENTION active mutants of IDH1 having a neoactivity that results in 25 the production of 2 -hydroxyglutarate , e.g., R - 2 -hydroxyglu The invention relates to methods and compositions for tarate and associated disorders are discussed in detail herein . evaluating and treating cell proliferation - related disorders, They are exemplary , but not limiting , examples of embodi e.g. , proliferative disorders such as cancer. ments of the invention . While not wishing to be bound by theory it is believed that BACKGROUND 30 the balance between the production and elimination of neoactive product , e.g. , 2HG , e.g. , R -2HG , is important in Isocitrate dehydrogenase , also known as IDH , is an disease . Neoactive mutants , to varying degrees for varying enzyme which participates in the citric acid cycle . It cata m ns, increase the level of neoactive product , while lyzes the third step of the cycle : the oxidative decarboxy other processes , e.g. , in the case of 2HG , e.g., R -2HG , lation of isocitrate, producing alpha- ketoglutarate ( a -keto- 35 enzymatic degradation of 2HG , e.g., by 2HG dehydroge glutarate or a - KG ) and CO2 while converting NAD + to nase, reduce the level of neoactive product. An incorrect NADH . This is a two - step process , which involves oxidation balance is associated with disease . In embodiments , the net of isocitrate ( a secondary alcohol) to oxalosuccinate ( a result of a neoactive mutation at IDH1 or IDH2 result in ketone ), followed by the decarboxylation of the carboxyl increased levels , in affected cells , of neoactive product, group beta to the ketone, forming alpha -ketoglutarate . 40 2HG , e.g., R -2HG , Another isoform of the enzyme catalyzes the same reaction ; Accordingly , in one aspect, the invention features , a however this reaction is unrelated to the citric acid cycle , is method of treating a subject having a cell proliferation carried out in the cytosol as well as the mitochondrion and related disorder, e.g., a disorder characterized by unwanted peroxisome, and uses NADP + as a cofactor instead of cell proliferation , e.g. , cancer , or a precancerous disorder . NAD + . 45 The cell proliferation - related disorder is characterized by a somatic mutation in a metabolic pathway enzyme. The SUMMARY OF THE INVENTION mutation is associated with a neoactivity that results in the production of a neoactivity product. The method comprises : Methods and compositions disclosed herein relate to the administering to the subject a therapeutically effective role played in disease by neoactive products produced by 50 amount of a therapeutic agent described herein , e.g. , a neoactive mutant enzymes , e.g. , mutantmetabolic pathway therapeutic agent that decreases the level of neoactivity enzymes . The inventors have discovered , inter alia , a neo product encoded by a selected or mutant somatic allele , e.g., activity associated with IDH mutants and that the product of an inhibitor of a neoactivity of the metabolic pathway the neoactivity can be significantly elevated in cancer cells . enzyme (the neoactive enzyme ), a therapeutic agent that Disclosed herein are methods and compositions for treating, 55 ameliorates an unwanted affect of the neoactivity product, or and methods of evaluating , subjects having or at risk for a a nucleic acid based inhibitor , e.g., a dRNA which targets the disorder , e.g. , a cell proliferation - related disorder character neoactive enzymemRNA , to thereby treat the subject. ized by a neoactivity in a metabolic pathway enzyme, e.g., In an embodiment the subject is a subject not having , or IDH neoactivity . Such disorders include e.g., proliferative not diagnosed as having , 2 -hydroxyglutaric aciduria . disorders such as cancer. The inventors have discovered and 60 In an embodiment the subject has a cell proliferation disclosed herein novel therapeutic agents for the treatment related disorder , e.g. , a cancer, characterized by the neoac of disorders , e.g., cancers , characterized by , e.g., by a tivity of the metabolic pathway enzyme encoded by selected neoactivity, neoactive protein , neoactive mRNA , or neoac or mutant allele . tive mutations . In embodiments a therapeutic agent reduces In an embodiment the subject has a cell proliferation levels of neoactivity or neoactive product or ameliorates an 65 related disorder, e.g., a cancer, characterized by the product effect of a neoactive product. Methods described herein also formed by the neoactivity of the metabolic pathway enzyme allow the identification of a subject , or identification of a encoded by selected or mutant allele . US 10,610,125 B2 3 4 In one embodiment, the metabolic pathway is selected e.g., an alpha hydroxyl, e.g. , a 2HG , neoactive enzyme, can from a metabolic pathway leading to fatty acid biosynthesis , act on more than one substrate , e.g., more than one alpha glycolysis , glutaminolysis , the pentose phosphate shunt, hydroxy substrate . nucleotide biosynthetic pathways , or the fatty acid biosyn The method comprises administering to the subject an thetic pathway. 5 effective amount of a therapeutic agent of type described In an embodiment the therapeutic agent is a therapeutic herein to thereby treat the subject. agent described herein . In an embodiment the therapeutic agent: results in low In an embodiment the method comprises selecting a ering the level of a neoactivity product , e.g., an alpha subject on the basis of having a cancer characterized by the hydroxy neoactivity product , e.g. , 2HG , e.g., R - 2HG . 10 In an embodiment the method comprises administering a ofselected neaoctivity ormutant product allele . , the neoactivity, or an elevated level therapeutic agent that lowers neoactivity , e.g. , 2HG neoac In an embodiment the method comprises selecting a tivity . In an embodiment the method comprises administer subject on the basis of having a cancer characterized by the ing an inhibitor of a mutant IDH protein , e.g., a mutant IDH1 product formed by the neoactivity of the protein encoded by 15 orhydroxy mutant neoactivity IDH2 protein , e.g. , 2HGhaving neoactivity a neoactivity . , e.g. , alpha selected or mutant allele , e.g., by the imaging and / or spec In an embodiment the therapeutic agent comprises a troscopic analysis , e.g. ,magnetic resonance -based analysis , compound from Table 24a or Table 24b or a compound e.g., MRI (magnetic resonance imaging ) and/ or MRS (mag having the structure of Formula ( X ) or (Formula (XI ) netic resonance spectroscopy ) , to determine the presence , described herein . distribution or level of the product of the neoactivity , e.g. , in 20 In an embodiment the therapeutic agent comprises nucleic the case of an IDH1 allele described herein , 2 - hydroxyglu acid -based therapeutic agent, e.g. , a dsRNA , e.g. , a dsRNA tarate ( sometimes referred to herein as 2HG ) , e.g., R - 2 described herein . hydro glutarate ( sometimes referred to herein as R - 2HG ) . In an embodiment the therapeutic agent is an inhibitor, In an embodiment the method comprises confirming or e.g. , a polypeptide, peptide , or small molecule ( e.g., a determining , e.g. , by direct examination or evaluation of the 25 molecule of less than 1,000 daltons) , or aptomer, that binds subject, or sample e.g. , tissue , product ( e.g., feces , sweat, to an IDH1mutant or wildtype subunit and inhibits neoac semen , exhalation , hair or nails ) , or bodily fluid ( e.g. , blood tivity , e.g., by inhibiting formation of a dimer, e.g. , a ( e.g., blood plasma) , urine , lymph , or cerebrospinal fluid or homodimer of mutant IDH1 subunits or a heterodimer of a mutant and a wildtype subunit. In an embodiment the other sample sourced disclosed herein ) therefrom , ( e.g. , by 30 inhibitor is a polypeptide. In an embodiment the polypeptide DNA sequencing, immuno analysis, or assay for enzymatic acts as a dominant negative with respect to the neoactivity activity ) , or receiving such information about the subject, of the mutant enzyme. The polypeptide can correspond to that the cancer is characterized by the selected or mutant full length IDH1 or a fragment thereof. The polypeptide allele . need not be identical with the corresponding residues of In an embodiment the method comprises confirming or 35 wildtype IDH1, but in embodiments has at least 60 , 70 , 80 , determining , e.g., by direct examination or evaluation of the 90 or 95 % homology with wildtype IDH1 . subject, the level of neoactivity or the level of the product of In an embodiment the therapeutic agent decreases the the neoactivity , or receiving such information about the affinity of an IDH , e.g., IDH1 or IDH2 neoactive mutant subject . In an embodiment the presence, distribution or level protein for NADH , NADPH or a divalent metal ion , e.g. , of the productof the neoactivity , e.g., in the case of an IDH1 40 Mg2 + or Mn2+ , or decreases the levels or availability of allele described herein , 2HG , e.g. , R -2HG , is determined NADH , NADPH or divalent metal ion , e.g. ,Mg2 + or Mn2 +, non - invasively , e.g. , by imaging methods, e.g., by magnetic e.g., by competing for binding to the mutant enzyme. In an resonance - based methods . embodiment the enzyme is inhibited by replacing Mg2 + or In an embodiment the method comprises administering a Mn2 + with Ca2 + . second anti -cancer agent or therapy to the subject , e.g., 45 In an embodiment the therapeutic agent is an inhibitor that surgical removal or administration of a chemotherapeutic . reduces the level a neoactivity of an IDH , e.g., IDH1 or In another aspect , the invention features , a method of IDH2, e.g., 2HG neoactivity . treating a subject having a cell proliferation - related disorder , In an embodiment the therapeutic agent is an inhibitor that e.g., a precancerous disorder, or cancer. In an embodiment reduces the level of the product of a mutant having a the subject does not have, or has not been diagnosed as 50 neoactivity of an IDH , e.g., IDH1 or IDH2 mutant , e.g., it having , 2 -hydroxyglutaric aciduria . The cell proliferation reduces the level of 2HG , e.g. , R -2HG . related disorder is characterized by a somatic allele , e.g., a In an embodiment the therapeutic agent is an inhibitor preselected allele , or mutant allele, of an IDH , e.g., IDH1 or that: IDH2, which encodes a mutant IDH , e.g., IDH1 or IDH2 , inhibits , e.g., specifically , a neoactivity of an IDH , e.g., enzyme having a neoactivity . 55 IDH1 or IDH2, e.g. , a neoactivity described herein , e.g. , In embodiments the neoactivity is alpha hydroxy neoac 2HG neoactivity ; or tivity . As used herein , alpha hydroxy neoactivity refers to the inhibits both the wildtype activity and a neoactivity of an ability to convert an alpha ketone to an alpha hydroxy . In IDH , e.g., IDH1 or IDH2, e.g. , a neoactivity described embodiments alpha hydroxy neoactivity proceeds with a herein , e.g. , 2HG neoactivity . reductive cofactor, e.g. , NADPH or NADH . In embodiments 60 In an embodiment the therapeutic agent is an inhibitor that the alpha hydroxyl neoactivity is 2HG neoactivity . 2HG is selected on the basis that it: neoactivity , as used herein , refers to the ability to convert inhibits , e.g., specifically , a neoactivity of an IDH , e.g. , alpha ketoglutarate to 2- hydroxyglutarate (sometimes IDH1 or IDH2 , e.g., a neoactivity described herein e.g., 2HG referred to herein as 2HG ), e.g., R - 2 -hydroxyglutarate neoactivity ; or ( sometimes referred to herein as R -2HG ) . In embodiments 65 inhibits both the wildtype activity and a neoactivity of an 2HG neoactivity proceeds with a reductive cofactor , e.g., IDH1 , e.g. , IDH1 or IDH2, e.g., a neoactivity described NADPH or NADH . In an embodiment a neoactive enzyme, herein , e.g., 2HG neoactivity . US 10,610,125 B2 5 6 In an embodiment the therapeutic agent is an inhibitor that ment, to determine if it is characterized by presence of an reduces the amount of a mutant IDH , e.g., IDH1 or IDH2, alpha hydroxy neoactivity product, e.g. , 2HG , e.g., R -2HG . protein or mRNA . In an embodiment the method comprises evaluating , e.g., In an embodiment the therapeutic agent is an inhibitor that by direct examination or evaluation of the subject , or a interacts directly with , e.g. , it binds to , the mutant IDH , e.g., 5 sample from the subject, or receiving such information IDH1 or IDH2 mRNA . about the subject, the IDH , e.g. , IDH1 or IDH2, genotype , In an embodiment the therapeutic agent is an inhibitor that or an alpha hydroxy neoactivity product, e.g. , 2HG , e.g., interacts directly with , e.g., it binds to , the mutant IDH , e.g., R -2HG phenotype of , the subject , e.g. , of a cell , e.g., a IDH1 or IDH2, protein . cancer cell , characterized by the cell proliferation -related In an embodiment the therapeutic agent is an inhibitor that 10 disorderevaluation . ( As can described be, e.g., byin DNAmore sequencingdetail elsewhere, immuno herein analy the reduces the amount of neoactive enzyme activity , e.g., by sis , evaluation of the presence , distribution or level of an interacting with , e.g., binding to , mutant IDH , e.g. , IDH1 or alpha hydroxy neoactivity product , e.g., 2HG , e.g., R -2HG , IDH2, protein . In an embodiment the inhibitor is other than e.g., from spectroscopic analysis , e.g. , magnetic resonance an antibody. 15 based analysis , e.g., MRI and/ or MRS measurement, sample In an embodiment the therapeutic agent is an inhibitor that analysis such as serum or spinal cord fluid analysis , or by is a smallmolecule and interacts with , e.g., binds, the mutant analysis of surgical material, e.g., by mass - spectroscopy ) . In RNA , e.g., mutant IDH1 or IDH2 mRNA ( e.g. ,mutant IDH1 embodiments this information is used to determine or con mRNA ). firm that a proliferation - related disorder , e.g. , a cancer, is In an embodiment the therapeutic agent is an inhibitor that 20 characterized by an alpha hydroxy neoactivity product, e.g., interacts directly with , e.g., binds , either the mutant IDH , 2HG , e.g., R -2HG . In embodiments this information is used e.g., IDH1 or IDH2 , protein or interacts directly with , e.g., to determine or confirm that a cell proliferation - related binds, the mutant IDH mRNA , e.g., IDH1 or IDH2mRNA . disorder, e.g., a cancer , is characterized by an IDH , e.g., In an embodiment the IDH is IDH1 and the neoactivity is IDH1 or IDH2 , allele described herein , e.g., an IDH1 allele alpha hydroxy neoactivity , e.g. , 2HG neoactivity . Mutations 25 having a mutation , e.g. , a His , Ser , Cys, Gly , Val , Pro or Leu in IDH1 associated with 2HG neoactivity include mutations ( e.g., His, Ser , Cys, Gly , Val, or Leu at residue 132, more at residue 132 , e.g., R132H , R132C , R132S , R132G , specifically , His or Cys, or an IDH2 allele having a mutation R132L , or R132V ( e.g. , R132H or R132C ) . at residue 172 , e.g., a K , M , S , G , or W. In an embodiment the IDH is IDH2 and the neoactivity of In an embodiment, before and /or after treatment has the IDH2 mutant is alpha hydroxy neoactivity , e.g., 2HG 30 begun , the subject is evaluated or monitored by a method neoactivity . Mutations in IDH2 associated with 2HG neo described herein , e.g., the analysis of the presence , distri activity include mutations at residue 172 , e.g., R172K , bution , or level of an alpha hydroxy neoactivity product, R172M , R172S , R172G , or R172W . e.g., 2HG , e.g., R - 2HG , e.g., to select, diagnose or prognose Treatment methods described herein can comprise evalu the subject, to select an inhibitor, or to evaluate response to ating a neoactivity genotype or phenotype . Methods of 35 the treatment or progression of disease . obtaining and analyzing samples , and the in vivo analysis in In an embodiment the cell proliferation - related disorder is subjects , described elsewhere herein , e.g., in the section a tumor of the CNS , e.g., a glioma, a leukemia , e.g. , AML entitled , “Methods of evaluating samples and / or subjects ,” or ALL , e.g. , B -ALL or T - ALL , prostate cancer, fibrosar can be combined with this method . coma , paraganglioma, ormyelodysplasia or myelodysplastic In an embodiment, prior to or after treatment , the method 40 syndrome ( e.g., B - ALL or T - ALL , prostate cancer, ormyelo includes evaluating the growth , size , weight , invasiveness, dysplasia or myelodysplastic syndrome) and the evaluation stage or other phenotype of the cell proliferation - related is : evaluation of the presence , distribution , or level of an disorder. alpha hydroxy neoactivity product, e.g., 2HG , e.g., R - 2HG ; In an embodiment, prior to or after treatment, the method or evaluation of the presence, distribution , or level of a includes evaluating the IDH , e.g., IDH1 or IDH2, alpha 45 neoactivity , e.g., an alpha hydroxy neoactivity , e.g., 2HG hydroxyl neoactivity genotype , e.g., 2HG , genotype , or neoactivity, of an IDH1 or IDH2, mutant protein . alpha hydroxy neoactivity phenotype, e.g., 2HG , e.g., In an embodiment the disorder is other than a solid tumor. R - 2HG , phenotype . Evaluating the alpha hydroxyl, e.g., In an embodiment the disorder is a tumor that, at the time of 2HG , genotype can comprise determining if an IDH1 or diagnosis or treatment, does not have a necrotic portion . In IDH2mutation having alpha hydroxy neoactivity , e.g. , 2HG 50 an embodiment the disorder is a tumor in which at least 30 , neoactivity , is present, e.g. , a mutation disclosed herein 40, 50 , 60 , 70 , 80 or 90 % of the tumor cells carry an IHD , having alpha hydroxy neoactivity , e.g. , 2HG neoactivity . e.g., IDH1 or IDH2, mutation having 2HG neoactivity , at the Alpha hydroxy neoactivity phenotype, e.g., 2HG , e.g., time of diagnosis or treatment. R - 2HG , phenotype , as used herein , refers to the level of In an embodiment the cell proliferation - related disorder is alpha hydroxy neoactivity product, e.g., 2HG , e.g., R -2HG , 55 a cancer, e.g., a cancer described herein , characterized by an level of alpha hydroxy neoactivity , e.g., 2HG neoactivity , or IDH1 somatic mutant having alpha hydroxy neoactivity , level of mutant enzyme having alpha hydroxy neoactivity , e.g., 2HG neoactivity , e.g., a mutant described herein . In an e.g., 2HG neoactivity (or corresponding mRNA ) . The evalu embodiment the tumor is characterized by increased levels ation can be by a method described herein . of an alpha hydroxy neoactivity product , 2HG , e.g., R -2HG , In an embodiment the subject can be evaluated , before or 60 as compared to non -diseased cells of the same type . after treatment, to determine if the cell proliferation - related In an embodiment the method comprises selecting a disorder is characterized by an alpha hydroxy neoactivity subject having a glioma, on the basis of the cancer being product, e.g., 2HG , e.g. , R - 2HG . characterized by unwanted ( i.e. , increased ) levels of an alpha In an embodiment a cancer, e.g., a glioma or brain tumor hydroxy neoactivity , product, e.g., 2HG , e.g. , R - 2HG . in a subject , can be analyzed , e.g., by imaging and / or 65 In an embodiment the cell proliferation - related disorder is spectroscopic analysis , e.g. , magnetic resonance -based a tumor of the CNS, e.g., a glioma, e.g. , wherein the tumor analysis , e.g. , MRI and / or MRS, e.g., before or after treat is characterized by an IDH1 somatic mutant having alpha US 10,610,125 B2 7 8 hydroxy neoactivity , e.g. , 2HG neoactivity , e.g. , a mutant ( see also FIG . 21) ( e.g. , His , Ser , Cys, Gly , Val, or Leu ). In described herein . Gliomas include astrocytic tumors , oligo an embodiment the allele encodes an IDH1 having His or dendroglial tumors , oligoastrocytic tumors, anaplastic astro Cys at residue 132 . cytomas , and glioblastomas . In an embodiment the tumor is In an embodiment the IDH1 allele has a T (or any other characterized by increased levels of an alpha hydroxy neo 5 nucleotide other than C ) at nucleotide position 394 , or an A activity product, e.g. , 2HG , e.g. , R - 2HG , as compared to (or any other nucleotide other than G ) at nucleotide position 395. In an embodiment the allele is a C394T or a G395A non - diseased cells of the same type. E.g., in an embodiment, mutation according to the sequence of SEQ ID NO : 5 . the IDH1 allele encodes an IDH1 having other than an Arg In an embodiment the method comprises selecting a at residue 132. E.g. , the allele encodes His , Ser , Cys, Gly , 10 subject having prostate cancer , e.g., prostate adenocarci Val, Pro or Leu ( e.g. , His , Ser , Cys, Gly , Val , or Leu ), or any noma, wherein the cancer is characterized by an IDH1 allele residue described in Yan et al ., at residue 132 , according to described herein , e.g., an IDH1 allele having His or Cys at the sequence of SEQ ID NO : 8 ( see also FIG . 21 ) . In an residue 132 (SEQ ID NO : 8 ). embodiment the allele encodes an IDH1 having His at In an embodiment the method comprises selecting a residue 132. In an embodiment the allele encodes an IDH1 15 subject having prostate cancer, e.g., prostate adenocarci having Ser at residue 132 . noma, on the basis of the cancer being characterized by an In an embodiment the IDH1 allele has an A ( or any other IDH1 allele described herein , e.g. , an IDH1 allele having nucleotide other than C ) at nucleotide position 394 , or an A His or Cys at residue 132 (SEQ ID NO : 8 ). ( or any other nucleotide other than G ) at nucleotide position In an embodiment the method comprises selecting a 395. In an embodiment the allele is a C394A , a C394G , a 20 subject having prostate cancer , on the basis of the cancer C3947, a G395C , a G395T or a G395A mutation ; specifi being characterized by increased levels of an alpha hydroxy cally a C394A or a G395A mutation according to the neoactivity product, e.g., 2HG , e.g., R -2HG . sequence of SEQ ID NO :5 . In an embodiment the cell proliferation - related disorder is In an embodiment the method comprises selecting a a hematological cancer , e.g., a leukemia , e.g. , AML , or ALL , subject having a glioma, wherein the cancer is characterized 25 wherein the hematological cancer is characterized by an by having an IDH1 allele described herein , e.g. , an IDH1 IDH1 somatic mutant having alpha hydroxy neoactivity , allele having His , Ser , Cys , Gly , Val, Pro or Leu at residue e.g. , 2HG neoactivity , e.g., a mutant described herein . In an 132 (SEQ ID NO :8 ), more specifically His , Ser , Cys , Gly , embodiment the cancer is characterized by increased levels Val, or Leu ; or His or Cys. of an alpha hydroxy neoactivity product, e.g., 2HG , e.g. , In an embodiment the method comprises selecting a 30 R -2HG , as compared to non - diseased cells of the same type . subject having a glioma, on the basis of the cancer being In an embodiment the cell proliferation - related disorder is characterized by an IDH1 allele described herein , e.g. , an acute lymphoblastic leukemia ( e.g. , an adult or pediatric IDH1 allele having His , Ser , Cys , Gly , Val, Pro or Leu at form ), e.g. , wherein the acute lymphoblastic leukemia residue 132 (SEQ ID NO : 8 ), more specifically His , Ser , Cys , ( sometimes referred to herein as ALL ) is characterized by an Gly , Val, or Leu ; or His or Cys . 35 IDH1 somatic mutant having alpha hydroxy neoactivity , In an embodiment the method comprises selecting a e.g., 2HG neoactivity , e.g., a mutant described herein . The subject having a glioma, on the basis of the cancer being ALL can be, e.g., B -ALL or T - ALL. In an embodiment the characterized by increased levels of an alpha hydroxy neo cancer is characterized by increased levels of 2 an alpha activity , product, e.g., 2HG , e.g., R - 2HG . hydroxy neoactivity product, e.g., HG , e.g. , R -2HG , as In an embodiment the method comprises selecting a 40 compared to non -diseased cells of the same type . E.g., in an subjecthaving a fibrosarcoma or paraganglioma wherein the embodiment, the IDH1 allele is an IDH1having other than cancer is characterized by having an IDH1 allele described an Arg at residue 132 (SEQ ID NO : 8 ) . E.g., the allele herein , e.g. , an IDH1 allele having Cys at residue 132 (SEQ encodes His , Ser, Cys , Gly , Val, Pro or Leu , or any residue ID NO : 8 ) . described in Kang et al. , at residue 132, according to the In an embodiment the method comprises selecting a 45 sequence of SEQ ID NO :8 (see also FIG . 21 ), more spe subject having a fibrosarcoma or paraganglioma, on the cifically His, Ser, Cys, Gly , Val, or Leu . In an embodiment basis of the cancer being characterized by an IDH1 allele the allele encodes an IDH1 having Cys at residue 132 . described herein , e.g., an IDH1 allele having Cys at residue In an embodiment the IDH1 allele has a T (or any other 132 (SEQ ID NO :8 ). nucleotide other than C ) at nucleotide position 394. In an In an embodiment the method comprises selecting a 50 embodiment the allele is a C394T mutation according to the subject having a fibrosarcoma or paraganglioma, on the sequence of SEQ ID NO :5 . basis of the cancer being characterized by increased levels of In an embodiment the method comprises selecting a an alpha hydroxy neoactivity , product, e.g., 2HG , e.g., subject having ALL , e.g. , B -ALL or T - ALL , characterized R - 2HG . by an IDH1 allele described herein , e.g. , an IDH1 allele In an embodiment the cell proliferation - related disorder is 55 having Cys at residue 132 according to the sequence of SEQ localized or metastatic prostate cancer, e.g., prostate adeno ID NO : 8 . carcinoma, e.g. , wherein the cancer is characterized by an In an embodiment the method comprises selecting a IDH1 somatic mutant having alpha hydroxy neoactivity , subject ALL , e.g. , B - ALL or T - ALL , on the basis of cancer e.g. , 2HG neoactivity , e.g., a mutant described herein . In an being characterized by having an IDH1 allele described embodiment the cancer is characterized by increased levels 60 herein , e.g., an IDH1 allele having Cys at residue 132 (SEQ of an alpha hydroxy neoactivity product , e.g., 2HG , e.g., ID NO :8 ) . R -2HG , as compared to non -diseased cells of the same type . In an embodiment the method comprises selecting a E.g. , in an embodiment, the IDH1 allele encodes an IDH1 subject having ALL , e.g., B -ALL or T -ALL , on the basis of having other than an Arg at residue 132. E.g., the allele the cancer being characterized by increased levels of an encodes His , Ser , Cys, Gly , Val , Pro or Leu , or any residue 65 alpha hydroxy neoactivity product , e.g., 2HG , e.g. , R - 2HG . described in Kang et al , 2009, Int. J. Cancer , 125 : 353-355 In an embodiment the cell proliferation - related disorder is at residue 132, according to the sequence of SEQ ID NO : 8 acute myelogenous leukemia ( e.g. , an adult or pediatric US 10,610,125 B2 9 10 form ) , e.g. , wherein the acute myelogenous leukemia ( some an IDH1 allele described herein , e.g., an IDH1 allele having times referred to herein as AML ) is characterized by an Cys, His , or Gly at residue 132 (SEQ ID NO :8 ) , more IDH1 somatic mutant having alpha hydroxy neoactivity , specifically , Cys at residue 132 . e.g. , 2HG neoactivity , e.g. , a mutant described herein . In an In an embodiment the method comprises selecting a embodiment the cancer is characterized by increased levels 5 subject having myelodysplasia or myelodysplastic syn of an alpha hydroxy neoactivity product, e.g. , 2HG , e.g., drome, on the basis of the cancer being characterized by R -2HG , as compared to non -diseased cells of the same type . increased levels of an alpha hydroxy neoactivity product , E.g., in an embodiment , the IDH1 allele is an IDH1having e.g. , 2HG , e.g., R - 2HG . other than an Arg at residue 132 (SEQ ID NO : 8 ). E.g., the In an embodiment the cell proliferation - related disorder is allele encodes His , Ser, Cys , Gly , Val, Pro or Leu , or any 10 a glioma, characterized by a mutation , or preselected allele , residue described in Kang et al ., at residue 132 , according to of IDH2 associated with an alpha hydroxy neoactivity , e.g. , the sequence of SEQ ID NO :8 ( see also FIG . 21 ) . In an 2HG neoactivity . E.g., in an embodiment, the IDH2 allele embodiment the allele encodes an IDH1 having Cys , His or encodes an IDH2 having other than an Arg at residue 172 . Gly at residue 132 , more specifically , Cys at residue 132 . E.g., the allele encodes Lys, Gly , Met , Trp , Thr, Ser , or any In an embodiment the IDH1 allele has a T ( or any other 15 residue described in described in Yan et al. , at residue 172 , nucleotide other than C ) at nucleotide position 394. In an according to the sequence of SEQ ID NO : 10 ( see also FIG . embodiment the allele is a C394Tmutation according to the 22 ), more specifically Lys , Gly , Met, Trp , or Ser . In an sequence of SEQ ID NO :5 . embodiment the allele encodes an IDH2 having Lys at In an embodiment the method comprises selecting a residue 172. In an embodiment the allele encodes an IDH2 subject having acute myelogenous lymphoplastic leukemia 20 having Met at residue 172 . ( AML ) characterized by an IDH1 allele described herein , In an embodiment the method comprises selecting a e.g., an IDH1 allele having Cys, His, or Gly at residue 132 subject having a glioma, wherein the cancer is characterized according to the sequence of SEQ ID NO :8 , more specifi by having an IDH2 allele described herein , e.g., an IDH2 cally , Cys at residue 132 . allele having Lys, Gly , Met, Trp , Thr, or Ser at residue 172 In an embodiment the method comprises selecting a 25 (SEQ ID NO : 10 ) , more specifically Lys , Gly , Met, Trp , or subject having acute myelogenous lymphoplastic leukemia Ser ; or Lys or Met . (AML ) on the basis of cancer being characterized by having In an embodiment the method comprises selecting a an IDH1 allele described herein , e.g., an IDH1 allele having subject having a glioma, on the basis of the cancer being Cys, His , or Gly at residue 132 (SEQ ID NO : 8 ), more characterized by an IDH2 allele described herein , e.g., an specifically , Cys at residue 132 . 30 IDH2 allele having Lys, Gly , Met , Trp , Thr, or Ser at residue In an embodiment the method comprises selecting a 172 (SEQ ID NO : 10 ), more specifically Lys , Gly, Met, Trp , subject having acute myelogenous lymphoplastic leukemia or Ser ; or Lys or Met . ( AML ), on the basis of the cancer being characterized by In an embodiment the method comprises selecting a increased levels of an alpha hydroxy neoactivity product, subject having a glioma, on the basis of the cancer being e.g. , 2HG , e.g., R - 2HG . 35 characterized by increased levels of an alpha hydroxy neo In an embodiment the method further comprises evaluat activity product , e.g. , 2HG , e.g., R - 2HG . ing the subject for the presence of a mutation in the NRAS In an embodiment the cell proliferation -related disorder is or NPMc gene . a prostate cancer, e.g. , prostate adenocarcinoma, character In an embodiment the cell proliferation - related disorder is ized by a mutation , or preselected allele, of IDH2 associated myelodysplasia or myelodysplastic syndrome, e.g., wherein 40 with an alpha hydroxy neoactivity , e.g., 2HG neoactivity . the myelodysplasia or myelodysplastic syndrome is charac E.g., in an embodiment, the IDH2 allele encodes an IDH2 terized by having an IDH1 somatic mutant having alpha having other than an Arg at residue 172. E.g. , the allele hydroxy neoactivity , e.g. , 2HG neoactivity , e.g., a mutant encodes Lys, Gly , Met , Trp , Thr, Ser , or any residue described herein . In an embodiment the disorder is charac described in described in Yan et al ., at residue 172 , according terized by increased levels of an alpha hydroxy neoactivity 45 to the sequence of SEQ ID NO : 10 ( see also FIG . 22 ), more product, e.g., 2HG , e.g., R -2HG , as compared to non specifically Lys , Gly , Met, Trp , or Ser . In an embodiment the diseased cells of the same type. E.g., in an embodiment, the allele encodes an IDH2 having Lys at residue 172. In an IDH1 allele is an IDH1 having other than an Arg at residue embodiment the allele encodes an IDH2 having Met at 132 (SEQ ID NO : 8 ). E.g., the allele encodes His , Ser , Cys, residue 172 . Gly , Val, Pro or Leu, or any residue described in Kang et al. , 50 In an embodiment the method comprises selecting a according to the sequence of SEQ ID NO : 8 ( see also FIG . subject having a prostate cancer, e.g., prostate adenocarci 21 ), more specifically His , Ser, Cys , Gly , Val , or Leu . In an noma, wherein the cancer is characterized by having an embodiment the allele encodes an IDH1 having Cys at IDH2 allele described herein , e.g., an IDH2 allele having residue 132 . Lys or Met at residue 172 (SEQ ID NO :10 ). In an embodiment the IDH1 allele has a T (or any other 55 In an embodiment the method comprises selecting a nucleotide other than C ) at nucleotide position 394. In an subject having a prostate cancer, e.g., prostate adenocarci embodiment the allele is a C394Tmutation according to the noma, on the basis of the cancer being characterized by an sequence of SEQ ID NO :5 . IDH2 allele described herein , e.g., an IDH2 allele having In an embodiment the method comprises selecting a Lys or Met at residue 172 (SEQ ID NO : 10 ) . subject having myelodysplasia or myelodysplastic syn- 60 In an embodiment the method comprises selecting a drome characterized by an IDH1 allele described herein , subject having a prostate cancer, e.g., prostate adenocarci e.g. , an IDH1 allele having Cys, His, or Gly at residue 132 noma, on the basis of the cancer being characterized by according to the sequence of SEQ ID NO : 8 , more specifi increased levels of an alpha hydroxy neoactivity product , cally , Cys at residue 132 . e.g. , 2HG , e.g., R - 2HG . In an embodiment the method comprises selecting a 65 In an embodiment the cell proliferation - related disorder is subject having myelodysplasia or myelodysplastic syn ALL , e.g., B -ALL or T -ALL , characterized by a mutation , or drome on the basis of cancer being characterized by having preselected allele , of IDH2 associated with an alpha hydroxy US 10,610,125 B2 11 12 neoactivity , e.g., 2HG neoactivity. E.g. , in an embodiment , In an embodiment the method comprises selecting a the IDH2 allele encodes an IDH2 having other than an Arg subject having myelodysplasia or myelodysplastic syn at residue 172. E.g., the allele encodes Lys, Gly , Met, Trp , drome, on the basis of the cancer being characterized by an Thr , Ser, or any residue described in described in Yan et al ., IDH2 allele described herein , e.g. , an IDH2 allele having at residue 172, according to the sequence of SEQ ID NO : 10 5 Lys, Gly , or Met at residue 172 ( SEQ ID NO : 10 ) , in specific ( see also FIG . 22 ). In an embodiment the allele encodes an embodiments , Lys or Met. IDH2having Lys at residue 172. In an embodiment the allele In an embodiment the method comprises selecting a encodes an IDH2 having Met at residue 172 . subject having myelodysplasia or myelodysplastic syn In an embodiment the method comprises selecting a subject having ALL , e.g., B -ALL or T- ALL , wherein the 10 dromeincreased, on levelsthe basis of anof alpha the cancer hydroxy being neoactivity characterized product by , cancer is characterized by having an IDH2 allele described e.g., 2HG , e.g., R - 2HG . herein , e.g., an IDH2 allele having Lys or Met at residue 172 In an embodiment a product of the neoactivity is 2HG ( SEQ ID NO : 10 ) . ( e.g. , R -2HG ) which acts as a metabolite . In another subjectIn an havingembodiment ALL , e.g. the, B method -ALL or comprisesT- ALL , on theselecting basis ofa 15 embodiment a product of the neoactivity is 2HG (e.g. , the cancer being characterized by an IDH2 allele described R -2HG ) which acts as a toxin , e.g. , a carcinogen . herein , e.g., an IDH2 allele having Lys or Met at residue 172 In some embodiments , the methods described herein can ( SEQ ID NO : 10 ) . result in reduced side effects relative to other known meth In an embodiment the method comprises selecting a ods of treating cancer. subject having ALL , e.g., B - ALL or T - ALL , on the basis of 20 Therapeutic agents and methods of subject evaluation the cancer being characterized by increased levels of an described herein can be combined with other therapeutic alpha hydroxy neoactivity product , e.g., 2HG , e.g., R -2HG . mocalities , e.g. , with art -known treatments . In an embodiment the cell proliferation -related disorder is In an embodiment the method comprises providing a AML , characterized by a mutation , or preselected allele , of second treatment , to the subject , e.g., surgical removal , IDH2 associated with an alpha hydroxy neoactivity , e.g. , 25 irradiation or administration of a chemotherapeutitc agent, 2HG neoactivity . E.g., in an embodiment, the IDH2 allele e.g., an administration of an alkylating agent. Administration encodes an IDH2 having other than an Arg at residue 172 . (or the establishment of therapeutic levels ) of the second E.g. , the allele encodes Lys , Gly , Met , Trp , Thr, Ser, or any treatment can : begin prior to the beginning or treatment with residue described in described in Yan et al. , at residue 172 , ( or prior to the establishment of therapeutic levels of) the according to the sequence of SEQ ID NO : 10 ( see also FIG . 30 inhibitor ; begin after the beginning or treatment with ( or 22 ) , more specifically Lys, Gly , Met, or Ser . In an embodi after the establishment of therapeutic levels of) the inhibitor, ment the allele encodes an IDH2 having Lys at residue 172 . or can be administered condoncurrently with the inhibitor, e.g. , In an embodiment the allele encodes an IDH2 having Met to achieve therapeutic levels of both concurrently . residue 172. In an embodiment the allele encodes an IDH2 In an embodiment the cell proliferation - related disorder is having Gly at residue 172. 35 a CNS tumor , e.g., a glioma, and the second therapy com In an embodiment the method comprises selecting a prises administration of one or more of : radiation ; an alky subjecthaving AML , wherein the cancer is characterized by lating agent, e.g. , temozolomide , e.g., Temoader® , or having an IDH2 allele described herein , e.g., an IDH2 allele BCNU ; or an inhibitor of HER1/ EGFR tyrosine kinase , e.g., having Lys , Gly or Met at residue 172 (SEQ ID NO : 10 ) , erlotinib , e.g. , Tarceva® . more specifically Lys or Met . 40 The second therapy , e.g., in the case of glioma, can In an embodiment the method comprises selecting a comprise implantation of BCNU or carmustine in the brain , subject having AML , on the basis of the cancer being e.g., implantation of a Gliadel® wafer. characterized by an IDH2 allele described herein , e.g. , an The second therapy, e.g. , in the case of glioma, can IDH2 allele having Lys ,Gly , or Met at residue 172 (SEQ ID comprise administration of imatinib , e.g. , Gleevec® . NO : 10 ), more specifically Lys or Met . 45 In an embodiment the cell proliferation - related disorder is In an embodiment the method comprises selecting a prostate cancer and the second therapy comprises one or subject having AML , on the basis of the cancer being more of: androgen ablation ; administration of a microtubule characterized by increased levels of an alpha hydroxy neo stabilizer, e.g. , docetaxol, e.g. , Taxotere? ; or administration activity product, e.g. , 2HG , e.g., R - 2HG . of a topoisomerase II inhibitor, e.g., mitoxantrone. In an embodiment the cell proliferation - related disorder is 50 In an embodiment the cell proliferation - related disorder is myelodysplasia or myelodysplastic syndrome, characterized ALL , e.g., B - ALL or T -ALL , and the second therapy com by a mutation , or preselected allele, of IDH2 . E.g. , in an prises one or more of: embodiment, the IDH2 allele encodes an IDH2 having other induction phase treatment comprising the administration than an Arg at residue 172. E.g. , the allele encodes Lys, Gly , of one or more of: a steroid ; an inhibitor of microtubule Met, Trp , Thr, Ser , or any residue described in described in 55 assembly , e.g., vincristine; an agent that reduces the avail Yan et al. , at residue 172, according to the sequence of SEQ ability of asparagine , e.g. , asparaginase; an anthracycline; or ID NO : 10 ( see also FIG . 22 ) , more specifically Lys , Gly , an antimetabolite , e.g. , methotrexate , e.g. , intrathecal Met , Trp or Ser. In an embodiment the allele encodes an methotrexate , or 6 -mercaptopurine ; IDH2having Lys at residue 172. In an embodiment the allele consolidation phase treatment comprising the administra encodes an IDH2 having Met at residue 172. In an embodi- 60 tion of one or more of: a drug listed above for the induction ment the allele encodes an IDH2 having Gly at residue 172 . phase; an antimetabolite , e.g., a guanine analog , e.g., 6 -thio In an embodiment the method comprises selecting a guanine; an alkylating agent, e.g. , cyclophosphamide ; an subject having myelodysplasia or myelodysplastic syn antimetabolite , e.g., AraC or cytarabine; or an inhibitor of drome, wherein the cancer is characterized by having an topoisomerase I, e.g., etoposide ; or IDH2 allele described herein , e.g., an IDH2 allele having 65 maintenance phase treatment comprising the administra Lys, Gly , or Met at residue 172 (SEQ ID NO : 10 ), in specific tion of one or more of the drugs listed above for induction embodiments , Lys or Met . or consolidation phase treatment . US 10,610,125 B2 13 14 In an embodiment the cell proliferation - related disorder is staging the subject , e.g., determining the stage of a cell AML and the second therapy comprises administration of proliferation -related disorder , e.g., a disorder characterized one or more of an inhibitor of topoisomerase II, e.g., by unwanted cell proliferation , e.g., cancer, or a precancer daunorubicin , idarubicin , topotecan or mitoxantrone ; an ous disorder ; inhibitor of topoisomerase I, e.g., etoposide; or an anti- 5 providing a prognosis for the subject , e.g. , providing a metabolite , e.g. , AraC or cytarabine . prognosis for a cell proliferation -related disorder , e.g. , a In another aspect, the invention features , a method of disorder characterized by unwanted cell proliferation , e.g., evaluating , e.g. diagnosing, a subject, e.g., a subject not cancer, or a precancerous disorder ; having , or not diagnosed as having , 2 -hydroxyglutaric aci determining the efficacy of a treatment, e.g., the efficacy duria . The method comprises analyzing a parameter related 10 of a chemotherapeutic agent, irradiation or surgery ; to the neoactivity genotype or phenotype of the subject, e.g., determining the efficacy of a treatment with a therapeutic analyzing one or more of: agent, e.g., an inhibitor, described herein ; a ) the presence, distribution , or level of a neoactive selecting the subject for a treatment for a cell prolifera product, e.g., the product of an alpha hydroxy neoactivity, 15 tionunwanted -related cell disorder proliferation , e.g. , e.g.a disorder, cancer, orcharacterized a precancerous by e.g., 2HG , e.g., R -2HG , e.g., an increased level of product, disorder. The selection can be based on the need for a 2HG , e.g., R -2HG (as used herein , an increased level of a reduction in neoactivity or on the need for amelioration of a product of an alpha hydroxy neoactivity , e.g., 2HG , e.g. , condition associated with or resulting from neoactivity . For R - 2HG , or similar term , e.g. , an increased level of neoactive example , if it is determined that the subject has a cell product or neoactivity product, means increased as com- 20 proliferation -related disorder, e.g. , e.g., cancer , or a precan pared with a reference , e.g., the level seen in an otherwise cerous disorder characterized by increased levels of an alpha similar cell lacking the IDH mutation , e.g. , IDH1 or IDH2 hydroxy neoactivity product, e.g., 2HG , e.g., R -2HG , or by mutation , or in a tissue or product from a subjectnoth having a mutant IDH1 or IDH2, having alpha hydroxyl neoactivity , the mutation ( the terms increased and elevated as refered to e.g. , 2HG , neaoctivity , selecting the subject for treatment the level of a product of alpha hydroxyl neoactivity as used 25 with a therapeutic agent described herein , e.g. , an inhibitor herein , are used interchangably ) ; ( e.g., a small molecule or a nucleic acid -based inhibitor) of b ) the presence, distribution ,or level of a neoactivity , e.g. , the neoactivity of that mutant (e.g. , conversion of alpha alpha hydroxy neoactivity , e.g., 2HG neoactivity, of an ketoglutarate to 2HG , e.g., R -2HG ) ; IDH1 or IDH2, mutant protein ; correlating the analysis with an outcome or a prognosis ; 30 providing a value for an analysis on which the evaluation c ) the presence , distribution , or level of a neoactive is based , e.g., the value for a parameter correlated to the mutant protein , e.g., an IDH , e.g., an IDH1 or IDH2, mutant presence , distribution , or level of an alpha hydroxyl neoac protein which has a neoactivity , e.g., alpha hydroxy neoac tivity product , e.g., 2HG , e.g., R -2HG ; tivity , e.g., 2HG neoactivity , or a corresponding RNA ; or providing a recommendation for treatmentof the subject; d ) the presence of a selected somatic allele or mutation 35 or conferring neoactivity, e.g., an IDH , e.g., IDH1 or IDH2 , memorializing a result of, or output from , the method , which encodes a protein with a neoactivity , e.g. , alpha e.g., a measurement made in the course of performing the hydroxy neoactivity , e.g., 2HG neoactivity , e.g., an allele method , and optionally transmitting the memorialization to disclosed herein , in cells characterized by a cell prolifera a party , e.g. , the subject , a healthcare provider, or an entity tion - related disorder from the subject , thereby evaluating the 40 that pays for the subject's treatment, e.g., a government, subject . insurance company , or other third party payer. In an embodiment analyzing comprises performing a As described herein , the evaluation can provide informa procedure , e.g., a test, to provide data or information on one tion on which a number of decisions or treatments can be or more of a -d , e.g., performing a method which results in based . a physical change in a sample , in the subject , or in a device 45 Thus, in an embodiment the result of the evaluation , e.g., or reagent used in the analysis , or which results in the an increased level of an alpha hydroxyl neoactivity product, formation of an image representative of the data .Methods of e.g. , 2HG , e.g. , R -2HG , the presence of an IDH , e.g. , IDH1 obtaining and analyzing samples , and the in vivo analysis in or IDH2 , neoactivity , e.g., alpha hydroxyl neoactivity , e.g., subjects , described elsewhere herein , e.g. , in the section 2HG neoactivity , the presence of an IDH , e.g. , IDH1 or entitled , “ Methods of evaluating samples and / or subjects, ” 50 IDH2 , mutant protein ( or corresponding RNA ) which has can be combined with this method . In another embodiment alpha hydroxyl neoactivity , e.g. , 2HG neoactivity , the pres analyzing comprises receiving data or information from ence of a mutant allele of IDH , e.g., IDH1 or IDH2, having such test from another party . In an embodiment the analyz alpha hydroxyl neoactivity , 2HG neoactivity , e.g., an allele ing comprises receiving data or information from such test disclosed herein , is indicative of: from another party and , the method comprises, responsive to 55 a cell proliferation - related disorder , e.g., cancer, e.g., it is that data or information , administering a treatment to the indicative of a primary or metastatic lesion ; subject . the stage of a cell proliferation - related disorder ; As described herein , the evaluation can be used in a a prognosis or outcome for a cell proliferation - related number of applications, e.g. , for diagnosis , prognosis, stag disorder, e.g. , it is indicative of a less aggressive form of the ing , determination of treatment efficacy, patent selection , or 60 disorder , e.g., cancer . E.g., in the case of glioma, presence of drug selection . an alpha hydroxyl neoactivity product , e.g., 2HG , e.g., Thus , in an embodiment method further comprises, e.g. , R -2HG , can indicate a less aggressive form of the cancer ; responsive to the analysis of one or more of a - d : the efficacy of a treatment, e.g., the efficacy of a chemo diagnosing the subject, e.g. , diagnosing the subject as therapeutic agent, irradiation or surgery ; having a cell proliferation - related disorder, e.g., a disorder 65 the need of a therapy disclosed herein , e.g. , inhibition a characterized by unwanted cell proliferation , e.g., cancer, or neoactivity of an IDH , e.g., IDH1 or IDH2, neoactive mutant a precancerous disorder ; described herein . In an embodiment relatively higher levels US 10,610,125 B2 15 16 ( or the presence of the mutant ) is correlated with need of In an embodiment evaluation comprises selecting the inhibition a neoactivity of an IDH , e.g., IDH1 or IDH2, subject, e.g., for treatment. mutant described herein ; or In embodiments the treatment is the administration of a responsiveness to a treatment. The result can be used as a therapeutic agent described herein . noninvasive biomarker for clinical response . E.g. , elevated 5 The methods can also include treating a subject , e.g , with levels can be predictive on better outcome in glioma patients a treatment selected in response to , or on the basis of, an (e.g. , longer life expectancy ). evaluation made in the method . As described herein , the evaluation can provide for the Thus, in an embodiment the method comprises, e.g. , selection of a subject. responsive to the analysis of one or more of a - d, adminis Thus , in an embodiment the method comprises , e.g., 10 tering a treatment to the subject, e.g., the administration of responsive to the analysis of one or more of a -d , selecting a a therapeutic agent of a type described herein . subject , e.g. , for a treatment. The subject can be selected on In an embodiment the therapeutic agent comprises a a basis described herein , e.g. , on the basis of: compound from Table 24a or Table 24b or a compound normalsaid levelssubject of being an alpha at hydroxyrisk for, neoactivityor having ,product higher , thane.g., 15 having the structure of Formula (X ) or (XI ) described below . 2 -hydroxyglurarate ( e.g., R - 2HG ) in cell having a cell In an embodiment the therapeutic agent comprises nucleic proliferation - related disorder , e.g., a leukemia such as AML acid , e.g. , dsRNA , e.g. , a dsRNA described herein . or ALL , e.g., B - ALL or T - ALL , or a tumor lesion , e.g. , a In an embodiment the therapeutic agent is an inhibitor, glioma or a prostate tumor; e.g., a polypeptide, peptide , or small molecule ( e.g. , a said subject having a proliferation - related disorder char- 20 molecule of less than 1,000 daltons) , or aptomer , that binds acterized by a selected IDH , e.g. , IDH1 or IDH2 allele , e.g. , to an IDH1 or IDH2 mutant ( e.g., an aptomer that binds to an IDH1 or IDH2 mutation , having alpha hydroxyl neoac an IDH1mutant ) or wildtype subunit and inhibits neoactiv tivity , e.g., 2HG neoactivity ; ity , e.g., by inhibiting formation of a dimer , e.g. , a homodi said subject having a selected IDH allele , e.g. , a selected mer ofmutant IDH1 or IDH2 subunits (e.g. , a homodimer of IDH1 or IDH2 allele ; 25 mutant IDH1 subunits ) or a heterodimer of a mutant and a having alpha hydroxyl neoactivity , e.g. , 2HG neoactivity ; wildtype subunit . In an embodiment the inhibitor is a said subject having a proliferation - related disorder; polypeptide. In an embodiment the polypeptide acts as a said subject being in need of, or being able to benefit dominant negative with respect to the neoactivity of the from , a therapeutic agent of a type described herein ; mutant enzyme. The polypeptide can correspond to full said subject being in need of, or being able to benefit 30 length IDH1 or IDH2 or a fragment thereof ( e.g. , the from , a compound that inhibits alpha hydroxyl neoactivity , polypeptide corresponds to full length IDH1 or a fragment e.g. , 2HG neoactivity ; thereof) . The polypeptide need not be identical with the said subject being in need of, or being able benefit corresponding residues of wildtype IDH1 or IDH2 ( e.g. , from , a compound that lowers the level of an alpha hydroxyl wildtype IDH1) , but in embodiments has at least 60 , 70, 80 , neoactivity product , e.g. , 2HG , e.g., R -2HG . 35 90 or 95 % homology with wildtype IDH1 or IDH2 ( e.g., In an embodiment evaluation comprises selecting the wildtype IDH1) . subject , e.g. , for treatment with an anti- neoplastic agent, on In an embodiment the therapeutic agent decreases the the establishment of, or determination that, the subject has affinity of an IDH , e.g., IDH1 or IDH2 neoactive mutant increased alpha hydroxyl neoactivity product , e.g., 2HG , protein for NADH , NADPH or a divalent metal ion , e.g., e.g., R - 2HG , or increased alpha hydroxyl neoactivity , e.g., 40 Mg2+ or Mn2+ , or decreases the levels or availability of 2HG neoactivity , or that the subject is in need of inhibition NADH , NADPH or divalent metal ion , e.g. ,Mg2 + or Mn2 +, of a neoactivity of an IDH , e.g., IDH1 or IDH2, mutant e.g., by competing for binding to the mutant enzyme. In an described herein . embodiment the enzyme is inhibited by replacing Mg2 + or As described herein , the evaluations provided for by Mn2 + with Ca2 + . methods described herein allow the selection of optimal 45 In an embodiment the therapeutic agent is an inhibitor that treatment regimens. reduces the level a neoactivity of an IDH , e.g., IDH1 or Thus, in an embodiment the method comprises , e.g., IDH2, e.g., 2HG neoactivity . responsive to the analysis of one or more of a -d , selecting a In an embodiment the therapeutic agent is an inhibitor that treatment for the subject, e.g., selecting a treatment on a reduces the level of the product of a mutant having a basis disclosed herein . The treatment can be the adminis- 50 neoactivity of an IDH , e.g. , IDH1 or IDH2mutant , e.g., it tration of a therapeutic agent disclosed herein . The treatment reduces the level of 2HG , e.g. , R -2HG . can be selected on the basis that : In an embodiment the therapeutic agent is an inhibitor it us useful in treating a disorder characterized by one or that: more of alpha hydroxyl neoactivity , e.g. , 2HG neoactivity , inhibits , e.g., specifically , a neoactivity of an IDH , e.g., an IDH1 or IDH2 , mutant protein having alpha hydroxyl 55 IDH1 or IDH2, e.g., a neoactivity described herein , e.g. , neoactivity, e.g., 2HG neoactivity ( or a corresponding 2HG neoactivity ; or RNA ) ; inhibits both the wildtype activity and a neoactivity of an it is useful in treating a disorder characterized by a IDH , e.g., IDH1 or IDH2, e.g. , a neoactivity described selected somatic allele or mutation of an IDH , e.g., IDH1 or herein , e.g. , 2HG neoactivity . IDH2, which encodes a protein with alpha hydroxyl neoac- 60 In an embodiment the therapeutic agent is an inhibitor that tivity , e.g. , 2HG neoactivity , e.g. , an allele disclosed herein , is selected on the basis that it: in cells characterized by a cell proliferation - related disorder inhibits , e.g., specifically , a neoactivity of an IDH , e.g. , from the subject; IDH1 or IDH2 , e.g., a neoactivity described herein e.g., 2HG it reduces the level of an alpha hydroxyl neoactivity neoactivity ; or product, e.g., 2HG , e.g. , R - 2HG ; 65 inhibits both the wildtype activity and a neoactivity of an it reduces the level of alpha hydroxyl neoactivity, e.g. , IDH1 , e.g. , IDH1 or IDH2, e.g., a neoactivity described 2HG neoactivity . herein , e.g., 2HG neoactivity . US 10,610,125 B2 17 18 In an embodiment the therapeutic agent is an inhibitor that evaluation or to what would be administered in the absence reduces the amount of a mutant IDH , e.g. , IDH1 or IDH2, of elevated levels of an alpha hydroxy neoactivity product , protein or mRNA . e.g., 2HG , e.g., R - 2HG ) ; In an embodiment the therapeutic agent is an inhibitor that a treatment which includes administration of a therapeutic interacts directly with , e.g. , it binds to , the mutant IDH , e.g. , 5 agent at a different frequency , e.g. , more or less frequently , IDH1 or IDH2 mRNA . or not at all ( e.g. , different as compared to what was In an embodiment the therapeutic agent is an inhibitor that administered prior to the evaluation or to what would be interacts directly with , e.g., it binds to , the mutant IDH , e.g., administered in the absence of elevated levels of an alpha IDH1 or IDH2, protein . hydroxy neoactivity product , e.g. , 2HG , e.g., R -2HG ) ; or 10 a treatment which includes administration of a therapeutic In an embodiment the therapeutic agent is an inhibitor that agent in a different therapeutic setting ( e.g., adding or reduces the amount of neoactive enzyme activity , e.g., by deleting a second treatment from the treatment regimen ) interacting with , e.g., binding to , mutant IDH , e.g. , IDH1 or ( e.g., different as compared to what was administered prior IDH2, protein . In an embodiment the inhibitor is other than to the evaluation or to what would be administered in the an antibody. 15 absence of elevated levels of an alpha hydroxy neoactivity In an embodiment the therapeutic agent is an inhibitor that product , e.g., 2HG , e.g. , R -2HG ) . is a small molecule and interacts with , e.g. , binds, the mutant Methods of evaluating a subject described herein can RNA , e.g. , mutant IDH1 mRNA . comprise evaluating a neoactivity genotype or phenotype. In an embodiment the therapeutic agent is an inhibitor that Methods of obtaining and analyzing samples , and the in vivo interacts directly with , e.g. , binds , either the mutant IDH , 20 analysis in subjects , described elsewhere herein , e.g., in the e.g., IDH1 or IDH2, protein or interacts directly with , e.g., section entitled , “ Methods of evaluating samples and / or binds , the mutant IDH mRNA , e.g. , IDH1 or IDH2 mRNA . subjects , " can be combined with this method . In an embodiment the therapeutic agent is administered . In an embodiment the method comprises : In an embodiment the treatment : inhibits , e.g., specifi subjecting the subject (e.g. , a subject not having 2 -hy cally , a neoactivity of IDH1 or IDH2 ( e.g., a neoactivity of 25 droxyglutaric aciduria ) to imaging and / or spectroscopic IDH1) , e.g. , a neoactivity described herein ; or inhibits both analysis , e.g. ,magnetic resonance - based analysis , e.g., MRI the wildtype and activity and a neoactivity of IDH1 or IDH2 and/ or MRS e.g., imaging analysis , to provide a determina ( e.g., a neoactivity of IDH1) , e.g., a neoactivity described tion of the presence , distribution , or level of an alpha herein In an embodiment, the subject is subsequently evalu hydroxy neoactivity product , e.g., 2HG , e.g., R - 2HG , e.g., as ated or monitored by a method described herein , e.g., the 30 associated with a tumor, e.g., a glioma, in the subject ; analysis of the presence , distribution , or level of an alpha optionally storing a parameter related to the determina hydroxy neoactivity product, e.g. , 2HG , e.g., R - 2HG , e.g., to tion , e.g., the image or a value related to the image from the evaluate response to the treatment or progression of disease . imaging analysis , in a tangible medium ; and In an embodiment the treatment is selected on the basis responsive to the determination , performing one or more that it : inhibits , e.g., specifically , a neoactivity of IDH1 or 35 of: correlating the determination with outcome or with a IDH2 ( e.g., a neoactivity of IDH1) , e.g., alpha hydroxy prognosis ; providing an indication of outcome or prognosis ; neoactivity , e.g. , 2HG neoactivity ; or inhibits both the providing a value for an analysis on which the evaluation is wildtype and activity and a neoactivity of IDH1 or IDH2 based , e.g. , the presence , distribution , or level of an alpha ( e.g. , a neoactivity of IDH1) , e.g. , a neoactivity described hydroxy neoactivity product, e.g., 2HG , e.g., R -2HG ; pro herein . 40 viding a recommendation for treatment of the subject ; In an embodiment , the method comprises determining the selecting a course of treatment for the subject , e.g. , a course possibility of a mutation other than a mutation in IDH1 or in of treatment described herein , e.g., selecting a course of IDH2. In embodiments a relatively high level of 2HG , e.g., treatment that includes inhibiting a neoactivity of a mutant R -2HG is indicative of another mutation . IDH , e.g., IDH1 or IDH2, allele , e.g. , a neoactivity described In an embodiment, which embodiment includes selecting 45 herein ; administering a course of treatment to the subject , or administering a treatment for the subject, the subject : e.g., a course of treatment described herein , e.g., a course of has not yet been treated for the subject the cell prolifera treatment that includes inhibiting a neoactivity of a mutant tion - related disorder and the selected or administered treat IDH , e.g., IDH1 or IDH2, allele , e.g. , a neoactivity described ment is the initial or first line treatment; herein ; and memorializing memorializing a result of the has already been treated for the cell proliferation -related 50 method or a measurement made in the course of the method , and the selected or administered treatment results in an e.g. , one or more of the above and /or transmitting memo alteration of the existing treatment; rialization of one or more of the above to a party , e.g. , the has already been treated for the cell proliferation - related , subject, a healthcare provider , or an entity that pays for the and the selected treatment results in continuation of the subject's treatment, e.g. , a government, insurance company , existing treatment; or 55 or other third party payer. has already been treated for the cell proliferation - related In an embodiment the method comprises confirming or disorder and the selected or administered treatment is dif determining, e.g., by direct examination or evaluation of the ferent, e.g., as compared to what was administered prior to subject, or sample e.g. , tissue or bodily fluid ( e.g., blood the evaluation or to what would be administered in the ( e.g., blood plasma) , urine, lymph , or cerebrospinal fluid ) absence of elevated levels of an alpha hydroxy neoactivity 60 therefrom , ( e.g., by DNA sequencing or immuno analysis or product, e.g., 2HG , e.g. , R - 2HG . evaluation of the presence , distribution or level of an alpha In an embodiment , which embodiment includes selecting hydroxy neoactivity product, e.g., 2HG , e.g. , R -2HG ) , or or administering a treatment for the subject , the selected or receiving such information about the subject, that the subject administered treatment can comprise : has a cancer characterized by an IDH , e.g., IDH1 or IDH2, a treatment which includes administration of a therapeutic 65 allele described herein , e.g. , an IDH1 allele having His , Ser , agent at different , e.g., a greater ( or lesser ) dosage ( e.g., Cys, Gly , Val , Pro or Leu at residue 132 (SEQ ID NO :8 ), in different as compared to what was administered prior to the specific embodiments , an IDH1 allele having His , Ser, Cys, US 10,610,125 B2 19 20 Gly , Val, or Leu at residue 132 or an IDH1 allele having His e.g. , IDH1 or IDH2, mutation having 2HG neoactivity , at the or Cys at residue 132 ; or an IDH2 allele having Lys, Gly , time of diagnosis or treatment . Met , Trp , Thr, or Ser at residue 172 (SEQ ID NO : 10 ) . In an embodiment the cell proliferation - related disorder is In an embodiment , prior to or after treatment, the method a cancer, e.g. , a cancer described herein , characterized by an includes evaluating the growth , size , weight , invasiveness , 5 IDH1 somatic mutant having alpha hydroxy neoactivity , stage or other phenotype of the cell proliferation -related e.g., 2HG neoactivity , e.g., a mutant described herein . In an disorder. embodiment the tumor is characterized by increased levels In an embodiment the cell proliferation -related disorder is of an alpha hydroxy neoactivity product, 2HG , e.g., R - 2HG , a tumor of the CNS, e.g. , a glioma, a leukemia , e.g., AML as compared to non -diseased cells of the same type. or ALL , e.g., B - ALL or T - ALL , prostate cancer , or myelo- 10 In an embodiment the method comprises selecting a dysplasia or myelodysplastic syndrome and the evaluation is subject having a glioma, on the basis of the cancer being a or b . In an embodiment the method comprises evaluating characterized by increased levels of an alpha hydroxy neo a sample , e.g., a sample described herein , e.g., a tissue , e.g., activity , product , e.g. , 2HG , e.g., R -2HG . a cancer sample , or a bodily fluid , e.g., serum or blood , for In an embodiment the cell proliferation - related disorder is increased alpha neoactivity product, e.g., 2HG , e.g., R -2HG . 15 a tumor of the CNS, e.g., a glioma, e.g., wherein the tumor In an embodiment, a subject is subjected to MRS and the is characterized by an IDH1 somatic mutant having alpha evaluation comprises evaluating the presence or elevated hydroxy neoactivity , e.g. , 2HG neoactivity , e.g. , a mutant amount of a peak correlated to or corresponding to 2HG , described herein . Gliomas include astrocytic tumors, oligo e.g. , R -2HG , as determined by magnetic resonance . For dendroglial tumors, oligoastrocytic tumors, anaplastic astro example , a subject can be analyzed for the presence and / or 20 cytomas, and glioblastomas . In an embodiment the tumor is strength of a signal at about 2.5 ppm to determine the characterized by increased levels of an alpha hydroxy neo presence and / or amount of 2HG , e.g., R - 2HG in the subject . activity product, e.g. , 2HG , e.g. , R -2HG , as compared to In an embodiment the method comprises obtaining a non - diseased cells of the same type. E.g., in an embodiment, sample from the subject and analyzing the sample , or the IDH1 allele encodes an IDH1 having other than an Arg analyzing the subject , e.g. , by imaging the subject and 25 at residue 132. E.g., the allele encodes His, Ser , Cys, Gly , optionally forming a representation of the image on a Val , Pro or Leu , or any residue described in Yan et al. , at computer. residue 132 , according to the sequence of SEQ ID NO : 8 (see In an embodiment the results of the analysis is compared also FIG . 21 ) . In an embodiment the allele encodes an IDH1 to a reference . having His at residue 132. In an embodiment the allele In an embodiment a value for a parameter correlated to the 30 encodes an IDH1 having Ser at residue 132 . presence, distribution , or level, e.g. , of 2HG , e.g., R - 2HG , is In an embodiment the IDH1 allele has an A (or any other determined . It can be compared with a reference value , e.g. , nucleotide other than C ) at nucleotide position 394 , or an A the value for a reference subject not having abnormal (or any other nucleotide other than G ) at nucleotide position presence , level, or distribution , e.g., a reference subject cell 395. In an embodiment the allele is a C394A , a C394G , a not having a mutation in IDH , e.g. , IDH1 or IDH2, having 35 C3947, a G395C , a G395T or a G395A mutation , specifi a neoactivity described herein . cally C394A or a G395A mutation according to the sequence In an embodiment the method comprises determining if of SEQ ID NO : 5 . an IDH , e.g., IDH1 or IDH2, mutant allele that is associated In an embodiment the method comprises selecting a with 2HG neoactivity is present. E.g., in the case of IDHI, subject having a glioma, wherein the cancer is characterized the presence of a mutation at residue 132 associated with 40 by having an IDH1 allele described herein , e.g., an IDH1 2HG neoactivity can be determined . In the case of IDH2 , the allele having His , Ser , Cys, Gly , Val, Pro or Leu at residue presence of a mutation at residue 172 associated with 2HG 132 ( SEQ ID NO :8 ) ( e.g. , His , Ser , Cys, Gly , Val, or Leu ; or neoactivity can be determined . The determination can com His or Cys ). prise sequencing a nucleic acid , e.g. , genomic DNA or In an embodiment the method comprises selecting a cDNA , from an affected cell, which encodes the relevant 45 subject having a glioma, on the basis of the cancer being amino acid ( s ). The mutation can be a deletion , insertion , characterized by an IDH1 allele described herein , e.g., an rearrangement, or substitution . The mutation can involve a IDH1 allele having His , Ser , Cys , Gly , Val, Pro or Leu at single nucleotide, e.g., a single substitution , or more than residue 132 (SEQ ID NO : 8 ) (e.g. , His , Ser, Cys ,Gly , Val, or one nucleotide , e.g., a deletion ofmore than one nucleotides . Leu ; or His or Cys ) . In an embodiment themethod comprises determining the 50 In an embodiment the method comprises selecting a sequence at position 394 or 395 of the IDH1 gene, or subject having a glioma, on the basis of the cancer being determining the identity of amino acid residue 132 (SEQ ID characterized by increased levels of an alpha hydroxy neo NO : 8 ) in the IDH1 gene in a cell characterized by the cell activity , product , e.g., 2HG , e.g., R -2HG . proliferation related disorder. In an embodiment, the cell proliferation disorder is fibro In an embodiment the method comprises determining the 55 sarcoma or paraganglioma wherein the cancer is character amino acid sequence , e.g. , by DNA sequencing , at position ized by having an IDH1 allele described herein , e.g. , an 172 of the IDH2 gene in a cell characterized by the cell IDH1 allele having Cys at residue 132 (SEQ ID NO : 8 ). proliferation related disorder. In an embodiment, the cell proliferation disorder is fibro In an embodiment a product of the neoactivity is 2 - HG , sarcoma or paraganglioma wherein the cancer is character e.g. , R - 2HG , which acts as a metabolite . In another embodi- 60 ized by an IDH1 allele described herein , e.g., an IDH1 allele ment a product of the neoactivity is 2HG , e.g. , R - 2HG , having Cys at residue 132 (SEQ ID NO : 8 ) . which acts as a toxin , e.g. , a carcinogen . In an embodiment, the cell proliferation disorder is fibro In an embodiment the disorder is other than a solid tumor . sarcoma or paraganglioma wherein the cancer is character In an embodiment the disorder is a tumor that, at the time of ized by increased levels of an alpha hydroxy neoactivity , diagnosis or treatment, does not have a necrotic portion . In 65 product, e.g., 2HG , e.g. , R - 2HG . an embodiment the disorder is a tumor in which at least 30 , In an embodiment the cell proliferation - related disorder is 40 , 50 , 60 , 70 , 80 or 90 % of the tumor cells carry an IHD , localized or metastatic prostate cancer, e.g., prostate adeno US 10,610,125 B2 21 22 carcinoma, e.g. , wherein the cancer is characterized by an by an IDH1 allele described herein , e.g. , an IDH1 allele IDH1 somatic mutant having alpha hydroxy neoactivity , having Cys at residue 132 according to the sequence of SEQ e.g., 2HG neoactivity , e.g., a mutant described herein . In an ID NO : 8 . embodiment the cancer is characterized by increased levels In an embodiment the method comprises selecting a of an alpha hydroxy neoactivity product, e.g. , 2HG , e.g., 5 subject ALL , e.g. , B - ALL or T -ALL , on the basis of cancer R - 2HG , as compared to non -diseased cells of the same type. being characterized by having an IDH1 allele described E.g. , in an embodiment, the IDH1 allele encodes an IDH1 herein , e.g., an IDH1 allele having Cys at residue 132 (SEQ having other than an Arg at residue 132. E.g., the allele ID NO :8 ). encodes His , Ser, Cys, Gly , Val, Pro or Leu , or any residue In an embodiment the method comprises selecting a described in Kang et al, 2009, Int. J. Cancer, 125 : 353-355 10 subject having ALL , e.g. , B - ALL or T -ALL , on the basis of at residue 132 , according to the sequence of SEQ ID NO :8 the cancer being characterized by increased levels of an ( see also FIG . 21 ) ( e.g., His , Ser , Cys, Gly , Val, or Leu ). In alpha hydroxy neoactivity product, e.g., 2HG , e.g., R - 2HG . an embodiment the allele encodes an IDH1 having His or In an embodiment the cell proliferation -related disorder is Cys at residue 132 . acute myelogenous leukemia ( e.g., an adult or pediatric In an embodiment the IDH1 allele has a T (or any other 15 form ), e.g., wherein the acute myelogenous leukemia ( some nucleotide other than C ) at nucleotide position 394 , or an A times referred to herein as AML ) is characterized by an ( or any other nucleotide other than G ) at nucleotide position IDH1 somatic mutant having alpha hydroxy neoactivity , 395. In an embodiment the allele is a C394T or a G395A e.g. , 2HG neoactivity , e.g. , a mutant described herein . In an mutation according to the sequence of SEQ ID NO :5 . embodiment the cancer is characterized by increased levels In an embodiment the method comprises selecting a 20 of an alpha hydroxy neoactivity product, e.g., 2HG , e.g., subject having prostate cancer, e.g. , prostate adenocarci R - 2HG , as compared to non -diseased cells of the same type . noma, wherein the cancer is characterized by an IDH1 allele E.g., in an embodiment, the IDH1 allele is an IDH1 having described herein , e.g. , an IDH1 allele having His or Cys at other than an Arg at residue 132 ( SEQ ID NO : 8 ). E.g., the residue 132 (SEQ ID NO : 8 ) . allele encodes His , Ser , Cys, Gly , Val , Pro or Leu , or any In an embodiment the method comprises selecting a 25 residue described in Kang et al. , at residue 132 , according to subject having prostate cancer, e.g. , prostate adenocarci the sequence of SEQ ID NO : 8 ( see also FIG . 21 ) ( e.g., His , noma, on the basis of the cancer being characterized by an Ser, Cys, Gly , Val or Leu ) . In an embodiment the allele IDH1 allele described herein , e.g., an IDH1 allele having encodes an IDH1 having Cys , His or Gly at residue 132 , His or Cys at residue 132 (SEQ ID NO : 8 ) . specifically , Cys. In an embodiment the method comprises selecting a 30 In an embodiment the IDH1 allele has a T (or any other subject having prostate cancer , on the basis of the cancer nucleotide other than C ) at nucleotide position 394. In an being characterized by increased levels of an alpha hydroxy embodiment the allele is a C394T mutation according to the neoactivity product , e.g., 2HG , e.g. , R - 2HG . sequence of SEQ ID NO :5 . In an embodiment the cell proliferation -related disorder is In an embodiment the method comprises selecting a a hematological cancer , e.g., a leukemia , e.g. , AML , or ALL , 35 subject having acute myelogenous lymphoplastic leukemia wherein the hematological cancer is characterized by an ( AML ) characterized by an IDH1 allele described herein , IDH1 somatic mutant having alpha hydroxy neoactivity , e.g. , an IDH1 allele having Cys, His or Gly at residue 132 e.g. , 2HG neoactivity , e.g., a mutant described herein . In an according to the sequence of SEQ ID NO : 8 , specifically , embodiment the cancer is characterized by increased levels Cys . of an alpha hydroxy neoactivity product, e.g., 2HG , e.g., 40 In an embodiment the method comprises selecting a R - 2HG , as compared to non -diseased cells of the same type . subject having acute myelogenous lymphoplastic leukemia In an embodiment the method comprises evaluating a serum ( AML ) on the basis of cancer being characterized by having or blood sample for increased alpha neoactivity product , an IDH1 allele described herein , e.g., an IDH1 allele having e.g., 2HG , e.g., R - 2HG . Cys , His or Gly at residue 132 (SEQ ID NO : 8 ), specifically , In an embodiment the cell proliferation -related disorder is 45 Cys . acute lymphoblastic leukemia ( e.g. , an adult or pediatric In an embodiment the method comprises selecting a form ), e.g., wherein the acute lymphoblastic leukemia subject having acute myelogenous lymphoplastic leukemia ( sometimes referred to herein as ALL ) is characterized by an (AML ), on the basis of the cancer being characterized by IDH1 somatic mutant having alpha hydroxy neoactivity , increased levels of an alpha hydroxy neoactivity product , e.g., 2HG neoactivity , e.g., a mutant described herein . The 50 e.g., 2HG , e.g. , R -2HG . In an embodiment the method ALL can be , e.g., B - ALL or T -ALL . In an embodiment the comprises evaluating a serum or blood sample for increased cancer is characterized by increased levels of 2 an alpha alpha neoactivity product , e.g., 2HG , e.g., R - 2HG . hydroxy neoactivity product, e.g., HG , e.g. , R - 2HG , as In an embodiment the method further comprises evaluat compared to non -diseased cells of the same type . E.g., in an ing the subject for the presence of a mutation in the NRAS embodiment, the IDH1 allele is an IDH1 having other than 55 or NPMc gene . an Arg at residue 132 (SEQ ID NO : 8 ) . E.g. , the allele In an embodiment the cell proliferation - related disorder is encodes His , Ser, Cys, Gly , Val, Pro or Leu , or any residue myelodysplasia or myelodysplastic syndrome, e.g., wherein described in Kang et al. , at residue 132 , according to the the myelodysplasia or myelodysplastic syndrome is charac sequence of SEQ ID NO : 8 ( see also FIG . 21 ) ( e.g. , His , Ser , terized by having an IDH1 somatic mutant having alpha Cys , Gly , Val, or Leu ). In an embodiment the allele encodes 60 hydroxy neoactivity , e.g., 2HG neoactivity , e.g., a mutant an IDH1 having Cys at residue 132 . described herein . In an embodiment the disorder is charac In an embodiment the IDH1 allele has a T ( or any other terized by increased levels of an alpha hydroxy neoactivity nucleotide other than C ) at nucleotide position 394. In an product , e.g., 2HG , e.g. , R -2HG , as compared to non embodiment the allele is a C394Tmutation according to the diseased cells of the same type . E.g., in an embodiment, the sequence of SEQ ID NO :5 . 65 IDH1 allele is an IDH1 having other than an Arg at residue In an embodiment the method comprises selecting a 132 (SEQ ID NO : 8 ) . E.g. , the allele encodes His , Ser , Cys, subject having ALL , e.g., B -ALL or T- ALL , characterized Gly , Val, Pro or Leu, or any residue described in Kang et al. , US 10,610,125 B2 23 24 according to the sequence of SEQ ID NO : 8 ( see also FIG . In an embodiment the method comprises selecting a 21 ), specifically , His, Ser, Cys, Gly , Val, or Leu . In an subject having a prostate cancer , e.g., prostate adenocarci embodiment the allele encodes an IDH1 having Cys at noma, wherein the cancer is characterized by having an residue 132 . IDH2 allele described herein , e.g., an IDH2 allele having In an embodiment the IDH1 allele has a T (or any other 5 Lys or Met at residue 172 (SEQ ID NO :10 ) . In an embodiment the method comprises selecting a nucleotide other than C ) at nucleotide position 394. In an subject having a prostate cancer , e.g., prostate adenocarci embodiment the allele is a C394T mutation according to the noma, on the basis of the cancer being characterized by an sequence of SEQ ID NO :5 . IDH2 allele described herein , e.g. , an IDH2 allele having In an embodiment the method comprises selecting a 10 Lys or Met at residue 172 (SEQ ID NO : 10 ) . subject having myelodysplasia or myelodysplastic syn In an embodiment the method comprises selecting a drome characterized by an IDH1 allele described herein , subject having a prostate cancer, e.g., prostate adenocarci e.g., an IDH1 allele having Cys at residue 132 according to noma, on the basis of the cancer being characterized by the sequence of SEQ ID NO :8 . increased levels of an alpha hydroxy neoactivity product , In an embodiment the method comprises selecting a 15 e.g., 2HG , e.g., R -2HG . subject having myelodysplasia or myelodysplastic syn In an embodiment the cell proliferation - related disorder is drome on the basis of cancer being characterized by having ALL , e.g., B -ALL or T - ALL , characterized by a mutation , or an IDH1 allele described herein , e.g., an IDH1 allele having preselected allele , of IDH2 associated with an alpha hydroxy Cys at residue 132 ( SEQ ID NO :8 ). neoactivity , e.g., 2HG neoactivity . E.g., in an embodiment, In an embodiment the method comprises selecting a 20 the IDH2 allele encodes an IDH2 having other than an Arg subject having myelodysplasia or myelodysplastic syn at residue 172. E.g., the allele encodes Lys, Gly , Met , Trp , drome, on the basis of the cancer being characterized by Thr, Ser, or any residue described in described in Yan et al ., increased levels of an alpha hydroxy neoactivity product , at residue 172 , according to the sequence of SEQ ID NO :10 e.g., 2HG , e.g., R - 2HG . In an embodiment the method ( see also FIG . 22) , specifically Lys , Gly , Met , Trp , or Ser. In comprises evaluating a serum or blood sample for increased 25 an embodiment the allele encodes an IDH2 having Lys at alpha neoactivity product, e.g., 2HG , e.g. , R - 2HG . residue 172. In an embodiment the allele encodes an IDH2 In an embodiment the cell proliferation - related disorder is having Met at residue 172 . a glioma , characterized by a mutation , or preselected allele , In an embodiment the method comprises selecting a of IDH2 associated with an alpha hydroxy neoactivity , e.g. , subject having ALL , e.g., B -ALL or T -ALL , wherein the 2HG neoactivity . E.g. , in an embodiment, the IDH2 allele 30 cancer is characterized by having an IDH2 allele described encodes an IDH2 having other than an Arg at residue 172 . herein , e.g., an IDH2 allele having Lys or Met at residue 172 E.g., the allele encodes Lys , Gly , Met, Trp , Thr, Ser, or any (SEQ ID NO : 10 ) . residue described in described in Yan et al. , at residue 172 , In an embodiment the method comprises selecting a according to the sequence of SEQ ID NO :10 (see also FIG . subject having ALL , e.g., B -ALL or T- ALL , on the basis of 22 ), specifically , Lys , Gly , Met, Trp or Ser . In an embodi- 35 the cancer being characterized by an IDH2 allele described ment the allele encodes an IDH2 having Lys at residue 172 . herein , e.g., an IDH2 allele having Lys or Met at residue 172 In an embodiment the allele encodes an IDH2 having Met at (SEQ ID NO : 10 ) . residue 172 . In an embodiment the method comprises selecting a In an embodiment the method comprises selecting a subject having ALL , e.g., B - ALL or T - ALL , on the basis of subject having a glioma, wherein the cancer is characterized 40 the cancer being characterized by increased levels of an by having an IDH2 allele described herein , e.g. , an IDH2 alpha hydroxy neoactivity product , e.g. , 2HG , e.g. , R -2HG . allele having Lys , Gly , Met , Trp , Thr, or Ser at residue 172 In an embodiment the method comprises evaluating a serum ( SEQ ID NO :10 ) , specifically Lys , Gly , Met, Trp , or Ser ; or or blood sample for increased alpha neoactivity product , Lys or Met. e.g., 2HG , e.g., R - 2HG . In an embodiment the method comprises selecting a 45 In an embodiment the cell proliferation -related disorder is subject having a glioma, on the basis of the cancer being AML , characterized by a mutation , or preselected allele , of characterized by an IDH2 allele described herein , e.g., an IDH2 associated with an alpha hydroxy neoactivity , e.g. , IDH2 allele having Lys ,Gly , Met, Trp , Thr, or Ser at residue 2HG neoactivity . E.g., in an embodiment, the IDH2 allele 172 (SEQ ID NO :10 ), specifically Lys, Gly , Met, Trp, or Ser ; encodes an IDH2 having other than an Arg at residue 172 . or Lys or Met . 50 E.g., the allele encodes Lys, Gly , Met , Trp , Thr, Ser, or any In an embodiment the method comprises selecting a residue described in described in Yan et al . , at residue 172 , subject having a glioma, on the basis of the cancer being according to the sequence of SEQ ID NO : 10 (see also FIG . characterized by increased levels of an alpha hydroxy neo 22 ), specifically Lys , Gly, Met , Trp , or Ser . In an embodi activity product, e.g. , 2HG , e.g., R -2HG . ment the allele encodes an IDH2 having Lys at residue 172 . In an embodiment the cell proliferation - related disorder is 55 In an embodiment the allele encodes an IDH2 havingMet at a prostate cancer , e.g. , prostate adenocarcinoma, character residue 172 . ized by a mutation , or preselected allele , of IDH2 associated In an embodiment the method comprises selecting a with an alpha hydroxy neoactivity , e.g., 2HG neoactivity . subject having AML , wherein the cancer is characterized by E.g., in an embodiment, the IDH2 allele encodes an IDH2 having an IDH2 allele described herein , e.g., an IDH2 allele having other than an Arg at residue 172. E.g. , the allele 60 having Lys or Met at residue 172 (SEQ ID NO : 10 ) . encodes Lys, Gly , Met , Trp , Thr , Ser , or any residue In an embodiment the method comprises selecting a described in described in Yan et al ., at residue 172 , according subject having AML , on the basis of the cancer being to the sequence of SEQ ID NO : 10 (see also FIG . 22 ), characterized by an IDH2 allele described herein , e.g. , an specifically Lys , Gly , Met , Trp , or Ser . In an embodiment the IDH2 allele having Lys or Met at residue 172 (SEQ ID allele encodes an IDH2 having Lys at residue 172. In an 65 NO : 10 ). embodiment the allele encodes an IDH2 having Met at In an embodiment the method comprises selecting a residue 172 . subject having AML , on the basis of the cancer being US 10,610,125 B2 25 26 characterized by increased levels of an alpha hydroxy neo mutations at residue 132 , e.g., R132H , R132C , R132S , activity product , e.g. , 2HG , e.g., R -2HG . In an embodiment R132G , R132L , or R132V , more specifically , R132H or the method comprises evaluating a serum or blood sample R132C . for increased alpha neoactivity product , e.g., 2HG , e.g., In an embodiment the mutant enzyme is a mutant IDH2, R - 2HG . 5 e.g., an IDH2 mutant described herein , and the neoactivity In an embodiment the cell proliferation - related disorder is is an alpha hydroxy neoactivity , e.g., 2HG neoactivity . myelodysplasia or myelodysplastic syndrome, characterized Mutations associated with 2HG neoactivity in IDH2 include by a mutation , or preselected allele , of IDH2. E.g., in an mutations at residue 172, e.g., R172K , R172M , R172S , embodiment, the IDH2 allele encodes an IDH2 having other R172G , or R172W . than an Arg at residue 172. E.g., the allele encodes Lys, Gly , 10 In an embodiment the method includes evaluating the Met , Trp , Thr, Ser, or any residue described in described in ability of the candidate compound to inhibit the neoactivity Yan et al ., at residue 172 , according to the sequence of SEQ or the proxy activity . ID NO :10 ( see also FIG . 22 ), specifically Lys, Gly , Met, Trp , In an embodiment the method further comprises evaluat or Ser . In an embodiment the allele encodes an IDH2 having ing the ability of the candidate compound to inhibit the Lys at residue 172. In an embodiment the allele encodes an 15 forward reaction of non -mutant or wild type enzyme activ IDH2 having Met at residue 172 . ity , e.g. , in the case of IDH , e.g., IDH1 or IDH2 , the In an embodiment the method comprises selecting a conversion of isocitrate to a -ketoglutarate (or an interme subject having myelodysplasia or myelodysplastic syn diate thereof, including the reduced hydroxyl intermediate) . drome, wherein the cancer is characterized by having an In an embodiment, the contacting step comprises contact IDH2 allele described herein , e.g., an IDH2 allele having 20 ing the candidate compound with a cell, or a cell lysate Lys or Met at residue 172 (SEQ ID NO : 10 ) . thereof, wherein the cell comprises a mutant enzyme having In an embodiment the method comprises selecting a the neoactivity or an enzyme having the activity . subject having myelodysplasia or myelodysplastic syn In an embodiment, the cell comprises a mutation , or drome, on the basis of the cancer being characterized by an preselected allele , of a mutant IDH1 gene. E.g., in an IDH2 allele described herein , e.g., an IDH2 allele having 25 embodiment, the IDH1 allele encodes an IDH1 having other Lys or Met at residue 172 (SEQ ID NO :10 ). than an Arg at residue 132. E.g., the allele can encode His , In an embodiment the method comprises selecting a Ser, Cys, Gly , Val, Pro or Leu , or any other residue described subject having myelodysplasia or myelodysplastic syn in Yan et al. , at residue 132 , according to the sequence of drome , on the basis of the cancer being characterized by SEQ ID NO : 8 ( see also FIG . 21 ), specifically His , Ser , Cys , increased levels of an alpha hydroxy neoactivity product, 30 Gly , Val, or Leu . e.g., 2HG , e.g., R - 2HG . In an embodiment the method In an embodiment the allele encodes an IDH1 having His comprises evaluating a serum or blood sample for increased at residue 132 . alpha neoactivity product , e.g. , 2HG , e.g., R -2HG . In an embodiment the allele encodes an IDH1 having Ser In another aspect the invention features a pharmaceutical at residue 132 . composition of an inhibitor ( e.g. , a small molecule or a 35 In an embodiment the allele is an Arg132His mutation , or nucleic acid - based inhibitor ) described herein . an Arg132Ser mutation , according to the sequence of SEQ In an embodiment a mutant protein specific reagent, e.g. , ID NO :8 ( see FIGS . 2 and 21 ). an antibody that specifically binds an IDH mutant protein , In an embodiment, the cell comprises a mutation , or e.g. , an antibody that specifically binds an IDH1- R132H preselected allele , of a mutant IDH2 gene. E.g., in an mutant protein , can be used to detect neoactive mutant 40 embodiment, the IDH2 allele encodes an IDH2 having other enzyme see , for example , that described by Y. Kato et al. , “ A than an Arg at residue 172. E.g., the allele encodes Lys ,Gly , monoclonal antibody Mab - 1 specifically recognizes Met, Trp , Thr, Ser , or any residue described in described in IDH1R132H . the most common glioma - derived mutation : Yan et al. , at residue 172 , according to the sequence of SEQ (Kato , Biochem . Biophys. Res . Commun . ( 2009 ) , which is ID NO : 10 (see also FIG . 22 ), specifically , Lys, Gly , Met , hereby incorporated by reference in its entirety. 45 Trp , or Ser. In an embodiment the allele encodes an IDH2 In another aspect, the invention features , a method of having Lys at residue 172. In an embodiment the allele evaluating a candidate compound , e.g., for the ability to encodes an IDH2 having Met at residue 172 . inhibit a neoactivity of a mutant enzyme, e.g., for use as an In an embodiment, the cell includes a heterologous copy anti - proliferative or anti - cancer agent. In an embodiment the of a mutant IDH gene , e.g., a mutant IDH1 or IDH2 gene . mutant enzyme is an IDH , e.g. , an IDH1 or IDH2 mutant, 50 (Heterologous copy refers to a copy introduced or formed by e.g., a mutant described herein . In an embodiment the a genetic engineering manipulation . ) neaoctivity is alpha hydroxy neoactivity , e.g., 2HG neoac In an embodiment, the cell is transfected ( e.g. , transiently tivity . The method comprises : or stably transfected ) or transduced ( e.g. , transiently or optionally supplying the candidate compound ; stably transduced ) with a nucleic acid sequence encoding an contacting the candidate compound with a mutant enzyme 55 IDH , e.g., IDH1 or IDH2, described herein , e.g. , an IDH1 having a neoactivity , or with another enzyme, a referred to having other than an Arg at residue 132. In an embodiment, herein as a proxy enzyme, having an activity , referred to the IDH , e.g., IDH1 or IDH2, is epitope -tagged , e.g., myc herein as a proxy activity , which is the same as the neoac tagged tivity (or with a cell or cell lysate comprising the same ); and In an embodiment, the cell , e.g., a cancer cell, is non evaluating the ability of the candidate compound to 60 mutant or wild type for the IDH , e.g., IDH1 or IDH2 , allele . modulate , e.g. , inhibit or promote , the neoactivity or the The cell can include a heterologous IDH1 or IDH2mutant . proxy activity , In an embodiment, the cell is a cultured cell , e.g. , a thereby evaluating the candidate compound . primary cell , a secondary cell, or a cell line . In an embodi In an embodiment the mutant enzyme is a mutant IDH1, ment, the cell is a cancer cell , e.g., a glioma cell ( e.g., a e.g. , an IDH1 mutant described herein , and the neoactivity 65 glioblastoma cell) , a prostate cancer cell , a leukemia cell is an alpha hydroxy neoactivity , e.g., 2HG neoactivity . ( e.g., an ALL , e.g. , B -ALL or T - ALL , cell or AML cell ) or Mutations associated with 2HG neoactivity in IDH1 include a cell characterized by myelodysplasia or myelodysplastic US 10,610,125 B2 27 28 syndrome. In embodiment, the cell is a 293T cell, a U87MG ( e.g. , IDH1R132H ) or an IDH2 having other than an Arg at cell, or an LN - 18 cell (e.g. , ATCC HTB - 14 or CRL - 2610 ). residue 172 ( specifically an IDH1 having other than an Arg In an embodiment, the cell is from a subject, e.g. , a subject at residue 132 ) ; and having cancer , e.g., a cancer characterized by an IDH , e.g. , evaluating the presence and /or amount of an alpha IDH1 or IDH2, allele described herein , e.g., an IDH1 allele 5 hydroxy neoactivity product, e.g. , 2HG , e.g., R -2HG , in the having His , Ser, Cys , Gly , Val, Pro or Leu at residue 132 cell lysate or culturemedium , by LC -MS , thereby evaluating (SEQ ID NO :8 ); specifically His or Cys; or an IDH2 allele the compound . having Lys , Gly , Met , Trp , Thr, or Ser at residue 172 ( SEQ In an embodiment the result of the evaluation is compared ID NO :10 ) , specifically Lys , Gly , Met , Trp , or Ser . with a reference, e.g., the level of product , e.g. , an alpha In an embodiment, the evaluating step comprises evalu- 10 hydroxycontrol cell neoactivity , e.g., a cell product having, e.g. inserted, 2HG .therein e.g., R a- 2HGwild, typein a ating the presence and /or amount of an alpha hydroxy or non -mutant copy of IDH1 or IDH2 ( e.g., IDH1) . neoactivity product, e.g., 2HG , e.g., R -2HG , e.g., in the cell In another aspect, the invention features, a method of lysate or culture medium , e.g., by LC -MS . evaluating a candidate compound , e.g. , for the ability to In an embodiment, the evaluating step comprises evalu 15 inhibit an RNA encoding a mutant enzyme having a neoac ating the presence and/ or amount of an alpha hydroxy tivity , e.g., for use as an anti -proliferative or anti- cancer neoactivity , e.g., 2HG neoactivity , in the cell lysate or agent. In an embodiment the mutant enzyme is an IDH , e.g. , culture medium . an IDH1 or IDH2 mutant, e.g. , a mutant described herein . In In an embodiment, the method further comprises evalu an embodiment the neaoctivity is alpha hydroxy neoactivity , ating the presence/ amount one or more of TCA metabolite 20 e.g., 2HG neoactivity . The method comprises : (s ) , e.g., citrate , a -KG , succinate , fumarate , and /or malate , optionally supplying the candidate compound , e.g., a e.g. , by LC -MS , e.g. , as a control. nucleic acid based inhibitor ( e.g., a dsRNA (e.g. , siRNA or In an embodiment, the method further comprises evalu shRNA ), an antisense , or a microRNA ) ; ating the oxidation state of NADPH , e.g., the absorbance at contacting the candidate compound with an RNA , e.g., an 340 nm , e.g., by spectrophotometer. 25 mRNA , which encodes IDH , e.g. , an IDH1 or IDH2, e.g., an In an embodiment, the method further comprises evalu RNA that encode mutant enzyme having a neoactivity (or ating the ability of the candidate compound to inhibit a with a cell or cell lysate comprising the same) ; and second enzymatic activity , e.g. , the forward reaction of evaluating the ability of the candidate compound to non -mutant or wild type enzyme activity , e.g., in the case of inhibit the RNA , thereby evaluating the candidate com IDH1 or IDH2 ( e.g. , IDH1) , the conversion of isocitrate to 30 pound . By inhibit the RNA means, e.g. , to cleave or other a -ketoglutarate ( or an intermediate thereof, including the wise inactivate the RNA . reduced hydroxyl intermediate ). In an embodiment the RNA encodes a fusion of all or part In an mbodiment, the candidate compound is a small of the IDH , e.g., IDH1 or IDH2, wildtype or mutant protein molecule , a polypeptide, peptide , a carbohydrate based to a second protein , e.g., a reporter protein , e.g., a fluorescent molecule , or an aptamer ( e.g. , a nucleic acid aptamer , or a 35 protein , e.g. , a green or red fluorescent protein . peptide aptamer ) . The method can be used broadly and can , In an embodiment the mutant enzyme is a mutant IDH1, e.g., be used as one or more of a primary screen , to confirm e.g., an IDH1 mutant described herein , and the neoactivity candidates produced by this or other methods or screens, or is an alpha hydroxy neoactivity , e.g., 2HG neoactivity . generally to guide drug discovery or drug candidate opti In an embodiment the mutant enzyme is a mutant IDH2, mization . 40 e.g. , an IDH2 mutant described herein , and the neoactivity In an embodiment, the method comprises evaluating , e.g. , is an alpha hydroxy neoactivity , e.g., 2HG neoactivity . confirming , the ability of a candidate compound (e.g. , a In an embodiment, the contacting step comprises contact candidate compound which meets a predetermined level of ing the candidate compound with a cell , or a cell lysate inhibition in the evaluating step ) to inhibit the neoactivity or thereof , wherein the cell comprises RNA encoding IDH , proxy activity in a second assay. 45 e.g., IDH1 or IDH2 , e.g. , a mutant IDH , e.g. , IDH1 or IDH2, In an embodiment, the second assay comprises repeating enzyme having the neoactivity . one or more of the contacting and /or evaluating step ( s ) of the In an embodiment, the cell comprises a mutation , or basic method . preselected allele , of a mutant IDH1 gene . E.g., in an In another embodiment, the second assay is different from embodiment, the IDH1 allele encodes an IDH1 having other the first . E.g. , where the first assay can use a cell or cell 50 than an Arg at residue 132. E.g. , the allele can encode His , lysate or other non -whole animal model the second assay Ser , Cys, Gly , Val, Pro or Leu , or any other residue described can use an animal model , e.g. , a tumor transplant model , in Yan et al ., at residue 132 , according to the sequence of e.g. , a mouse having an IDH , e.g., IDH1 or IDH2, mutant SEQ ID NO :8 ( see also FIG . 21) , specifically His , Ser, Cys, cell or tumor transplanted in it . E.g., a U87 cell , or glioma, Gly , Val, or Leu . e.g. , glioblastoma, cell, harboring a transfected IDH , e.g. , 55 In an embodiment the allele encodes an IDH1having His IDH1 or IDH2, neoactive mutant can be implanted as a at residue 132 . xenograft and used in an assay . Primary human glioma or In an embodiment the allele encodes an IDH1having Ser AML tumor cells can be grafted into mice to allow propo at residue 132 . gation of the tumor and used in an assay . A genetically In an embodiment the allele is an Arg132His mutation , or engineered mouse model (GEMM ) harboring an IDH1 or 60 an Arg132Ser mutation , according to the sequence of SEQ IDH2 mutation and /or other mutation , e.g., a p53 null ID NO :8 ( see FIGS. 2 and 21 ). mutation , can also be used in an assay . In an embodiment, the cell comprises a mutation , or In an embodiment the method comprises : preselected allele , of a mutant IDH2 gene . E.g., in an optionally supplying the candidate compound ; embodiment, the IDH2 allele encodes an IDH2 having other contacting the candidate compound with a cell comprising 65 than an Arg at residue 172. E.g., the allele encodes Lys, Gly , a nucleic acid sequence, e.g., a heterologous sequence , Met, Trp , Thr, Ser, or any residue described in described in encoding an IDH1 having other than an Arg at residue 132 Yan et al. , at residue 172 , according to the sequence of SEQ US 10,610,125 B2 29 30 ID NO : 10 ( see also FIG . 22 ), specifically Lys, Gly , Met , Trp wildtype or mutant protein to a second protein , e.g., a or Ser . In an embodiment the allele encodes an IDH2 having reporter protein , e.g. , a fluorescent protein , e.g. , a green or Lys at residue 172. In an embodiment the allele encodes an red fluorescent protein . IDH2 having Met at residue 172 . In an embodiment the mutant enzyme is a mutant IDHI, In an embodiment, the cell includes a heterologous copy 5 e.g., an IDH1 mutant described herein , and the neoactivity of a wildtype ormutant IDH gene, e.g. , a wildtype ormutant is alpha hydroxy neoactivity , e.g., 2HG neoactivity . IDH1 or IDH2 gene. (Heterologous copy refers to a copy In an embodiment the mutant enzyme is a mutant IDH2, introduced or formed by a genetic engineering manipula e.g., an IDH2 mutant described herein , and the neoactivity tion .) In an embodiment the heterologous gene comprises a is alpha hydroxy neoactivity , e.g., 2HG neoactivity. fusion to a reporter protein , e.g., a fluorescent protein , e.g. , 10 In an embodiment, the system includes a heterologous a green or red fluorescent protein . copy of a wildtype or mutant IDH gene, e.g. , a wildtype or In an embodiment , the cell is transfected ( e.g., transiently mutant IDH1 or IDH2 gene. (Heterologous copy refers to a or stably transfected ) or transduced (e.g. , transiently or copy introduced or formed by a genetic engineering manipu stably transduced ) with a nucleic acid sequence encoding an 15 lation .) In an embodiment the heterologous gene comprises IDH , e.g. , IDH1 or IDH2 , described herein , e.g., an IDH1 a fusion to a reporter protein , e.g. , a fluorescent protein , e.g., having other than an Arg at residue 132 or an IDH2 having a green or red fluorescent protein . other than an Arg at residue 172 ( e.g. , an IDH1 having other In an embodiment the cell, e.g. , a cancer cell , is non than an Arg at residue 132 ) . In an embodiment, the IDH , e.g. , mutant or wild type for the IDH , e.g., IDH1 or IDH2, allele . IDH1 or IDH2, is epitope -tagged , e.g., myc- tagged . 20 The cell can include a heterologous IDH1 or IDH2 mutant. In an embodiment, the cell , e.g. , a cancer cell , is non In an embodiment, the cell is a cultured cell , e.g., a mutant or wild type for the IDH , e.g., IDH1 or IDH2, allele. primary cell, a secondary cell , or a cell line. In an embodi The cell can include a heterologous IDH1 or IDH2 mutant. ment, the cell is a cancer cell, e.g. , a glioma cell ( e.g., a In an embodiment, the cell is a cultured cell, e.g. , a glioblastoma cell) , a prostate cancer cell, a leukemia cell primary cell , a secondary cell , or a cell line . In an embodi- 25 ( e.g., an ALL , e.g. , B - ALL or T- ALL , cell or AML cell) or ment, the cell is a cancer cell, e.g. , a glioma cell ( e.g. , a a cell characterized by myelodysplasia or myelodysplastic glioblastoma cell) , a prostate cancer cell , a leukemia cell syndrome. In embodiment, the cell is a 293T cell, a U87MG ( e.g., an ALL , e.g., B -ALL or T - ALL cell or AML cell) or a cell, or an LN - 18 cell ( e.g. , ATCC HTB - 14 or CRL - 2610 ). cell characterized by myelodysplasia or myelodysplastic In an embodiment, the cell is from a subject, e.g., a subject syndrome. In embodiment, the cell is a 293T cell, a U87MG 30 having cancer , e.g., a cancer characterized by an IDH , e.g., cell , or an LN - 18 cell ( e.g. , ATCC HTB - 14 or CRL - 2610 ) . IDH1 or IDH2, allele described herein , e.g. , an IDH1 allele In an embodiment, the cell is from a subject , e.g., a subject having His , Ser, Cys, Gly , Val, Pro or Leu at residue 132 having cancer , e.g., a cancer characterized by an IDH , e.g., (SEQ ID NO : 8 ) ; specifically His , Cys, Gly , Val, or Leu . IDH1 or IDH2 , allele described herein , e.g. , an IDH1 allele In an embodiment, the cancer is characterized an IDH2 having His , Ser, Cys, Gly , Val , Pro or Leu at residue 132 35 allele having Lys, Gly , Met , Trp , Thr, or Ser at residue 172 (SEQ ID NO : 8 ) ; specifically His or Cys. In an embodiment, (SEQ ID NO : 10 ). the cancer is characterized by an IDH2 allele having Lys , In an embodiment , the method comprises a second assay Gly , Met , Trp , Thr, or Ser at residue 172 (SEQ ID NO : 10 ), and the second assay comprises repeating the method . specifically Lys, Gly , Met , Trp , or Ser . In another embodiment, the second assay is different from In an embodiment, the method comprises a second assay 40 the first. E.g. , where the first assay can use a cell or cell and the second assay comprises repeating one or more of the lysate or other non -whole animal model the second assay contacting and /or evaluating step (s ) of the basic method . can use an animal model. In another embodiment, the second assay is different from In an embodiment the efficacy of the candidate is evalu the first. E.g. , where the first assay can use a cell or cell ated by its effect on reporter protein activity . lysate or other non -whole animal model the second assay 45 In another aspect, the invention features, a method of can use an animal model evaluating a candidate compound, e.g., a therapeutic agent, In an embodiment the efficacy of the candidate is evalu or inhibitor , described herein in an animal model . The ated by its effect on reporter protein activity . candidate compound can be, e.g., a small molecule , poly In another aspect, the invention features, a method of peptide , peptide , aptomer , a carbohydrate -based molecule or evaluating a candidate compound , e.g. , for the ability to 50 nucleic acid based molecule . The method comprises, con inhibit transcription of an RNA encoding a mutant enzyme tacting the candidate with the animal model and evaluating having a neoactivity , e.g., for use as an anti -proliferative or the animal model. anti- cancer agent. In an embodiment the mutant enzyme is In an embodiment evaluating comprises ; an IDH , e.g., an IDH1 or IDH2 mutant, e.g., a mutant determining an effect of the compound on the general described herein . In an embodiment the neaoctivity is alpha 55 health of the animal; hydroxy neoactivity, e.g. , 2HG neoactivity . The method determining an effect of the compound on the weight of comprises : the animal; optionally supplying the candidate compound , e.g., a determining an effect of the compound on liver function , small molecule , polypeptide , peptide , aptomer , a carbohy e.g , on a liver enzyme; drate -based molecule or nucleic acid based molecule ; 60 determining an effect of the compound on the cardiovas contacting the candidate compound with a system com cular system of the animal; prising a cell or cell lysate ; and determining an effect of the compound on neurofunction , evaluating the ability of the candidate compound to e.g., on neuromuscular control or response ; inhibit the translation of IDH , e.g., IDH1 or IDH2, RNA , determining an effect of the compound on eating or e.g , thereby evaluating the candidate compound . 65 drinking ; In an embodiment the system comprises a fusion gene determining the distribution of the compound in the encoding of all or part of the IDH , e.g. , IDH1 or IDH2, animal; US 10,610,125 B2 31 32 determining the persistence of the compound in the ani points ). In an embodiment , the test value , or an indication of mal or in a tissue or organ of the animal, e.g., determining whether the preselected criterion is met, can be memorial plasma half - life ; or ized , e.g., in a computer readable record . determining an effect of the compound on a selected cell In an embodiment, a decision or step is taken , e.g., a in the animal; 5 sample containing the therapeutic agent, or a batch of the determining an effect of the compound on the growth , therapeutic agent, is classified , selected , accepted or dis size, weight, invasiveness or other phenotype of a tumor, carded , released or withheld , processed into a drug product, e.g. , an endogenous tumor or a tumor arising from intro shipped , moved to a different location , formulated , labeled , duction of cells from the same or a different species. packaged, contacted with , or put into , a container , e.g. , a gas In an embodiment the animal is a non -human primate , 10 or liquid tight container, released into commerce, or sold or e.g., a cynomolgus monkey or chimpanzee . offered for sale, or a record made or altered to reflect the In an embodiment the animal is a rodent, e.g., a rat or determination , depending on whether the preselected crite mouse . rion is met. E.g., based on the result of the determination or In an embodiment the animal is a large animal, e.g., a dog whether an activity is present, or upon comparison to a or pig , other than a non -human primate . 15 reference standard , the batch from which the sample is taken In an embodiment the evaluation is memorialized and can be processed , e.g. , as just described . optionally transmitted to another party . The evaluation of the presence or level of activity can In one aspect, the invention provides , a method of evalu show if the therapeutic agentmeets a reference standard . ating or processing a therapeutic agent, e.g., a therapeutic In an embodiment, methods and compositions disclosed agent referred to herein , e.g., a therapeutic agent that results 20 herein are useful from a process standpoint , e.g., to monitor in a lowering of the level of a product of an IDH , e.g., IDH1 or ensure batch -to - batch consistency or quality , or to evalu or IDH2, mutant having a neoactivity . In an embodiment the ate a sample with regard to a reference , e.g., a preselected neoactivity is an alpha hydroxy neoactivity , e.g., 2HG value . neoactivity , and the level of an alpha hydroxy neoactivity In an embodiment, the method can be used to determine product , e.g., 2HG , e.g. , R -2HG , is lowered . 25 if a test batch of a therapeutic agent can be expected to have The method includes: one or more of the properties . Such properties can include a providing , e.g. , by testing a sample , a value ( e.g., a test property listed on the product insert of a therapeutic agent, value ) for a parameter related to a property of the therapeutic a property appearing in a compendium , e.g., the US Phar agent, e.g., the ability to inhibit the conversion of alpha macopea , or a property required by a regulatory agency, e.g. , ketoglutarate to 2 hydroxyglutarate ( i.e., 2HG ) , e.g., R - 2 30 the FDA , for commercial use . hydroxyglutarate (i.e. , R -2HG ) , and , In an embodiment the method includes testing the thera optionally , providing a determination of whether the value peutic agent for its effect on the wildtype activity of an IDH , determined for the parameter meets a preselected criterion , e.g. , IDH1 or IDH2, protein , and providing a determination e.g., is present , or is present within a preselected range , of whether the value determined meets a preselected crite thereby evaluating or processing the therapeutic agent. 35 rion , e.g. , is present, or is present within a preselected range . In an embodiment the therapeutic agent is approved for In an embodiment the method includes : use in humans by a government agency, e.g. , the FDA . contacting a therapeutic agent that is an inhibitor of IDH1 In an embodiment the parameter is correlated to the an alpha hydroxy neoactivity, e.g. , 2HG neoactivity , with an ability to inhibit 2HG neoactivity, and, e.g. , the therapeutic IDH1 mutant having an alpha hydroxy neoactivity , e.g. , agent is an inhibitor which binds to IDH1 or IDH2 protein 40 2HG neoactivity , and reduces an alpha hydroxy neoactivity , e.g. , 2HG neo determining a value related to the inhibition of an alpha activity. hydroxy neoactivity , e.g., 2HG neoactivity , and In an embodiment the parameter is correlated to the level comparing the value determined with a reference value , ofmutant IDH , e.g. , IDH1 or IDH2, protein , and, e.g. , the e.g. , a range of values , for the inhibition of an alpha hydroxy therapeutic agent is an inhibitor which reduces the level of 45 neoactivity , e.g. , 2HG neoactivity . In an embodiment the IDH1 or IDH2 mutant protein . reference value is an FDA required value, e.g., a release In an embodiment the parameter is correlated to the level criteria . of an RNA that encodes a mutant IDH , e.g., IDH1 or IDH2, In an embodiment the method includes : protein , and , e.g., the therapeutic agent reduces the level of contacting a therapeutic agent that is an inhibitor of RNA , e.g., mRNA , that encodes IDH1 or IDH2 mutant 50 mRNA which encodes a mutant IDH1 having an alpha protein . hydroxy neoactivity , e.g. , 2HG neoactivity , with an mRNA In an embodiment the method includes contacting the that encodes an IDH1 mutant having an alpha hydroxy therapeutic agent with a mutant IDH , e.g. , IDH1 or IDH2, neoactivity , e.g. , 2HG neoactivity , protein (or corresponding RNA ). determining a value related to the inhibition of the In an embodiment, the method includes providing a 55 mRNA , and , comparison of the value determined for a parameter with a comparing the value determined with a reference value , reference value or values , to thereby evaluate the therapeutic e.g., a range of values for inhibition of the mRNA . In an agent. In an embodiment , the comparison includes deter embodiment the reference value is an FDA required value , mining if a test value determined for the therapeutic agent e.g., a release criteria . has a preselected relationship with the reference value , e.g., 60 In one aspect, the invention features a method of evalu determining if it meets the reference value . The value need ating a sample of a therapeutic agent , e.g., a therapeutic not be a numerical value but, e.g. , can be merely an agent referred to herein , that includes receiving data with indication of whether an activity is present . regard to an activity of the therapeutic agent; providing a In an embodiment the method includes determining if a record which includes said data and optionally includes an test value is equal to or greater than a reference value , if it 65 identifier for a batch of therapeutic agent ; submitting said is less than or equal to a reference value , or if it falls within record to a decision -maker , e.g., a government agency , e.g. , a range ( either inclusive or exclusive of one or both end the FDA ; optionally , receiving a communication from said US 10,610,125 B2 33 34 decision maker ; optionally , deciding whether to release specifically leukemia , e.g., AML ( e.g. , an adult or pediatric market the batch of therapeutic agent based on the commu form ) or ALL, e.g., B - ALL or T -ALL ( e.g. , an adult or nication from the decision maker. In one embodiment, the pediatric form ), localized or metastatic prostate cancer , e.g., method further includes releasing, or other wise processing , prostate adenocarcinoma. Examples of disorders character e.g., as described herein , the sample . 5 ized by a predisposition to lead to unwanted cell prolifera In another aspect, the invention features , a method of tion include myelodysplasia or myelodysplastic syndrome, selecting a payment class for treatment with a therapeutic which are a diverse collection of hematological conditions agent described herein , e.g. , an inhibitor of IDH , e.g., IDH1 marked by ineffective production (or dysplasia ) of myeloid or IDH2, neoactivity , for a subject having a cell prolifera blood cells and risk of transformation to AML . tion -related disorder. The method includes: 10 providing (e.g. , receiving ) an evaluation of whether the As used herein , specifically inhibits a neoactivity (and subject is positive for increased levels of an alpha hydroxy similar language ) , means the neoactivity of the mutant neoactivity product, e.g. , 2HG , e.g., R -2HG , or neoactivity , enzyme is inhibited to a significantly greater degree than is e.g., an alpha hydroxy neoactivity , e.g. , 2HG neoactivity , a the wildtype enzyme activity . By way of example , “ specifi mutant IDH1 or IDH2 having neoactivity, e.g., an alpha 15 cally inhibits the 2HG neoactivity of mutant IDH1 (or hydroxy neoactivity , e.g. , 2HG neoactivity , (or a corre IDH2) ” means the 2HG neoactivity is inhibited to a signifi sponding RNA ), or a mutant IDH , e.g. , IDH1 or IDH2 , cantly greater degree than is the forward reaction ( the somatic gene , e.g. , a mutant described herein , and conversion of isocitrate to alpha ketoglutarate ) of wildtype performing at least one of ( 1) if the subject is positive IDH1 (or IDH2) activity . In embodiments the neoactivity is selecting a first payment class , and ( 2 ) if the subject is a not 20 inhibited at least 2 , 5 , 10 , or 100 fold more than the wildtype positive selecting a second payment class . activity . In embodiments an inhibitor that is specific for the In an embodiment the selection is memorialized , e.g., in 2HG neaoctivity of IDH , e.g., IDH1 or IDH2 , will also a medical records system . inhibit another dehydrogenase, e.g., malate dehydrogenase . In an embodiment the method includes evaluation of In other embodiments the specific inhibitor does inhibit whether the subject is positive for increased levels of an 25 other dehydrogenases , e.g., malate dehydrogenase . alpha hydroxy neoactivity product, e.g., 2HG , e.g., R -2HG , As used herein , a cell proliferation - related disorder , e.g., or neoactivity , e.g. , an alpha hydroxy neoactivity , e.g. , 2HG a cancer, characterized by a mutation or allele , means a cell neoactivity . proliferation - related disorder having a substantial number of In an embodiment the method includes requesting the cells which carry that mutation or allele . In an embodiment evaluation . 30 at least 10 , 25 , 50 , 75 , 90 , 95 or 99 % of the cell proliferation In an embodiment the evaluation is performed on the related disorder cells , e.g. , the cells of a cancer, or a subject by a method described herein . representative , average or typical sample of cancer cells , In an embod ent, the method comprises communicating e.g. , from a tumor or from affected blood cells , carry at least the selection to another party , e.g., by computer , compact one copy of the mutation or allele . A cell proliferation disc , telephone , facsimile , email , or letter . 35 related disorder, characterized by a mutant IDH , e.g., a In an embodiment, the method comprises making or mutant IDH1 or mutant IDH2 , having 2HG neoactivity is authorizing payment for said treatment. exemplary. In an embodiment the mutation or allele is In an embodiment, payment is by a first party to a second present as a heterozygote at the indicated frequencies . party . In some embodiments , the first party is other than the As used herein , a “ SNP ” is a DNA sequence variation subject. In some embodiments , the first party is selected 40 occurring when a single nucleotide ( A , T, C , or G ) in the from a third party payor, an insurance company , employer, genome ( or other shared sequence ) differs between members employer sponsored health plan , HMO , or governmental of a species ( or between paired chromosomes in an indi entity . In some embodiments , the second party is selected vidual) . from the subject , a healthcare provider, a treating physician , As used herein , a subject can be a human or non - human an HMO , a hospital, a governmental entity , or an entity 45 subject . Non -human subjects include non -human primates , which sells or supplies the drug. In some embodiments , the rodents , e.g., mice or rats , or other non -human animals . first party is an insurance company and the second party is The details of one or more embodiments of the invention selected from the subject, a healthcare provider , a treating are set forth in the description below . Other features , objects , physician , an HMO , a hospital, a governmental entity , or an and advantages of the invention will be apparent from the entity which sells or supplies the drug . In some embodi- 50 description and the drawings, and from the claims. ments , the first party is a governmental entity and the second party is selected from the subject, a healthcare provider, a BRIEF DESCRIPTION OF THE DRAWINGS treating physician , an HMO , a hospital, an insurance com pany, or an entity which sells or supplies the drug . FIG . 1 depicts DNA sequence verification of pET41a As used herein , a cell proliferation -related disorder is a 55 IDH1 and alignment against published IDH1 CDS. The disorder characterized by unwanted cell proliferation or by sequence of IDH1 (CDS ) corresponds to SEQ ID NO : 5 . The a predisposition to lead to unwanted cell proliferation sequence of pET41a -IDH1 corresponds to SEQ ID NO :6 , ( sometimes referred to as a precancerous disorder) . and the “ consensus ” sequence corresponds to SEQ ID NO : 7 . Examples of disorders characterized by unwanted cell pro FIG . 2 depicts DNA sequence verification ofR132S and liferation include cancers , e.g., tumors of the CNS , e.g., a 60 R132H mutants according to the SEQ ID NO :8 . The amino glioma. Gliomas include astrocytic tumors, oligodendroglial acid sequence of IDH1 (SEQ ID NO : 8 ) is provided in FIG . tumors , oligoastrocytic tumors , anaplastic astrocytomas , and 21. glioblastomas. Other examples include hematological can FIG . 3 depicts separation of wild type IDH1 protein on cers , e.g., a leukemia , e.g., AML ( e.g., an adult or pediatric Ni- Sepharose column form ) or ALL , e.g., B -ALL or T- ALL (e.g. , an adult or 65 FIG . 4 depicts protein analysis of wild type IDH1 on SDS pediatric form ), localized or metastatic prostate cancer, e.g., gel pre and post Ni column fractionation . T : total protein ; I : prostate adenocarcinoma, fibrosarcoma, and paraganglioma; insoluble fractions ; S : soluble fraction ; L : sample for load US 10,610,125 B2 35 36 ing on Ni- column . The numbers in the figure indicates the FIG . 19 depicts LC -MS / MS analysis of alpha- hydroxy fraction numbers . Fractions # 17 ~ # 27 were collected for glutarate . further purification . FIG . 20 depicts LC -MS / MS analysis showing that R132H FIG . 5A depicts separation of wild type IDH1 protein consumes a - KG to produce 2 -hydroxyglutaric acid . through SEC column S - 200 . 5 FIG . 21 depicts the amino acid sequence of IDH1 (SEQ FIG . 5B depicts protein analysis of wild type IDH1 on ID NO : 13 ) as described in GenBank Accession No. SDS gel pre and post S - 200 column fractionation . M : NP_005887.2 (GI No. 28178825 ) ( record dated May 10 , molecular weightmarker ; Ni: nickel column fraction prior to 2009 ) . S - 200 ; S200 : fraction from SEC column . FIG . 6 depicts separation of mutant R132S protein on 10 GenBankFIG . 21A Accession is the cDNA No. NM_005896.2sequence of IDH1 (Record as presented dated May at Ni- Sepharose column . 10 , 2009 ; GI No. 28178824 ) (SEQ ID NO : 8 ) . FIG . 7 depicts protein analysis ofmutant R132S on SDS FIG . 21B depicts the mRNA sequence of IDH1 as gel pre and post Ni column fractionation . M : protein marker described in GenBank Accession No. NM_005896.2 (Re So(KDa : soluble ): 116,66.2 fraction , 45 ;, In 35 : ,insoluble 25 , 18.4 , fraction14.4 ; T: ; Fttotal: flow cell throughprotein ;. 15 cord dated May 10 , 2009; GI No. 28178824) (SEQ ID # 3-# 7 indicate the corresponding eluted fraction numbers . NO : 9 ). FIG . 8A depicts separation of mutant R132S protein FIG . 22 is the amino acid sequence of IDH2 as presented through SEC column S - 200 . at GenBank Accession No. NM_002168.2 ( Record dated FIG . 8B depicts protein analysis ofmutant R132S on SDS Aug. 16 , 2009 ; G128178831) (SEQ ID NO : 10 ) . gel post S - 200 column fractionation . M : molecular weight 20 FIG . 22A is the cDNA sequence of IDH2 as presented at marker; R132S : fraction from SEC column . GenBank Accession No. NM_002168 (Record dated Aug. FIG . 9 depicts separation of mutant R132H protein on 16 , 2009 ; G128178831 ) (SEQ ID NO : 11) . Ni- Sepharose column FIG . 22B is the mRNA sequence of IDH2 as presented at FIG . 10 depicts protein analysis ofmutant R132H on SDS GenBank Accession No. NM_002168.2 (Record dated Aug. gel pre and post Ni column fractionation . M : protein marker 25 16 , 2009 ; G128178831) (SEQ ID NO : 12 ) . (KDa ): 116,66.2 , 45, 35, 25 , 18.4 , 14.4 ; T : total cell protein ; FIG . 23 depicts the progress of forward reactions (isoci So : soluble fraction ; In : insoluble fraction ; Ft: flow through ; trate to a -KG ) for the mutant enzyme R132H and R132S . # 5-# 10 indicate the corresponding eluted fraction numbers ; FIG . 24A depicts LC -MS /MS analysis of derivitized Ni: sample from Ni- Sepharose column , pool # 5-# 10 2 -HG racemic mixture . together. 30 FIG . 24B depicts LC -MS / MS analysis of derivitized FIG . 11A depicts separation of mutant R132H protein R - 2HG standard . through SEC column S - 200 . FIG . 24C depicts LC -MS / MS analysis of a coinjection of FIG . 11B depicts protein analysis of mutant R132H on derivitized 2 -HG racemate and R - 2 -HG standard . SDS gel post S -200 column fractionation . M : molecular FIG . 24D depicts LC -MS /MS analysis of the deriviatized weight marker; R132H : fraction from SEC column . 35 neoactivity reaction product . FIG . 12A depicts Michaelis -Menten plot of IDH1 wild FIG . 24E depicts LC -MS / MS analysis of a coinjection of type in the oxidative decarboxylation of ioscitrate to a -ke the neoactivity enzyme reaction product and the R - 2- HG toglutarate. standard . FIG . 12B depicts Michaelis -Menten plot of R132H FIG . 24F depicts LC -MS / MS analysis of a coinjection of mutant enzyme in the oxidative decarboxylation of ioscitrate 40 the neoactivity enzyme reaction product and the 2 -HG to a -ketoglutarate . racemic mixture . FIG . 12C depicts Michaelis -Menten plot ofR132S mutant FIG . 25 depicts the inhibitory effect of 2 -HG derived from enzyme in the oxidative decarboxylation of ioscitrate to the reduction of a - KG by ICDH1 R132H on the wild - type a -ketoglutarate . ICDH1 catalytic oxidative decarboxylation of isocitrate to FIG . 13A depicts a -KG inhibition of IDH1 wild - type . 45 a -KG . FIG . 13B depicts a - KG inhibition of R132H mutant FIG . 26A depicts levels of 2 - HG in CRL -2610 cell lines enzyme . expressing wildtype or IDH - 1 R132H mutant protein . FIG . 13C depicts a - KG inhibition of R132S mutant FIG . 26B depicts levels of 2 - HG in HTB - 14 cell lines enzyme. expressing wildtype or IDH - 1 R132H mutant protein . FIG . 14 depicts IDH1 wt, R132H , and R132S in the 50 FIG . 27 depicts human IDH1 genomic DNA : intron / 2nd conversion a -ketoglutarate to 2 -hydroxyglutarate . exon sequence . FIG . 15A depicts Substrate - Concentration velocity plot FIG . 28 depicts concentrations of 2HG in human malig for R132H mutant enzyme . nant gliomas containing R132 mutations in IDH1. Human FIG . 15B depicts Substrate - Concentration velocity plot glioma samples obtained by surgical resection were snap for R132S mutant enzyme. 55 frozen , genotyped to stratify as wild -type (WT ) (N = 10 ) or FIG . 16 depicts IDH1 wt, R132H , and R132S in the carrying an R132 mutant allele (Mutant ) (n = 12 ) and metabo conversion a -ketoglutarate to 2- hydroxyglutarate with lites extracted for LC -MS analysis . Among the 12 mutant NADH . tumors, 10 carried a R132H mutation , one an R132S muta FIG . 17A depicts oxalomalate inhibition to IDH1 wt. tion , and one an R1326 mutation . Each symbol represents FIG . 17B depicts oxalomalate inhibition to R132H . 60 the amount of the listed metabolite found in each tumor FIG . 17C depicts oxalomalate inhibition to R132S . sample . Red lines indicate the group sample means. The FIG . 18A depicts LC -MS / MS analysis of the control difference in 2HG observed between WT and R132 mutant reaction . IDH1 mutant tumors was statistically significant by Stu FIG . 18B depicts LC -MS /MS analysis of the reaction dent's t- test (p <0.0001 ) . There were no statistically signifi containing enzyme. 65 cant differences in aKG , malate, fumarate, succinate , or FIG . 18C depicts LC -MS / MS analysis of the spiked isocitrate levels between the WT and R132 mutant IDH1 control reaction . tumors . US 10,610,125 B2 37 38 FIG . 29A depicts the structural analysis ofR132H mutant cate the mean . ( * ) indicates a statistically significant differ IDH1. On left is shown an overlay structure of R132H ence relative to wild - type patient cells (p < 0.05 ). ( C ) depicts mutant IDH1 and WT IDH1 in the closed ' conformation . In vitro growth curves of IDH1 R132 mutant and IDH1 On the right is shown an overlay structure of WT IDH1 in wild -type AML cells . the ' open ' conformation with mutant IDH1 for comparison . 5 FIG . 37 is a graph depicting the results of extracts from FIG . 29B depicts the close - up structural comparison of leukemia cells of AML patients carrying an IDH1/ 2 mutant the R132H IDH1 ( left ) and wild - type (WT ) IDH1 ( right ) ( n = 16 ) or wild - type ( n = 10 ) allele obtained at initial presen active - site containing both AKG and NADPH . In addition to tation and relapse assayed by LC -MS for levels of a - KG , changes at residue 132 , the position of the catalytic residues succinate , malate , and fumarate . Each point represents an Tyr 139 and Lys 212 are different and aKG is oriented 10 individual patient sample . Open circles represent wild - types, differently relative to NADPH for catalytic hydride transfer closed circles represent IDH1 mutants , and triangles repre in the WT versus R132H mutant enzymes . sent IDH2 mutants . Horizontal bars represent the mean . FIG . 30A depicts the enzymatic properties of IDH1 There were no statistically significant differences between R132H mutants when recombinant human wild - type (WT ) the wild - type and IDH1/ 2 mutant AML samples. and R132H mutant (R132H ) IDH1 enzymes were assessed 15 FIG . 38 depicts graphical representations of LC -MS for oxidative decarboxylation of isocitrate to aKG with analysis of in vitro reactions using recombinant IDH1 NADP as cofactor . Different concentrations of enzyme were R132C and IDH2 R172K confirming that 2- HG and not used to generate the curves . isocitrate is the end product of the mutant enzyme reactions . FIG . 30B depicts the enzymatic properties of IDH R132 FIGS. 39A and B depict ( A ) the wild -type IDH1 enzyme mutants when WT and R132H mutant IDH1 enzymes were 20 catalysis of the oxidative decarboxylation of isocitrate to assessed for reduction of aKG with NADPH as cofactor . alpha -ketoglutarate with the concomitant reduction of Different concentrations of enzyme were used to generate NADP to NADPH ; and ( B ) the IDH1 R132C mutant reduc the curves . tion of alpha -ketoglutarate to 2 -hydroxyglutarate while oxi FIG . 30C depicts kinetic parameters of oxidative and dizing NADPH to NADP. These are referred to as the reductive reactions as measured for WT and R132H IDH1 25 “ forward ” and “ partial reverse ” reactions, respectively . enzymes are shown . Km and kcat values for the reductive activity of the WT enzyme were unable to be determined as DETAILED DESCRIPTION no measurable enzyme activity was detectable at any sub strate concentration . The inventors have discovered that certain mutated forms FIG . 31A depicts the LC -MS / MS analysis identifying 30 of an enzyme ( e.g. , IDH1 or IDH2) have a gain of function , 2HG as the reductive reaction product of recombinant referred to herein as a neoactivity , which can be targeted in human R132H mutant IDH1. the treatment of a cell proliferation - related disorder , e.g., a FIG . 31B depicts the diacetyl- L -tartaric anhydride deriva proliferative disorder such as cancer. For example , in the tization and LC -MS / MS analysis of the chirality of 2HG case of a metabolic pathway enzyme, a gain of function or produced by R132H mutant IDH1 . Normalized LC -MS /MS 35 neoactivity can serve as a target for treatment of cancer . signal for the reductive reaction ( rxn ) product alone , an Described herein are methods and compositions for the R (- ) -2HG standard alone, and the two together (Rxn + R ( - ) treatment of a cell proliferation - related disorder , e.g. , a 2HG ) are shown as is the signal for a racemic mixture of proliferative disorder such as cancer . The methods include , R ( - ) and S ( + ) forms (2HG Racemate ) alone or with the e.g., treating a subject having a glioma or brain tumor reaction products (Rxn + Racemate ). 40 characterized by a preselected IDH1 allele , e.g., an allele FIG . 32A depicts SDS -PAGE and Western blot analyses having A at position 394 , such as a C394A , a C394G , a of C - terminal affinity -purification tagged IDH1 R132S pro C3947, a G395C , a G395T or a G395A mutation , ( e.g., a tein used for crystallization . C394A mutant) or an A at position 395 ( e.g. , a G395A FIG . 32B depicts the chromatogram of FPLC analysis of mutant) according to the sequence of SEQ ID NO : 5 , that the IDH1 R132S protein sample . 45 encodes an IDH1 having His , Ser, Cys , Gly , Val , Pro or Leu FIG . 33 depicts crystals obtained from a protein solution at position 132 ( e.g., His ); or a preselected IDH2 allele that contained 5 mM NADP , 5 mM isocitrate , 10 mM Ca2 + . encodes an IDH2 having Lys , Gly , Met , Trp , Thr, or Ser at Precipitant solution contained 100 mM MES ( pH 6.0 ) and position 172 and having a neoactivity disclosed herein , by 20 % PEG 6000 using a hanging drop method of crystalli administering to the subject a therapeutically effective zation . 50 amount of an inhibitor of IDH1 or IDH2 ( e.g. , IDH1) , e.g. , FIG . 34 depicts crystal obtained from a protein solution a small molecule or nucleic acid . The nucleic acid based contained 5 mM NADP, 5 mM a -ketoglutarate , 10 mm inhibitor is , for example , a dsRNA , e.g. , a dsRNA that Ca2 + . Precipitant contained 100 mM MES (pH 6.5 ) and comprises the primary sequences of the sense strand and 12 % PEG 20000 . antisense strands of Tables 7-14 . The dsRNA is composed of FIG . 35 is a bar graph depicting elevated NADPH reduc- 55 two separate strands , or a single strand folded to form a tive catalysis activity in IDH2- R172K mutant enzyme as hairpin structure (e.g. , a short hairpin RNA (shRNA ) ) . In compared to wildtype IDH2. some embodiments , the nucleic acid based inhibitor is an FIGS . 36A - C are graphs depicting the following : ( A ) antisense nucleic acid , such as an antisense having a Extracts from IDH1/ 2 wt ( n = 10 ), and IDH1/ 2 mutant ( n = 16 ) sequence that overlaps, or includes, an antisense sequence patient leukemia cells obtained at presentation and relapse , 60 provided in Tables 7-14 . and IDH1 R132 mutant leukemia cells grown in culture for Neoactivity of an Enzyme 14 days ( n = 14 ) analyzed by LC -MS to measure levels of Neoactivity , as used herein , means an activity that arises 2 -HG ; and (B ) 2 -HG measured in serum of patients with as a result of a mutation , e.g. , a point mutation , e.g., a IDH1 wt or IDH1 R132 mutant leukemia . In ( A ) and ( B ) , substitution , e.g., in the active site of an enzyme. In an each point represents an individual patient sample . Dia- 65 embodiment the neoactivity is substantially absent from monds represent wildtype, circles represent IDH1mutants , wild type or non -mutant enzyme. This is sometimes referred and triangles represent IDH2mutants . Horizontal bars indi to herein as a first degree neoactivity . An example of a first US 10,610,125 B2 39 40 degree neoactivity is a “ gain of function ” wherein the NADP( + ) -dependent isocitrate dehydrogenase found in the mutant enzyme gains a new catalytic activity . In an embodi mitochondria . It plays a role in intermediary metabolism and ment the neoactivity is present in wild type or non -mutant energy production . This protein may tightly associate or enzyme but at a level which is less than 10 , 5 , 1 , 0.1 , 0.01 interact with the pyruvate dehydrogenase complex . Human or 0.001 % of what is seen in the mutant enzyme. This is 5 IDH2 gene encodes a protein of 452 amino acids. The sometimes referred to herein as a second degree neoactivity . nucleotide and amino acid sequences for IDH2 can be found An example of a second degree neoactivity is a " gain of as GenBank entries NM 002168.2 and NP 002159.2 respec function ” wherein the mutant enzyme has an increase , for tively . The nucleotide and amino acid sequence for human example , a 5 fold increase in the rate of a catalytic activity IDH2 are also described in , e.g., Huh et al ., Submitted possessed by the enzyme when lacking the mutation . 10 (November - 1992 ) to the EMBL /GenBank /DDBJ databases ; In some embodiments , a non -mutant form the enzyme, and The MGC Project Team , Genome Res. 14 :2121-2127 e.g. , a wild type form , converts substance A ( e.g., isocitrate ) ( 2004 ). to substance B ( e.g., a -ketoglutarate ), and the neoactivity Non -mutant , e.g., wild type, IDH1 catalyzes the oxidative converts substance B ( e.g., a -ketoglutarate ) to substance C , decarboxylation of ioscitrate to a -ketoglutarate thereby sometimes referred to as the neoactivity product ( e.g., 2 - hy- 15 reducing NAD (NADP + ) to NADP (NADPH ), e.g., in the droxyglutarate , e.g., R - 2 -hydroxyglutarate ). In some forward reaction : embodiments , the enzyme is in a metabolic pathway, e.g., a metabolic pathway leading to fatty acid biosynthesis , gly Isocitrate + NAD + (NADP + ) ? - KG + CO2 +NADH colysis , glutaminolysis , the pentose phosphate shunt , the (NADPH ) + H + nucleotide biosynthetic pathway, or the fatty acid biosyn- 20 In some embodiments , the neoactivity of a mutant IDH1 thetic pathway, e.g., IDH1 or IDH2 . can have the ability to convert a -ketoglutarate to 2 -hydroxy In some embodiments , a non -mutant form the enzyme, glutarate , e.g., R - 2 -hydroxyglutarate : e.g. , a wild type form , converts substance A to substance B , and the neoactivity converts substance B to substance A. In a -KG + NADH (NADPH ) + H + 2 -hydroxyglutarate , some embodiments , the enzyme is in a metabolic pathway, 25 e.g., R - 2 -hydroxyglutarate +NAD + (NADP + ) . e.g. , a metabolic pathway leading to fatty acid biosynthesis , In some embodiments , the neoactivity can be the reduc glycolysis , glutaminolysis , the pentose phosphate shunt, the tion of pyruvate or malate to the corresponding a -hydroxyl nucleotide biosynthetic pathway, or the fatty acid biosyn compounds. thetic pathway . In some embodiments , the neoactivity of a mutant IDH1 Isocitrate Dehydrogenases 30 can arise from a mutant IDH1 having a His, Ser , Cys , Gly , Isocitrate dehydrogenases ( IDHs ) catalyze the oxidative Val, Pro or Leu , or any other mutations described in Yan et decarboxylation of isocitrate to 2 -oxoglutarate ( i.e., a -keto al. , at residue 132 ( e.g. , His , Ser , Cys, Gly , Val or Leu ; or glutarate ). These enzymes belong to two distinct subclasses, His , Ser , Cys or Lys ) . In some embodiments , the neoactivity one of which utilizes NAD ( + ) as the electron acceptor and of a mutant IDH2 can arise from a mutant IDH2 having a the other NADP (+ ) . Five isocitrate dehydrogenases have 35 Lys , Gly , Met, Trp , Thr, or Ser ( e.g., Lys, Gly, Met, Trp , or been reported : three NAD (+ ) -dependent isocitrate dehydro Ser ; or Gly , Met or Lys ), or any other mutations described genases, which localize to themitochondrial matrix , and two in Yan H et al. , at residue 172. Exemplary mutations include NADP ( + ) -dependent isocitrate dehydrogenases, one of the following : R132H , R132C , R132S , R132G , R132L , and which is mitochondrial and the other predominantly cyto R132V . solic . Each NADP ( + )- dependent isozyme is a homodimer . 40 In some embodiments , the mutant IDH1 and /or IDH2 IDH1 ( isocitrate dehydrogenase 1 (NADP + ) , cytosolic ) is ( e.g., a mutant IDH1 and / or IDH2 having a neoactivity also known as IDH ; IDP ; IDCD ; IDPC or PICD . The protein described herein ) could lead to an increased level of 2 -hy encoded by this gene is the NADP ( + ) -dependent isocitrate droxyglutarate , e.g. , R - 2- hydroxyglutarate in a subject . The dehydrogenase found in the cytoplasm and peroxisomes. It accumulation of 2 -hydroxyglutarate , e.g., R -2 -hydroxyglu contains the PTS - 1 peroxisomal targeting signal sequence . 45 tarate in a subject, e.g., in the brain of a subject, can be The presence of this enzyme in peroxisomes suggests roles harmful. For example , in some embodiments , elevated lev in the regeneration of NADPH for intraperoxisomal reduc els of 2 - hydroxyglutarate , e.g. , R - 2 -hydroxyglutarate can tions , such as the conversion of 2, 4 -dienoyl - CoAs to lead to and /or be predictive of cancer in a subject such as a 3 - enoyl- CoAs, as well as in peroxisomal reactions that cancer of the central nervous system , e.g. , brain tumor, e.g., consume 2 -oxoglutarate , namely the alpha -hydroxylation of 50 glioma, e.g. , glioblastomamultiforme (GBM ). Accordingly , phytanic acid . The cytoplasmic enzyme serves a significant in some embodiments , a method described herein includes role in cytoplasmic NADPH production . administering to a subject an inhibitor of the neoactivity . The human IDH1 gene encodes a protein of 414 amino Detection of 2 -hydroxyglutarate acids . The nucleotide and amino acid sequences for human 2 - hydroxyglutarate can be detected , e.g., by LC /MS . To IDH1 can be found as GenBank entries NM_005896.2 and 55 detect secreted 2 -hydroxyglutarate in culture media , 500 uL NP_005887.2 respectively . The nucleotide and amino acid aliquots of conditioned media can be collected ,mixed 80:20 sequences for IDH1 are also described in , e.g. , Nekrutenko with methanol, and centrifuged at 3,000 rpm for 20 minutes et al ., Mol. Biol. Evol. 15 : 1674-1684 ( 1998 ); Geisbrecht et at 4 degrees Celsius. The resulting supernatant can be al. , J. Biol. Chem . 274 :30527-30533 ( 1999 ) ; Wiemann et al . , collected and stored at -80 degrees Celsius prior to LC -MS / Genome Res . 11: 422-435 (2001 ) ; The MGC Project Team , 60 MS to assess 2 -hydroxyglutarate levels . To measure whole Genome Res. 14 :2121-2127 (2004 ) ; Lubec et al. , Submitted cell associated metabolites , media can be aspirated and cells ( December -2008 ) to UniProtKB ; Kullmann et al. , Submitted can be harvested , e.g. , at a non -confluent density . A variety ( June - 1996 ) to the EMBL /GenBank /DDBJ databases ; and of different liquid chromatography (LC ) separation methods Sjoeblom et al. , Science 314 :268-274 (2006 ) . can be used . Each method can be coupled by negative IDH2 ( isocitrate dehydrogenase 2 (NADP +) , mitochon- 65 electrospray ionization (ESI , -3.0 kV ) to triple -quadrupole drial) is also known as IDH ; IDP ; IDHM ; IDPM ; ICD - M ; or mass spectrometers operating in multiple reaction monitor mNADP - IDH . The protein encoded by this gene is the ing (MRM ) mode, with MS parameters optimized on US 10,610,125 B2 41 42 infused metabolite standard solutions. Metabolites can be 2HG , e.g., R -2HG , and allows detection . In an embodiment separated by reversed phase chromatography using 10 mm the sample is analyzed for the level of neoactivity , e.g. , an tributylamine as an ion pairing agent in the aqueous mobile alpha hydroxy neoactivity , e.g. , 2HG neoactivity . In an phase , according to a variant of a previously reported embodiment the sample is analysed for the presence of a method (Luo et al . J Chromatogr A 1147, 153-64 , 2007) . 5 mutant IDH , e.g., IDH1 or IDH2, protein having an alpha Onemethod allows resolution of TCA metabolites: t = 0 , 50 % hydroxy neoactivity , e.g. , 2HG neoactivity ( or a correspond B ; t = 5 , 95 % B ; t = 7 , 95 % B ; t = 8 , 0 % B , where B refers to ing RNA ). E.g. , a mutant protein specific reagent, e.g. , an an organic mobile phase of 100 % methanol . Anothermethod antibody that specifically binds an IDH mutant protein , e.g. , is specific for 2 -hydroxyglutarate , running a fast linear an antibody that specifically binds an IDH1- R132H mutant gradient from 50 % -95 % B (buffers as defined above) over 5 10 R132Hprotein mutantor an IDH2 protein -R172 ), can mutant be used protein to detect( e.g., anneoactive IDH1 minutes . A Synergi Hydro -RP , 100 mmx2 mm , 2.1 um mutant enzyme In an embodiment a nucleic acid from the particle size (Phenomonex ) can be used as the column, as sample is sequenced to determine if a selected allele or described above. Metabolites can be quantified by compari mutation of IDH1 or IDH2 disclosed herein is present. In an son of peak areas with pure metabolite standards at known 15 embodiment the analysis is other than directly determining concentration . Metabolite flux studies from 13C - glutamine the presence of a mutant IDH , e.g., IDH1 or IDH2 , protein can be performed as described , e.g. , in Munger et al. Nat (or corresponding RNA ) or sequencing of an IDH , e.g. , Biotechnol 26 , 1179-86 , 2008 . IDH1 or IDH2 gene . In an embodiment the analysis is other In an embodiment 2HG , e.g., R - 2HG , is evaluated and the than directly determining , e.g. , it is other than sequencing Ranalyte - 2HG . onIn whichan embodiment the determination the analyte is onbased which is 2HGthe deter, e.g., 20 genomic DNA or cDNA, the presence of a mutation at mination is based is a derivative of 2HG , e.g., R - 2HG , residue 132 of IDH1 and/ or a mutation at residue 172 of formed in process of performing the analytic method . By IDH2. E.g. , the analysis can be the detection of an alpha way of example such a derivative can be a derivative formed hydroxy neoactivity product , e.g., 2HG , e.g. , R - 2HG , or the in MS analysis . Derivatives can include a salt adduct, e.g., 25 measurementity, e.g., 2HG ofneoactivity the mutation's. In an anembodiment alpha hydroxy the neoactivsample is a Na adduct , a hydration variant, or a hydration variant removed from the patient and analyzed . In an embodiment which is also a salt adduct , e.g. , a Na adduct, e.g., as formed the evaluation can include one or more of performing the in MS analysis . Exemplary 2HG derivatives include dehy analysis of the sample , requesting analysis of the sample , drated derivatives such as the compounds provided below or requesting results from analysis of the sample , or receiving a salt adduct thereof : 30 the results from analysis of the sample . (Generally herein , analysis can include one or both of performing the under lying method or receiving data from another who has performed the underlying method. ) In an embodiment the evaluation , which can be performed 35 before and /or after treatment has begun , is based , at least in HO OH part, on analysis of a tissue ( e.g. , a tissue other than a tumor hahid sample ), or bodily fluid , or bodily product . Exemplary H tissues include lymph node , skin , hair follicles and nails . Exemplary bodily fluids include blood , plasma, urine , HO ?? : 40 lymph , tears , sweat , saliva, semen , and cerebrospinal fluid . 0 and 0 . Exemplary bodily products include exhaled breath . E.g. , the tissue, fluid or product can be analyzed for the presence or level of an alpha hydroxy neoactivity product , e.g., 2HG , Methods of Evaluating Samples and /or Subjects e.g., R - 2HG , by evaluating a parameter correlated to the This section providesmethods of obtaining and analyzing 45 presence or level of an alpha hydroxy neoactivity product , samples and of analyzing subjects . e.g. , 2HG , e.g. , R - 2HG . An alpha hydroxy neoactivity Embodiments of the method comprise evaluation of one product, e.g. , 2HG , e.g. , R -2HG , in the sample can be or more parameters related to IDH , e.g., IDH1 or IDH2, an determined by a chromatographic method , e.g. , by LC -MS alpha hydroxy neoactivity, e.g., 2HG neoactivity , e.g., to analysis . It can also be determined by contact with a specific evaluate the IDH1 or IDH2 2HG neoactivity genotype or 50 binding agent, e.g., an antibody , which binds the alpha phenotype . The evaluation can be performed , e.g., to select , hydroxy neoactivity product, e.g., 2HG , e.g. , R - 2HG , and diagnose or prognose the subject, to select a therapeutic allows detection . In embodiments where sufficient levels are agent , e.g. , an inhibitor, or to evaluate response to the present, the tissue , fluid or product can be analyzed for the treatment or progression of disease . In an embodiment the level of neoactivity , e.g. , an alpha hydroxy neoactivity , e.g., evaluation , which can be performed before and / or after 55 the 2HG neoactivity . In an embodiment the sample is treatment has begun , is based , at least in part , on analysis of analysed for the presence of a mutant IDH , e.g., IDH1 or a tumor sample , cancer cell sample , or precancerous cell IDH2 , protein having an alpha hydroxy neoactivity , e.g., sample , from the subject. E.g., a sample from the patient can 2HG neoactivity (or a corresponding RNA ). E.g., a mutant be analyzed for the presence or level of an alpha hydroxy protein specific reagent, e.g., an antibody that specifically neoactivity product , e.g., 2HG , e.g., R -2HG , by evaluating 60 binds an IDH mutant protein , e.g., an antibody that specifi a parameter correlated to the presence or level of an alpha cally binds an IDH1- R132H mutant protein or an IDH2 hydroxy neoactivity product, e.g., 2HG , e.g., R - 2HG . An R172 mutant protein ( e.g. , an IDH1- R132H mutant protein ), alpha hydroxy neoactivity product , e.g., 2HG , e.g., R -2HG , can be used to detect neoactive mutant enzyme. In an in the sample can be determined by a chromatographic embodiment a nucleic acid from the sample is sequenced to method , e.g., by LC -MS analysis . It can also be determined 65 determine if a selected allele or mutation of IDH1 or IDH2 by contact with a specific binding agent, e.g. , an antibody, disclosed herein is present. In an embodiment the analysis is which binds the alpha hydroxy neoactivity product, e.g., other than directly determining the presence of a mutant US 10,610,125 B2 43 44 IDH , e.g., IDH1 or IDH2 , protein ( or corresponding RNA ) surement of potential enzymatic activities, activity ( e.g., or sequencing of an IDH , e.g. , IDH1 or IDH2 gene . E.g., the wild type /non -mutant or neoactivity ) can be detected by the analysis can be the detection of an alpha hydroxy neoactivity quenching of protein fluorescence upon binding of a poten product , e.g., 2HG , e.g. , R -2HG , or the measurement of tial substrate , cofactor, or enzymatic activity modulator to 2HG neoactivity . In an embodiment the tissue , fluid or 5 the enzyme. product is removed from the patient and analyzed . In an In one embodiment , assay progress can be monitored by embodiment the evaluation can include one or more of changes in the OD340 or fluorescence of the NAD or NADP performing the analysis of the tissue, fluid or product , cofactor . In another embodiment, the reaction progress can requesting analysis of the tissue, fluid or product, requesting be coupled to a secondary enzyme assay system in continu results from analysis of the tissue , fluid or product, or 10 ous mode or endpoint mode for increasing the dynamic receiving the results from analysis of the tissue, fluid or range of the assay . For example , an endpoint assay can be product. performed by adding to the reaction an excess of diaphorase In an embodiment the evaluation , which can be performed and rezasarin . Diaphorase consumes the remaining NADPH before and / or after treatment has begun , is based , at least in or NADH while producing resorufin from rezasarin . Reso part , on alpha hydroxy neoactivity product, e.g., 2HG , e.g., 15 rufin is a highly fluorescent productwhich can be measured R - 2HG , imaging of the subject . In embodiments magnetic by fluorescence at Ex544 Em590 . This not only terminates resonance methods are is used to evaluate the presence , the reaction but also generates an easily detectable signal distribution , or level of an alpha hydroxy neoactivity prod with greater quantum yield than the fluorescence of the uct , e.g., 2HG , e.g., R - 2HG , in the subject . In an embodi cofactor. ment the subject is subjected to imaging and / or spectro- 20 A continuous assay can be implemented through coupling scopic analysis , e.g., magnetic resonance- based analysis , a product of the primary reaction to a secondary enzyme e.g., MRI and /or MRS e.g. , analysis , and optionally an reaction that yields detectable results of greater dynamic image corresponding to the presence , distribution , or level of range or more convenient detection mode. For example , an alpha hydroxy neoactivity product , e.g., 2HG , e.g., inclusion in the reaction mix of aldehyde dehydrogenase R - 2HG , or of the tumor, is formed . Optionally the image or 25 (ALDH ), which is an NADP + dependent enzyme, and a value related to the image is stored in a tangible medium 6 -methoxy - 2 - napthaldehye , a chromogenic substrate for and / or transmitted to a second site . In an embodiment the ALDH , will result in the production of the fluorescent evaluation can include one or more of performing imaging product 6 -methoxy - 2 - napthoate (Ex310 Em 360 ) at a rate analysis , requesting imaging analysis , requesting results dependent on the production of NADP + by isocitrate dehy from imaging analysis, or receiving the results from imaging 30 drogenase . The inclusion of a coupling enzyme such as analysis . aldehyde dehydrogenase has the additional benefit of allow Methods of Treating a Proliferative Disorder ing screening of neoactivity irrespective of whether NADP + Described herein are methods of treating a cell prolifera or NAD + is produced , since this enzyme is capable of tion -related disorder, e.g., a cancer, e.g. , a glioma, e.g., by utilizing both . Additionally , since the NADPH or NADH inhibiting a neoactivity of a mutant enzyme, e.g., an enzyme 35 cofactor required for the “ reverse ” assay is regenerated , a in a metabolic pathway , e.g., a metabolic pathway leading to coupled enzyme system which cycles the cofactor back to fatty acid biosynthesis , glycolysis, glutaminolysis , the pen the IDH enzyme has the further advantage of permitting tose phosphate shunt, the nucleotide biosynthetic pathway , continuous assays to be conducted at cofactor concentrations or the fatty acid biosynthetic pathway, e.g. , IDH1 or IDH2. much below Km for the purpose of enhancing the detection The cancer can be characterized by the presence of a 40 of competitive inhibitors of cofactor binding . neoactivity , such as a gain of function in one or more mutant In yet a third embodiment of an activity ( e.g., wild enzymes (e.g. , an enzyme in the metabolic pathway, e.g. , a type/ non -mutant or neoactivity ) screen , one or a number of metabolic pathway leading to fatty acid biosynthesis, gly IDH substrates , cofactors , or products can be isotopically colysis , glutaminolysis , the pentose phosphate shunt , the labeled with radioactive or “ heavy ” elements at defined nucleotide biosynthetic pathway, or the fatty acid biosyn- 45 atoms for the purpose of following specific substrates or thetic pathway e.g., IDH1 or IDH2) . In some embodiments , atoms of substrates through the chemical reaction . For the gain of function is the conversion of a -ketoglutarate to example , the alpha carbon of a -KG , isocitrate , or 2 -hy 2 -hydroxyglutarate , e.g. , R -2 -hydroxyglutarate . droxyglutarate , e.g. , R - 2 -hydroxyglutarate may be 14C or Compounds for the Treatment of Cancer 13C . Amount, rate , identity and structure of products formed A candidate compound can be evaluated for modulation 50 can be analyzed by means known to those of skill in the art , ( e.g. , inhibition ) of neoactivity , for example , using an assay for example mass spectroscopy or radiometric HPLC . described herein . A candidate compound can also be evalu Compounds that inhibit a neoactivity , e.g., a neoactivity ated for modulation (e.g. , inhibition ) of wild type or non described herein , can include, e.g. , small molecule , nucleic mutant activity . For example , the formation of a product or acid , protein and antibody. by - product of any activity ( e.g., enzymatic activity ) can be 55 Exemplary small molecules include, e.g , small molecules assayed , thus evaluating a candidate compound. In some that bind to enzymes and decrease their activity , e.g. , a embodiments , the activity ( e.g. , wild type /non -mutant or neoactivity described herein . The binding ofan inhibitor can neoactivity ) can be evaluated by measuring one or more stop a substrate from entering the enzyme's active site readouts from an enzymatic assay . For example , the change and / or hinder the enzyme from catalyzing its reaction . in nature and / or amount of substrate and / or product can be 60 Inhibitor binding is either reversible or irreversible . Irre measured , e.g. , using methods such as fluorescent or radio versible inhibitors usually react with the enzyme and change labeled substrates. Exemplary substrates and / or products it chemically . These inhibitors can modify key amino acid include a -ketoglutarate , CO2, NADP, NADPH , NAD , residues needed for enzymatic activity . In contrast, revers NADH , and 2 -hydroxyglutarate , e.g., R - 2 -hydroxyglutarate . ible inhibitors bind non - covalently and different types of In some embodiments , the rate of reaction of the enzyme can 65 inhibition are produced depending on whether these inhibi also be evaluated as can the nature and / or amount of a tors bind the enzyme, the enzyme- substrate complex, or product of the enzymatic reaction . In addition to the mea both . US 10,610,125 B2 45 46 In some embodiments , the smallmolecule is oxalomalate , Nucleic acids can be used to inhibit a neoactivity , e.g., a oxalofumarate, or oxalosuccinate . neoactivity described herein , e.g., by decreasing the expres In some embodiments , the small molecule is a compound sion of the enzyme. Exemplary nucleic acids include, e.g. , of formula ( X ) , or a compound as listed in Table 24a . The siRNA, shRNA , antisense RNA , aptamer and ribozyme. compound of formula ( X ) is provided below : 5 Art -known methods can be used to select inhibitory mol ecules, e.g., siRNA molecules, for a particular gene sequence . Formula (X ) Proteins can also be used to inhibit a neoactivity , e.g. , a neoactivity described herein , by directly or indirectly bind X 10 ing to the enzyme and / or substrate , or competing binding to S (R ') m the enzyme and/ or substrate . Exemplary proteins include , e.g., soluble receptors, peptides and antibodies. Exemplary HN antibodies include , e.g., whole antibody or a fragment thereof that retains its ability to bind to the enzyme or 15 substrate . Exemplary candidate compounds, which can be tested for wherein X is C , -Co alkylene ( e.g., methylene ) , C ( O ), or inhibition of a neoactivity described herein (e.g. , a neoac C ( O )C1 - C6 alkylene; wherein X is optionally substituted ; tivity associated with mutant IDH1) , are described in the Rí is halo (e.g. , fluoro ), C.-C. alkyl, C.-C. haloalkyl, 20 byfollowing reference references : Bioorganic , each &of Medicinalwhich are incorporatedChemistry (2008herein ) , hydroxyl, C.-C. alkoxy, cyano , nitro , amino, alkylamino , 16 ( 7 ) , 3580-3586 ; Free Radical Biology & Medicine ( 2007 ) , dialkylamino, amido , -C (O )OH , or C (O )OC , -Coalkyl; and 42 ( 1 ), 44-51 ; KR 2005036293 A ; Applied and Environmen m is 0 , 1, 2 , or 3 . tal Microbiology ( 2005 ) , 71 ( 9 ) , 5465-5475 ; KR In some embodiments , the compound is a compound of 2002095553 A ; U.S. Pat . Appl. US 2004067234 A1; PCT formula ( XI) or a pharmaceutically acceptable salt thereof or 25 Int. Appl. (2002 ), WO 2002033063 Al ; Journal of Organic a compound listed in Table 24b Chemistry ( 1996 ) , 61( 14 ) , 4527-4531 ; Biochimica et Bio physica Acta , Enzymology ( 1976 ) , 452 ( 2 ), 302-9 ; Journal of Biological Chemistry ( 1975) , 250 ( 16 ) , 6351-4 ; Bollettino ( XI) Societa Italiana di Biologia Sperimentale ( 1972 ) , 48 (23 ) , B B 30 1031-5 ; Journal of Biological Chemistry (1969 ), 244 ( 20 ) , 5709-12 . N Q- D Isomers Certain compounds may exist in one or more particular RI geometric , optical, enantiomeric , diasteriomeric , epimeric , (R3 35 atropic , stereoisomer, tautomeric , conformational, or ano meric forms, including but not limited to , cis- and trans wherein : forms; E- and Z -forms ; c-, t-, and r - forms; endo- and W , X , Y and Z are each independently selected from CH or exo -forms ; R-, S., and meso - forms; D- and L -forms ; d- and N ; 1 - forms; ( + ) and ( - ) forms; keto-, enol-, and enolate - forms; B and B ’ are independently selected from hydrogen , alkyl or 40 syn- and anti- forms; synclinal- and anticlinal- forms; a- and when taken together with the carbon to which they are B - forms; axial and equatorial forms; boat-, chair-, twist-, attached form a carbonyl group ; envelope-, and halfchair - forms; and combinations thereof, Q is C = O or SO2 ; hereinafter collectively referred to as “ isomers ” (or “ iso D and D ' are independently selected from a bond , oxygen or meric forms” ) . NR " ; 45 In one embodiment, a compound described herein , e.g. , A is optionally substituted aryl or optionally substituted an inhibitor of a neoactivity or 2 - HG is an enantiomerically heteroaryl; enriched isomer of a stereoisomer described herein . For R ! is independently selected from alkyl, acyl , cycloalkyl, example , the compound has an enantiomeric excess of at aryl, heteroaryl, heterocyclyl, heterocyclylalkyl , cycloalky least about 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , lalkyl, aralkyl, and heteroaralkyl; each of which may be 50 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , optionally substituted with 0-3 occurrences of Rd; 96 % , 97 % , 98 % , or 99 % . Enantiomer , when used herein , each R3 is independently selected from halo , haloalkyl, alkyl refers to either of a pair of chemical compounds whose and OR " ; molecular structures have a mirror- image relationship to each R is independently selected from alkyl, and haloalkyl; each other. each R is independently alkyl; 55 In one embodiment , a preparation of a compound dis each R is independently selected from hydrogen , alkyl and closed herein is enriched for an isomer of the compound alkenyl; having a selected stereochemistry , e.g., R or S , correspond each Rd is independently selected from halo , haloalkyl, ing to a selected stereocenter , e.g. , the 2 - position of 2 -hy alkyl, nitro , cyano , and OR " , or two Rd taken together droxyglutaric acid . 2HG can be purchased from commercial with the carbon atoms to which they are attached form an 60 sources or can be prepared using methods known in the art, optionally substituted heterocyclyl ; for example , as described in Org. Syn . Coll vol. , 7 , P -99 , n is 0 , 1, or 2 ; 1990. For example , the compound has a purity correspond h is 0 , 1, 2 ; and ing to a compound having a selected stereochemistry of a gis 0 , 1 or 2 . selected stereocenter of at least about60 % , 65 % , 70 % , 75 % , In some embodiments , the small molecule is a selective 65 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % . inhibitor of the neoactivity ( e.g., relative to the wild type In one embodiment, a composition described herein activity ) . includes a preparation of a compound disclosed herein that US 10,610,125 B2 47 48 is enriched for a structure or structures having a selected of pharmaceutically acceptable salts are discussed in Berge stereochemistry, e.g., R or S , at a selected stereocenter , e.g. , et al. , 1977 , “ Pharmaceutically Acceptable Salts .” J. Pharm . the 2 -position of 2 -hydroxyglutaric acid . Exemplary R / S ScL . Vol. 66 , pp . 1-19. configurations can be those provided in an example For example , if the compound is anionic , or has a func described herein . 5 tional group which may be anionic (e.g. , COOH may be An “ enriched preparation ,” as used herein , is enriched for COO " ), then a salt may be formed with a suitable cation . a selected stereoconfiguration of one , two , three or more Examples of suitable inorganic cations include , but are not limited to , alkali metal ions such as Na+ and K + , alkaline selected stereocenters within the subject compound. Exem earth cations such as Ca2 + and Mg2 + , and other cations such plary selected stereocenters and exemplary stereoconfigura 10 as Al+ 3 . Examples of suitable organic cations include , but tions thereof can be selected from those provided herein , are not limited to , ammonium ion ( i.e. , NH4 + ) and substi e.g., in an example described herein . By enriched is meant tuted ammonium ions ( e.g., NH3R + , NH2R2 + , NHR3 + , at least 60 % , e.g., of the molecules of compound in the NR4 + ). Examples of some suitable substituted ammonium preparation have a selected stereochemistry of a selected ions are those derived from : ethylamine , diethylamine , dicy stereocenter . In an embodiment it is at least 65 % , 70 % , 75 % , 15 clohexylamine , triethylamine , butylamine , ethylenediamine , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , or 99 % . Enriched ethanolamine , diethanolamine , piperazine , benzylamine , refers to the level of a subject molecule ( s ) and does not phenylbenzylamine , choline , meglumine , and trometh connote a process limitation unless specified . amine , as well as amino acids, such as lysine and arginine . Note that, except as discussed below for tautomeric An example of a common quaternary ammonium ion is forms, specifically excluded from the term “ isomers , " as 20 N (CH3 )4+ . used herein , are structural ( or constitutional) isomers ( i.e. , If the compound is cationic , or has a functional group that isomers which differ in the connections between atoms may be cationic ( e.g., -NH2may.be -NH3 + ), then a salt rather than merely by the position of atoms in space ) . For may be formed with a suitable anion . Examples of suitable example , a reference to a methoxy group , OCH3, is not to inorganic anions include, but are not limited to , those be construed as a reference to its structural isomer, a 25 derived from the following inorganic acids : hydrochloric , hydroxymethyl group , CH2OH . Similarly , a reference to hydrobromic , hydroiodic , sulfuric , sulfurous , nitric , nitrous, ortho - chlorophenyl is not to be construed as a reference to phosphoric , and phosphorous . its structural isomer , meta - chlorophenyl. However , a refer Examples of suitable organic anions include, but are not ence to a class of structures may well include structurally limited to , those derived from the following organic acids: isomeric forms falling within that class ( e.g., C1-7alkyl 30 2 - acetyoxybenzoic , acetic , ascorbic , aspartic , benzoic , cam includes n - propyl and iso -propyl ; butyl includes n-, iso-, phorsulfonic , cinnamic , citric , edetic , ethanedisulfonic , eth sec-, and tert -butyl ; methoxyphenyl includes ortho-, meta-, anesulfonic , fumaric , glucheptonic , gluconic , glutamic , gly and para -methoxyphenyl ) . colic , hydroxymaleic , hydroxynaphthalene carboxylic , The above exclusion does not pertain to tautomeric forms, 35 isethionicanesulfonic , ,lactic mucic , lactobionic , oleic , oxalic , lauric , palmitic , maleic , pamoic , malic , pantoth , meth for example , keto-, enol-, and enolate - forms, as in , for enic , phenylacetic , phenylsulfonic , propionic , pyruvic , sali example , the following tautomeric pairs : keto / enol (illus cylic , stearic , succinic , sulfanilic , tartaric , toluenesulfonic , trated below ) , imine / enamine, amide/ imino alcohol, ami and valeric . Examples of suitable polymeric organic anions dine / amidine , nitroso /oxime , thioketone / enethiol, N -nitroso / include , but are not limited to , those derived from the hydroxyazo , and nitro / aci -nitro . 40 following polymeric acids: tannic acid , carboxymethyl cel lulose . Unless otherwise specified , a reference to a particular H compound also includes salt forms thereof. Chemically Protected Forms 45 It may be convenient or desirable to prepare , purify , and / or handle the active compound in a chemically protected ketoK - enolX + )enolate form . The term " chemically protected form ” is used herein in the conventional chemical sense and pertains to a com pound in which one or more reactive functional groups are compoundsNote that withspecifically one or included more isotopic in the termsubstitutions “ isomer. ” Forare 50 protected from undesirable chemical reactions under speci example , H may be in any isotopic form , including 1H , 2H fied conditions ( e.g. , pH , temperature , radiation , solvent, and ( D ) , and 3H ( T ) ; C may be in any isotopic form , including the like ). In practice, well known chemical methods are 12C , 13C , and 14C ; O may be in any isotopic form , employed to reversibly render unreactive a functional group , including 160 and 180 ; and the like. Unless otherwise ss whichtions . Inotherwise a chemically would protected be reactive form , under, one orspecifiedmore reactive condi specified , a reference to a particular compound includes all functional groups are in the form of a protected or protecting such isomeric forms, including (wholly or partially ) racemic group ( also known as a masked or masking group or a and other mixtures thereof. Methods for the preparation blocked or blocking group ) . By protecting a reactive func ( e.g., asymmetric synthesis ) and separation ( e.g., fractional tional group, reactions involving other unprotected reactive crystallisation and chromatographic means ) of such iso- 60 functional groups can be performed , without affecting the meric forms are either known in the art or are readily protected group ; the protecting group may be removed , obtained by adapting the methods taught herein , or known usually in a subsequent step , without substantially affecting methods, in a known manner. the remainder of the molecule . See, for example , Protective Salts Groups in Organic Synthesis ( T. Green and P. Wuts ; 3rd It may be convenient or desirable to prepare , purify , 65 Edition ; John Wiley and Sons, 1999 ) . Unless otherwise and / or handle a corresponding salt of the active compound , specified , a reference to a particular compound also includes for example, a pharmaceutically -acceptable salt. Examples chemically protected forms thereof. US 10,610,125 B2 49 50 A wide variety of such “ protecting , ” “ blocking, ” or mediated activation of protein kinase PKR and 2 , 5 '- oligoad “ masking ” methods are widely used and well known in enylate synthetase resulting in non - specific cleavage of organic synthesis . For example , a compound which has two mRNA by ribonuclease L. nonequivalent reactive functional groups , both of which dsRNAs targeting an IDH , e.g., IDH1, enzyme, e.g., a would be reactive under specified conditions, may be deriva- 5 wildtype or mutant IDH1, can be unmodified or chemically tized to render one of the functional groups " protected, ” and modified . The dsRNA can be chemically synthesized , therefore unreactive, under the specified conditions; so pro expressed from a vector or enzymatically synthesized . The tected , the compound may be used as a reactant which has invention also features various chemically modified syn effectively only one reactive functional group . After the thetic dsRNA molecules capable of modulating IDH1 gene desired reaction (involving the other functional group) is 10 expressionThe use of orchemically activity in modified cells by RNAdsRNA interference improves ( RNAIvarious ) . complete , the protected group may be “ deprotected ” to properties of native dsRNA molecules, such as through return it to its original functionality . increased resistance to nuclease degradation in vivo and /or For example , a hydroxy group may be protected as an through improved cellular uptake . ether ( OR ) or an ester ( OCO) R ), for example , as : a 15 The dsRNAs targeting nucleic acid can be composed of t -butyl ether; a benzyl, benzhydryl (diphenylmethyl ) , or two separate RNAs, or of one RNA strand , which is folded trityl ( triphenylmethyl) ether, a trimethylsilyl or t- butyldim to form a hairpin structure . Hairpin dsRNAs are typically ethylsilyl ether; or an acetyl ester ( OCEO) CH3, referred to as shRNAS. OAc ). An shRNA that targets IDH , e.g., a mutant or wildtype For example , an aldehyde or ketone group may be pro- 20 IDH1 gene can be expressed from a vector, e.g., viral vector, tected as an acetal (R_CH (OR ) 2 ) or ketal (R2C (OR ) 2 ), such as a lentiviral or adenoviral vector. In certain embodi respectively , in which the carbonyl group ( > C = O ) is con ments , a suitable dsRNA for inhibiting expression of an verted to diether ( > C (OR ) 2 ) , by reaction with , for IDH1 gene will be identified by screening an siRNA library , example , a primary alcohol. The aldehyde or ketone group such as an adenoviral or lentiviral siRNA library . is readily regenerated by hydrolysis using a large excess of 25 In an embodiment, a dsRNA that targets IDH , e.g., IDH1, water in the presence of acid . is about 15 to about 30 base pairs in length ( e.g., about 16 , For example , an amine group may be protected , for 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28, or 29 ) basepairs example , as an amide NRCOR) or a urethane in length . In another embodiment, the dsRNA includes a methyl amide overhanging ends of about 1 to about 3 ( e.g. , about 1, 2 , or NRCO OR ), for example, as : 30 3 ) nucleotides. By “ overhang ” is meant that 3 '- end of one ( NHCO CH3) ; a benzyloxy amide ( NHCO strand of the dsRNA extends beyond the 5 ' -end of the other OCH2C6H5, -NH -Cbz ); as a t- butoxy amide ( NHCO strand , or vice versa . The dsRNA can have an overhang on OC (CH3 ) 3 , NH - Boc ) ; a 2 - biphenyl- 2 -propoxy amide one or both ends of the dsRNA molecule . In some embodi ( NHCO_OC ( CH3) 2C6H4C6H5, —NH -Bpoc ), as a ments , the single -stranded overhang is located at the 3 '- ter 9 - fluorenylmethoxy amide ( NH - Fmoc) , as a 6 -nitrovera 35 minal end of the antisense strand , or , alternatively , at the tryloxy amide -NH( -Nvoc ) , as a 2 -trimethylsilylethyloxy 3 '- terminal end of the sense strand . In some embodiments , amide ( NH - Teoc ), as a 2,2,2 -trichloroethyloxy amide the overhang is a TT or UU dinucleotide overhang, e.g. , a TT ( NH - Troc ) , as an allyloxy amide NH -Alloc ), as a or UU dinucleotide overhang . For example , in an embodi 2 (-phenylsulphonyl ) ethyloxy amide ( NH -Psec ) ; or , in ment, the dsRNA includes a 21 -nucleotide antisense strand , suitable cases ( e.g. , cyclic amines) , as a nitroxide radical 40 a 19 base pair duplex region , and a 3 '- terminal dinucleotide . ( >NO << ) . In yet another embodiment, a dsRNA includes a duplex For example , a carboxylic acid group may be protected as nucleic acid where both ends are blunt, or alternatively , an ester for example , as : an C?alkyl ester ( e.g., a methyl where one of the ends is blunt. ester ; a t- butyl ester ); a Cvrhaloalkyl ester ( e.g. , a C1-7tri In an embodiment, the dsRNA includes a first and a haloalkyl ester ) ; a triC1-7alkylsilyl -Ci.7alkyl ester; or a 45 second strand, each strand is about 18 to about 28 nucleo C5.2oaryl- C1-7alkyl ester ( e.g., a benzyl ester ; a nitrobenzyl tides in length , e.g. , about 19 to about 23 nucleotides in ester ) ; or as an amide , for example, as a methyl amide . length , the first strand of the dsRNA includes a nucleotide For example , a thiol group may be protected as a thioether sequence having sufficient complementarity to the IDH , e.g., ( SR ) , for example , as : a benzyl thioether; an acetamidom IDHI, RNA for the dsRNA to direct cleavage of the IDH , ethyl ether ( S CH2NHC (= O ) CH3) . 50 e.g., IDH1, mRNA via RNA interference , and the second Nucleic Acid Based Inhibitors strand of the dsRNA includes a nucleotide sequence that is Nucleic acid -based inhibitors for inhibition IDH , e.g. , complementary to the first strand . IDH1, can be , e.g. , double stranded RNA (dsRNA ) that In an embodiment, a dsRNA targeting an IDH , e.g. , IDH1, function , e.g., by an RNA interference (RNAi mechanism ) , gene can target wildtype and mutant forms of the gene , or an antisense RNA , or a microRNA ( miRNA ). In an embodi- 55 can target different allelic isoforms of the same gene . For ment the nucleic -acid based inhibitor binds to the target example , the dsRNA will target a sequence that is identical mRNA and inhibits the production of protein therefrom , e.g., in two ormore of the different isoforms. In an embodiment, by cleavage of the target mRNA . the dsRNA targets an IDH1 having G at position 395 or C Double Stranded RNA (dsRNA ) at position 394 (e.g. , a wildtype IDH1 RNA ) and an IDH1 A nucleic acid based inhibitor useful for decreasing IDH1 60 having A at position 395 or A at position 394, such as a or IDH2mutant function is , e.g. , a dsRNA , such as a dsRNA C394A , a C394G , a C394T , a G395C , a G395T or a G395A that acts by an RNAimechanism . RNAirefers to the process mutation , ( e.g. , an IDH1 RNA carrying a G395A and / or a of sequence- specific post - transcriptional gene silencing in C394A mutation ) (FIG . 2 ). animals mediated by short interfering RNAs (siRNAs ) . In an embodiment, a dsRNA will preferentially or spe dsRNAs as used herein are understood to include siRNAs. 65 cifically target a mutant IDH RNA, or a particular IDH Typically , inhibition of IDH , e.g. , IDH1, by dsRNAs does polymorphism . In some embodiments , the IDH has a muta not trigger the interferon response that results from dsRNA tion at position 394 or 395 such as a C394A , a C394G , a US 10,610,125 B2 51 52 C394T , a G395C , a G395T or a G395A mutation . For siRNA , post- transcriptional gene silencing RNA ( ptg example , in an embodiment, the dsRNA targets an IDH1 SRNA ) , and others . In addition , as used herein , the term RNA carrying an A at position 395, e.g., G395A , and in “ RNAI” is meant to include sequence specific RNA inter another embodiment, the dsRNA targets an IDH1 RNA ference , such as post transcriptional gene silencing, trans carrying an A at position 394 , e.g. , C394A mutation . 5 lational inhibition , or epigenetics . In an embodiment, a dsRNA targeting an IDH RNA Nucleic Acid - Based IDH Inhibitors includes one or more chemical modifications. Non - limiting examples of such chemical modifications include without In an embodiment the inhibitor is a nucleic acid -based limitation phosphorothioate internucleotide linkages , 2' - de inhibitor, such as a double stranded RNA (dsRNA ) or oxyribonucleotides , 2 '- O -methyl ribonucleotides , 2 '- deoxy- 10 antisense RNA that targets a mutant IDH , e.g., mutant IDH1 2 - fluoro ribonucleotides , " universal base ” nucleotides, or IDH2. " acyclic ” nucleotides , 5 - C -methyl nucleotides , and terminal In one embodiment, the nucleic acid based inhibitor , e.g., glyceryl and / or inverted deoxy abasic residue incorporation . a dsRNA or antisense molecule , decreases or inhibits Such chemical modifications have been shown to preserve expression of an IDH1 having other than an Arg, e.g., having RNAi activity in cells while at the same time, dramatically 15 a His , Ser, Cys , Gly , Val, Pro or Leu , or any residue increasing the serum stability of these compounds. Further described in Yan et al ., N. Eng . J. Med . 360 : 765-73 , at more , one or more phosphorothioate substitutions are well residue 132 , according to the sequence of SEQ ID NO : 8 (see tolerated and have been shown to confer substantial also FIG . 21 ) . In one embodiment, the nucleic acid based increases in serum stability for modified dsRNA constructs . inhibitor decreases or inhibits expression of an IDH1 In an embodiment, a dsRNA targeting an IDH , e.g. , IDH1, 20 enzyme having His at residue 132 RNA includes modified nucleotides while maintaining the In an embodiment the nucleic acid -based inhibitor is a ability to mediate RNAi. The modified nucleotides can be dsRNA that targets an mRNA that encodes an IDH1 allele used to improve in vitro or in vivo characteristics such as described herein , e.g. , an IDH1 allele having other than an stability , activity , and / or bioavailability . For example , the Arg at residue 132. E.g., the allele encodes His , Ser, Cys, dsRNA can include modified nucleotides as a percentage of 25 Gly , Val, Pro or Leu , or any residue described in Yan et al. , the total number of nucleotides present in the molecule . As at residue 132 , according to the sequence of SEQ ID NO : 8 such , the dsRNA can generally include about 5 % to about (see also FIG . 21 ) . 100 % modified nucleotides ( e.g., about 5 % , 10 % , 15 % , In an embodiment the allele encodes an IDH1having His 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , at residue 132 . 70 % , 75 % , 80 % , 85 % , 90 % , 95 % or 100 % modified nucleo- 30 In an embodiment the allele encodes an IDH1 having Ser tides ). at residue 132 . In some embodiments , the dsRNA targeting IDH , e.g., In an embodiment, the nucleic acid -based inhibitor is a IDH1, is about 21 nucleotides long . In another embodiment, dsRNA that targets IDH1, e.g., an IDH1 having an A or a T the dsRNA does not contain any ribonucleotides , and in (or a nucleotide other than C ) at nucleotide position 394 or another embodiment , the dsRNA includes one or more 35 an A ( or a nucleotide other than G ) at nucleotide position ribonucleotides . In an embodiment , each strand of the 395 , e.g. , a mutant allele carrying a C394T mutation or a dsRNA molecule independently includes about 15 to about G395A mutation according to the IDH1 sequence of SEQ ID 30 ( e.g., about 15 , 16 , 17, 18 , 19 , 20 , 21 , 22 , 23, 24 , 25 , 26 , NO : 8 (see also FIG . 21A ). 27 , 28 , 29 , or 30 ) nucleotides , wherein each strand includes In an embodiment, the dsRNA targets an IDH1 having about 15 to about 30 (e.g. , about 15, 16 , 17 , 18 , 19 , 20 , 21, 40 other than C , e.g., a Tor an A , at nucleotide position 394 or 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29, or 30 ) nucleotides that are and other than G , e.g., an A , at 395 ( e.g., a mutant) and an complementary to the nucleotides of the other strand . In an IDH1 having a C at nucleotide position 394 or a G at embodiment, one of the strands of the dsRNA includes a nucleotide position 395 ( e.g., a wildtype) , e.g., by targeting nucleotide sequence that is complementary to a nucleotide a region of the IDH1 mRNA that is identical between the sequence or a portion thereofof the IDH1 or IDH2 gene, and 45 wildtype and mutant transcripts . In yet another embodiment, the second strand of the dsRNA includes a nucleotide the dsRNA targets a particular mutant or polymorphism sequence substantially similar to the nucleotide sequence of (such as a single nucleotide polymorphism (SNP ) ) , but not the IDH1 or IDH2 gene or a portion thereof. a wildtype allele. In this case , the nucleic acid based In an embodiment, the dsRNA targeting IDH1 or IDH2 inhibitor , e.g., a dsRNA , targets the region of the IDH1 includes an antisense region having a nucleotide sequence 50 containing the mutation . that is complementary to a nucleotide sequence of the IDH1 In some embodiments , the nucleic acid based inhibitor, or IDH2 gene or a portion thereof, and a sense region having e.g., a dsRNA preferentially or specifically inhibits the a nucleotide sequence substantially similar to the nucleotide product of a mutant IDH1 as compared to the product of a sequence of the IDH1 or IDH2 gene or a portion thereof. In wildtype IDH1. In some embodiments , the IDH has a an embodiment, the antisense region and the sense region 55 mutation at position 394 or 395 such as a C394A , a C394G , independently include about 15 to about 30 ( e.g., about 15 , a C394T, a G395C , a G395T or a G395A mutation . For 16 , 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , or 30 ) example , in one embodiment, a dsRNA targets a region of an nucleotides , where the antisense region includes about 15 to IDH1 mRNA that carries the mutation ( e.g. , a C394A of about 30 ( e.g., about 15 , 16 , 17 , 18, 19 , 20, 21 , 22, 23 , 24 , C394T or a G395A mutation according to SEQ ID NO : 5 ) . 25 , 26 , 27 , 28 , 29, or 30 ) nucleotides that are complementary 60 In one embodiment, the nucleic acid -based inhibitor is a to nucleotides of the sense region . dsRNA including a sense strand and an antisense strand As used herein , the term “ dsRNA ” is meant to include having a primary sequence presented in Tables 7-14 . In nucleic acid molecules that are capable of mediating another embodiment, the nucleic acid based inhibitor is an sequence specific RNAi, such as short interfering RNA antisense oligonucleotide that includes all or a part of an ( siRNA ) , short hairpin RNA (shRNA ), short interfering 65 antisense primary sequence presented in Tables 7-14 or oligonucleotide, short interfering nucleic acid , short inter which targets the same or substantially the same region as fering modified oligonucleotide , chemically modified does a dsRNA from Tables 7-14 . US 10,610,125 B2 53 54 In one embodiment, the nucleic acid based inhibitor device . In an embodiment, the nucleic acid - based inhibitor decreases or inhibits expression of an IDH2having Lys, Gly , is delivered by infusion using , e.g. , a catheter , and option Met, Trp , Thr, Ser , or any residue described in Yan et al. , at ally, a pump. residue 172, according to the amino acid sequence of SEQ Antisense ID NO : 10 ( see also FIG . 22 ). In one embodiment, the 5 Suitable nucleic acid based inhibitors include antisense nucleic acids. While not being bound by theory it is believed nucleic acid based inhibitor decreases or inhibits expression that antisense inhibition is typically based upon hydrogen of an IDH2 enzyme having Lys at residue 172 . bonding -based hybridization of oligonucleotide strands or In an embodiment the nucleic acid - based inhibitor is a segments such that at least one strand or segment is cleaved , dsRNA that targets an mRNA that encodes an IDH2 allele 10 degraded , or otherwise rendered inoperable . described herein , e.g., an IDH2 allele having other than an An antisense agent can bind IDH1 or IDH2 DNA . In Arg at residue 172. E.g., the allele can have Lys, Gly , Met , embodiments it inhibits replication and transcription . While Trp , Thr, Ser, or any residue described in Yan et al. , at not being bound by theory it is believed that an antisense residue 172 , according to the sequence of SEQ ID NO : 10 agent can also function to inhibit target RNA translocation , (see also FIG . 22 ). 15 e.g., to a site of protein translation , translation of protein In an embodiment the allele encodes an IDH2 having Lys from the RNA , splicing of the RNA to yield one or more at residue 172 . RNA species , and catalytic activity or complex formation In an embodiment the allele encodes an IDH2 having Met involving the RNA . at residue 172 . An antisense agents can have a chemical modification In an embodiment , the nucleic acid -based inhibitor is a 20 described above as being suitable for dsRNA . dsRNA that targets IDH2, e.g., an IDH2 having a G or a T Antisense agents can include , for example , from about 8 ( or a nucleotide other than A or C ) at nucleotide position 514 to about 80 nucleobases (i.e. , from about 8 to about 80 or an Aor T or C (or a nucleotide other than G ) at nucleotide nucleotides ), e.g., about 8 to about 50 nucleobases , or about position 515 , e.g., a mutant allele carrying a A514G muta 12 to about 30 nucleobases. Antisense compounds include tion or a G515T or a G515A mutation according to the IDH2 25 ribozymes, external guide sequence (EGS ) oligonucleotides sequence of SEQ ID NO :10 (FIG . 22A ) . In one embodiment, (oligozymes ), and other short catalytic RNAs or catalytic the nucleic acid -based inhibitor is a dsRNA that targets oligonucleotides which hybridize to the target nucleic acid IDH2, e.g., an IDH2 having a C or a T (or a nucleotide other and modulate its expression . Anti -sense compounds can than G or A ) at nucleotide position 516 according to the include a stretch of at least eight consecutive nucleobases IDH2 sequence of SEQ ID NO : 10 . 30 that are complementary to a sequence in the target gene. An In an embodiment, the nucleic acid -based inhibitor is a oligonucleotide need not be 100 % complementary to its dsRNA that targets IDH2, e.g., an IDH2 having a G at target nucleic acid sequence to be specifically hybridizable . nucleotide position 514 or a T at nucleotide position 515 or An oligonucleotide is specifically hybridizable when bind an A at position 515 , according to the IDH2 sequence of ing of the oligonucleotide to the target interferes with the SEQ ID NO : 10 . 35 normal function of the target molecule to cause a loss of In an embodiment , the dsRNA targets an IDH2 having utility , and there is a sufficient degree of complementarity to other than A , e.g., a G or a T , at nucleotide position 514 , or avoid non - specific binding of the oligonucleotide to non other than G , e.g., an A or C or T at position 515 ( e.g., a target sequences under conditions in which specific binding mutant) , or other than G , e.g. , C or T , and an IDH2 having is desired , i.e., under physiological conditions in the case of an A at nucleotide position 514 or a G at nucleotide position 40 in vivo assays or therapeutic treatment or, in the case of in 515 or a G at position 516 ( e.g. , a wildtype) , e.g., by vitro assays, under conditions in which the assays are targeting a region of the IDH2 mRNA that is identical conducted . between the wildtype and mutant transcripts . In yet another Hybridization of antisense oligonucleotides with mRNA embodiment, the dsRNA targets a particular mutant or ( e.g., an mRNA encoding IDH1 or IDH2) can interfere with polymorphism (such as a single nucleotide polymorphism 45 one or more of the normal functions of mRNA . While not (SNP ) ) , but not a wildtype allele . In this case , the nucleic being bound by theory it is believed that the functions of acid based inhibitor , e.g., a dsRNA , targets the region of the mRNA to be interfered with include all key functions such IDH2 containing the mutation . as, for example , translocation of the RNA to the site of In some embodiments , the nucleic acid based inhibitor, protein translation , translation of protein from the RNA , e.g., a dsRNA , preferentially or specifically inhibits the 50 splicing of the RNA to yield one ormore mRNA species , and product of a mutant IDH2 as compared to the product of a catalytic activity which may be engaged in by the RNA . wildtype IDH2 . For example , in one embodiment , a dsRNA Binding of specific protein ( s ) to the RNA may also be targets a region of an IDH2 mRNA that carries the mutation interfered with by antisense oligonucleotide hybridization to ( e.g., an A514G or G515T or a G515U mutation according the RNA . to SEQ ID NO : 10 ) . 55 Exemplary antisense compounds include DNA or RNA In one embodiment, the nucleic acid -based inhibitor is a sequences that specifically hybridize to the target nucleic dsRNA including a sense strand and an antisense strand acid , e.g., the mRNA encoding IDH1 or IDH2 . The comple having a primary sequence presented in Tables 15-23. In mentary region can extend for between about 8 to about 80 another embodiment, the nucleic acid based inhibitor is an nucleobases. The compounds can include one or more antisense oligonucleotide that includes all or a part of an 60 modified nucleobases . Modified nucleobases may include, antisense primary sequence presented in Tables 15-23 or e.g. , 5 -substituted pyrimidines such as 5 - iodouracil , 5 - iodo which targets the same or substantially the same region as cytosine, and C5 - propynyl pyrimidines such as C5 - propy does a dsRNA from Tables 15-23 . nylcytosine and C5- propynyluracil . Other suitable modified In an embodiment, the nucleic acid based inhibitor is nucleobases include N4— (C1 - C12 ) alkylaminocytosines and delivered to the brain , e.g., directly to the brain , e.g., by 65 N4, N4— (C7 -C12 ) dialkylaminocytosines . Modified nucle intrathecal or intraventricular delivery . The nucleic acid obases may also include 7 - substituted - 5 - aza - 7 -deazapurines based inhibitor can also be delivered from an inplantable and 7 -substituted -7 - deazapurines such as, for example , US 10,610,125 B2 55 56 7 -iodo - 7 -deazapurines , 7 - cyano - 7 - deazapurines , 7 - amin Retroviral vectors and adeno -associated virus vectors can ocarbonyl- 7 -deazapurines . Examples of these include be used as a recombinant gene delivery system for the 6 - amino - 7 - iodo - 7 -deazapurines , 6 -amino - 7 -cyano -7 transfer of exogenous genes in vivo particularly into deazapurines, 6 -amino - 7 -aminocarbonyl - 7 - deazapurines , humans. These vectors provide efficient delivery of genes 2 - amino - 6 -hydroxy - 7 - iodo - 7 -deazapurines , 2 - amino - 6 -hy- 5 into cells, and the transferred nucleic acids are stably inte droxy - 7 -cyano - 7 -deazapurines , and 2- amino -6 -hydroxy -7 grated into the chromosomal DNA of the host . Protocols for aminocarbonyl- 7 -deazapurines . Furthermore , NØ_C - C12) producing recombinant retroviruses and for infecting cells in alkylaminopurines and N “ ,N_ ( C7 -C12 ) dialkylaminopu vitro or in vivo with such viruses can be found in Current rines , including Nº- methylaminoadenine and Nº, Nó - dimeth Protocols in Molecular Biology, Ausubel , F. M. et al. ( eds. ) ylaminoadenine, are also suitable modified nucleobases. 10 Greene Publishing Associates ( 1989 ), Sections 9.10-9.14 Similarly , other 6 -substituted purines including, for and other standard laboratory manuals . Examples of suitable example , 6 - thioguanine may constitute appropriate modified retroviruses include pLJ, PZIP , PWE , and pEM which are nucleobases . Other suitable nucleobases include 2 - thioura known to those skilled in the art. Examples of suitable cil , 8 - bromoadenine, 8 -bromoguanine , 2 - fluoroadenine , and packaging virus lines for preparing both ecotropic and 2 - fluoroguanine . Derivatives of any of the aforementioned 15 amphotropic retroviral systems include Crip , Cre , 2 , and modified nucleobases are also appropriate . Substituents of Am . Retroviruses have been used to introduce a variety of any of the preceding compounds may include C7 -C30 alkyl, genes into many different cell types , including epithelial C2 -C30 alkenyl, C2 -C30 alkynyl, aryl, aralkyl, heteroaryl, cells , in vitro and / or in vivo ( see, for example , Eglitis et al. halo , amino , amido , nitro , thio , sulfonyl, carboxyl, alkoxy , ( 1985 ) Science 230 : 1395-1398 ; Danos and Mulligan ( 1988 ) alkylcarbonyl, alkoxycarbonyl, and the like . 20 Proc . Natl. Acad . Sci. USA 85 :6460-6464 ; Wilson et al . MicroRNA (1988 ) Proc . Natl. Acad . Sci. USA 85 : 3014-3018 ; Armen In some embodiments , the nucleic acid -based inhibitor tano et al. ( 1990 ) Proc . Natl . Acad . Sci. USA 87 :6141-6145 ; suitable for targeting IDH , e.g. , IDH1, is a microRNA Huber et al. (1991 ) Proc . Natl. Acad . Sci. USA 88 : 8039 (miRNA ) . A miRNA is a single stranded RNA that regulates 8043 ; Ferry et al . ( 1991 ) Proc . Natl . Acad . Sci. USA the expression of target mRNAs either by mRNA cleavage , 25 88 :8377-8381 ; Chowdhury et al. ( 1991 ) Science 254 :1802 translational repression /inhibition or heterochromatic 1805 ; van Beusechem et al . (1992 ) Proc . Natl . Acad . Sci. silencing . The miRNA is 18 to 25 nucleotides , typically 21 USA 89 :7640-7644 ; Kay et al . ( 1992 ) Human Gene Therapy to 23 nucleotides in length . In some embodiments , the 3 :641-647 ; Dai et al. ( 1992 ) Proc . Natl. Acad . Sci. USA miRNA includes chemical modifications, such as one or 89 : 10892-10895 ; Hwu et al . (1993 ) J. Immunol. 150 :4104 more modifications described herein . 30 4115 ; U.S. Pat. Nos . 4,868,116 and 4,980,286 ; PCT Pub . In some embodiments , a nucleic acid based inhibitor Nos. WO 89/07136 , WO 89/02468 , WO 89/05345 , and WO targeting IDH has partial complementarity ( i.e., less than 92/07573 ). 100 % complementarity ) with the target IDH , e.g. , IDH1 or Another viral gene delivery system utilizes adenovirus IDH2, mRNA . For example , partial complementarity can derived vectors . See , for example , Berkner et al. ( 1988 ) include various mismatches or non -base paired nucleotides 35 BioTechniques 6 :616 ; Rosenfeld et al . (1991 ) Science 252 : ( e.g. , 1 , 2 , 3 , 4 , 5 or more mismatches or non -based paired 431-434 ; and Rosenfeld et al . ( 1992 ) Cell 68 : 143-155 . nucleotides, such as nucleotide bulges ), which can result in Suitable adenoviral vectors derived from the adenovirus bulges, loops, or overhangs that result between the antisense strain Ad type 5 d1324 or other strains of adenovirus ( e.g., strand or antisense region of the nucleic acid -based inhibitor Ad2 , Ad3 , Ad7 etc. ) are known to those skilled in the art . and the corresponding target nucleic acid molecule . 40 Yet another viral vector system useful for delivery of the The nucleic acid -based inhibitors described herein , e.g. , subject gene is the adeno - associated virus ( AAV ) . See , for antisense nucleic acid described herein , can be incorporated example , Flotte et al. ( 1992 ) Am . J. Respir. Cell .Mol . Biol . into a gene construct to be used as a part of a gene therapy 7 :349-356 ; Samulski et al. (1989 ) J. Virol. 63 :3822-3828 ; protocol to deliver nucleic acids that can be used to express and McLaughlin et al. ( 1989 ) J. Virol . 62: 1963-1973 . and produce agents within cells . Expression constructs of 45 Pharmaceutical Compositions such components may be administered in any biologically The compositions delineated herein include the com effective carrier , e.g., any formulation or composition pounds delineated herein , as well as additional therapeutic capable of effectively delivering the component gene to cells agents if present, in amounts effective for achieving a in vivo . Approaches include insertion of the subject gene in modulation of disease or disease symptoms, including those viral vectors including recombinant retroviruses , adenovi- 50 described herein . rus, adeno - associated virus, lentivirus, and herpes simplex The term “ pharmaceutically acceptable carrier or adju virus - 1 , or recombinant bacterial or eukaryotic plasmids . vant” refers to a carrier or adjuvant that may be administered Viral vectors transfect cells directly ; plasmid DNA can be to a patient, together with a compound of this invention , and delivered with the help of, for example , cationic liposomes which does not destroy the pharmacological activity thereof (lipofectin ) or derivatized (e.g. , antibody conjugated ) 55 and is nontoxic when administered in doses sufficient to polylysine conjugates , gramacidin S , artificial viral enve deliver a therapeutic amount of the compound . lopes or other such intracellular earners , as well as direct Pharmaceutically acceptable carriers , adjuvants and injection of the gene construct or CaPO4 precipitation car vehicles that may be used in the pharmaceutical composi ried out in vivo . tions of this invention include, but are not limited to , ion In an embodiment, in vivo introduction of nucleic acid 60 exchangers , alumina , aluminum stearate, lecithin , self -emul into a cell includes use of a viral vector containing nucleic sifying drug delivery systems ( SEDDS) such as d -a - tocoph acid , e.g., a cDNA . Infection of cells with a viral vector has erol polyethyleneglycol 1000 succinate , surfactants used in the advantage that a large proportion of the targeted cells can pharmaceutical dosage forms such as Tweens or other simi receive the nucleic acid . Additionally , molecules encoded lar polymeric delivery matrices , serum proteins, such as within the viral vector, e.g., by a cDNA contained in the viral 65 human serum albumin , buffer substances such as phos vector, are expressed efficiently in cells which have taken up phates , glycine, sorbic acid , potassium sorbate , partial glyc viral vector nucleic acid . eride mixtures of saturated vegetable fatty acids, water, salts US 10,610,125 B2 57 58 or electrolytes , such as protamine sulfate , disodium hydro tion or suspension in a non - toxic parenterally acceptable gen phosphate , potassium hydrogen phosphate , sodium diluent or solvent, for example , as a solution in 1,3 -butane chloride, zinc salts , colloidal silica , magnesium trisilicate , diol. Among the acceptable vehicles and solvents that may polyvinyl pyrrolidone , cellulose -based substances , polyeth be employed are mannitol , water, Ringer's solution and ylene glycol, sodium carboxymethylcellulose , polyacry- 5 isotonic sodium chloride solution . In addition , sterile , fixed lates, waxes , polyethylene -polyoxypropylene -block poly oils are conventionally employed as a solvent or suspending mers, polyethylene glycol and wool fat. Cyclodextrins such medium . For this purpose , any bland fixed oil may be as A-, B-, and y -cyclodextrin , or chemically modified deriva employed including synthetic mono- or diglycerides . Fatty tives such as hydroxyalkylcyclodextrins, including 2- and acids, such as oleic acid and its glyceride derivatives are 3 -hydroxypropyl - ß -cyclodextrins , or other solubilized 10 useful in the preparation of injectables , as are natural phar derivatives may also be advantageously used to enhance maceutically -acceptable oils , such as olive oil or castor oil, delivery of compounds of the formulae described herein . especially in their polyoxyethylated versions . These oil The pharmaceutical compositions containing inhibitors of solutions or suspensions may also contain a long -chain IDH , e.g. , IDH1, may be administered directly to the central alcohol diluent or dispersant, or carboxymethyl cellulose or nervous system , such as into the cerebrospinal fluid or into 15 similar dispersing agents which are commonly used in the the brain . Delivery can be, for example , in a bolus or by formulation of pharmaceutically acceptable dosage forms continuous pump infusion . In certain embodiments , delivery such as emulsions and or suspensions. Other commonly is by intrathecal delivery or by intraventricular injection used surfactants such as Tweens or Spans and /or other directly into the brain . A catheter and , optionally , a pump can similar emulsifying agents or bioavailability enhancers be used for delivery . The inhibitors can be delivered in and 20 which are commonly used in the manufacture of pharma released from an implantable device, e.g. , a device that is ceutically acceptable solid , liquid , or other dosage forms implanted in association with surgical removal of tumor may also be used for the purposes of formulation . tissue. E.g., for delivery to the brain , the delivery can be The pharmaceutical compositions of this invention may analogous to that with Gliadel , a biopolymer wafer designed be orally administered in any orally acceptable dosage form to deliver carmustine directly into the surgical cavity created 25 including, but not limited to , capsules , tablets , emulsions when a brain tumor is resected . The Gliadel wafer slowly and aqueous suspensions, dispersions and solutions. In the dissolves and delivers carmustine. case of tablets for oral use , carriers which are commonly The therapeutics disclosed herein , e.g., nucleic acid based used include lactose and corn starch . Lubricating agents , inhibitors, e.g. siRNAs can be administered directly to the such as magnesium stearate , are also typically added . For CNS , e.g., the brain , e.g., using a pump and /or catheter 30 oral administration in a capsule form , useful diluents include system . In one embodiment, the pump is implanted under lactose and dried corn starch . When aqueous suspensions the skin . In an embodiment and a catheter attached to a pump and /or emulsions are administered orally , the active ingre is inserted into the CNS , e.g., into the brain or spine . In one dient may be suspended or dissolved in an oily phase is embodiment, the pump (such as the Iso Med Drug Pump combined with emulsifying and /or suspending agents . If from Medtronic ) delivers dosing , e.g , constant dosing , of a 35 desired , certain sweetening and / or flavoring and / or coloring nucleic acid based inhibitor. In an embodiment, the pump is agents may be added . programmable to administer variable or constant doses at The pharmaceutical compositions of this invention may predetermined time intervals . For example , the IsoMed Drug also be administered in the form of suppositories for rectal pump from Medtronic (or a similar device ) can be used to administration . These compositions can be prepared by administer a constant supply of the inhibitor, or the Syn- 40 mixing a compound of this invention with a suitable non chroMedII Drug Pump (or a similar device ) can be used to irritating excipient which is solid at room temperature but administer a variable dosing regime. liquid at the rectal temperature and therefore will melt in the Methods and devices described in U.S. Pat. Nos. 7,044 , rectum to release the active components . Such materials 932 , 6,620,151 , 6,283949, and 6,685,452 can be used in include , but are not limited to , cocoa butter, beeswax and methods described herein . 45 polyethylene glycols . The pharmaceutical compositions of this invention may Topical administration of the pharmaceutical composi be administered orally, parenterally , by inhalation , topically, tions of this invention is useful when the desired treatment rectally , nasally , buccally , vaginally or via an implanted involves areas or organs readily accessible by topical appli reservoir, preferably by oral administration or administra cation. For application topically to the skin , the pharmaceu tion by injection . The pharmaceutical compositions of this 50 tical composition should be formulated with a suitable invention may contain any conventional non - toxic pharma ointment containing the active components suspended or ceutically - acceptable carriers , adjuvants or vehicles. In dissolved in a carrier. Carriers for topical administration of some cases, the pH of the formulation may be adjusted with the compounds of this invention include, but are not limited pharmaceutically acceptable acids, bases or buffers to to , mineral oil , liquid petroleum , white petroleum , propylene enhance the stability of the formulated compound or its 55 glycol, polyoxyethylene polyoxypropylene compound , delivery form . The term parenteral as used herein includes emulsifying wax and water . Alternatively , the pharmaceuti subcutaneous , intracutaneous, intravenous, intramuscular, cal composition can be formulated with a suitable lotion or intraarticular, intraarterial, intrasynovial , intrasternal, cream containing the active compound suspended or dis intrathecal , intralesional and intracranial injection or infu solved in a carrier with suitable emulsifying agents . Suitable sion techniques . 60 carriers include, but are not limited to , mineral oil , sorbitan The pharmaceutical compositions may be in the form of monostearate , polysorbate 60 , cetyl esters wax , cetearyl a sterile injectable preparation , for example , as a sterile alcohol, 2 - octyldodecanol, benzyl alcohol and water. The injectable aqueous or oleaginous suspension . This suspen pharmaceutical compositions of this invention may also be sion may be formulated according to techniques known in topically applied to the lower intestinal tract by rectal the art using suitable dispersing or wetting agents ( such as , 65 suppository formulation or in a suitable enema formulation . for example , Tween 80 ) and suspending agents . The sterile Topically - transdermal patches are also included in this injectable preparation may also be a sterile injectable solu invention . US 10,610,125 B2 59 60 The pharmaceutical compositions of this invention may tive , instructional, marketing or other material that relates to be administered by nasal aerosol or inhalation . Such com the methods described herein and / or the use of a compound positions are prepared according to techniques well -known described herein for the methods described herein . in the art of pharmaceutical formulation and may be pre In an embodiment the kit provides materials for evaluat pared as solutions in saline , employing benzyl alcohol or 5 ing a subject. The evaluation can be, e.g., for: identifying a other suitable preservatives , absorption promoters to subject having unwanted levels (e.g. , higher than present in enhance bioavailability , fluorocarbons, and / or other solubi normal or wildtype cells ) of any of 2HG , 2HG neoactivity , lizing or dispersing agents known in the art. ormutant IDH1 or IDH2 protein having 2HG neoactivity ( or When the compositions of this invention comprise a corresponding RNA ) , or having a somatic mutation in IDH1 combination of a compound of the formulae described 10 or IDH2 characterized by 2HG neoactivity ; diagnosing , herein and one or more additional therapeutic or prophylac prognosing , or staging , a subject , e.g., on the basis ofhaving tic agents , both the compound and the additional agent increased levels of 2HG , 2HG neoactivity , or mutant IDH1 should be present at dosage levels of between about 1 to or IDH2 protein having 2HG neoactivity ( or corresponding 100 % , and more preferably between about 5 to 95 % of the RNA ), or having a somatic mutation in IDH1 or IDH2 dosage normally administered in a monotherapy regimen . 15 characterized by 2HG neoactivity ; selecting a treatment for , The additional agents may be administered separately , as or evaluating the efficacy of, a treatment, e.g., on the basis part of a multiple dose regimen , from the compounds of this of the subject having increased levels of 2HG , 2HG neoac invention . Alternatively , those agents may be part of a single tivity , or mutant IDH1 or IDH2 protein having 2HG neo dosage form , mixed together with the compounds of this activity ( or corresponding RNA ) , or having a somatic muta invention in a single composition . 20 tion in IDH1 or IDH2 characterized by 2HG neoactivity . The The compounds described herein can , for example , be kit can include one or more reagent useful in the evaluation , administered by injection , intravenously , intraarterially , sub e.g. , reagents mentioned elsewhere herein . A detection dermally , intraperitoneally , intramuscularly , or subcutane reagent, e.g., an antibody or other specific binding reagent ously ; or orally, buccally , nasally , transmucosally , topically , can be included . Standards or reference samples , e.g., a in an ophthalmic preparation , or by inhalation , with a dosage 25 positive or negative control standard can be included . E.g., ranging from about 0.02 to about 100 mg/ kg of body weight, if the evaluation is based on the presence of 2HG the kit can alternatively dosages between 1 mg and 1000 mg/ dose , include a reagent, e.g , a positive or negative control stan every 4 to 120 hours , or according to the requirements of the dards for an assay , e.g., a LC -MS assay. particular drug. The methods herein contemplate adminis If the evaluation is based on the presence of 2HG neo tration of an effective amount of compound or compound 30 activity , the kit can include a reagent , e.g., one or more of composition to achieve the desired or stated effect. Typi those mentioned elsewhere herein , for assaying 2HG neo cally , the pharmaceutical compositions of this invention will activity . If the evaluation is based on sequencing, the kit can be administered from about 1 about 6 times per day or include primers or other materials useful for sequencing the alternatively , as a continuous infusion . Such administration relevant nucleic acids for identifying an IHD , e.g. , IDH1 or can be used as a chronic or acute therapy. The amount of 35 IDH2 , neoactive mutant . E.g. , the kit can contain a reagent active ingredient that may be combined with the carrier that provides for interrogation of the identity , i.e., sequenc materials to produce a single dosage form will vary depend ing of, residue 132 of IDH1 to determine if a neoactive ing upon the host treated and the particular mode of admin mutant is present. The kit can include nucleic acids, e.g. , an istration . A typical preparation will contain from about 5 % oligomer , e.g., primers, which allow sequencing of of the to about 95 % active compound ( w / w ) . Alternatively , such 40 nucleotides that encode residue 132 of IDH1. In an embodi preparations contain from about 20 % to about 80 % active ment the kit includes a nucleic acid whose hybridization , or compound . ability to be amplified , is dependent on the identity of Lower or higher doses than those recited above may be residue 132 of IDH1 . In other embodiments the kit includes required . Specific dosage and treatment regimens for any a reagent, e.g. , an antibody or other specific binding mol particular patient will depend upon a variety of factors , 45 ecule that can identify the presence of a neoactive mutant, including the activity of the specific compound employed , e.g. , a protein encoded by a neoactive mutant at 132 of the age , body weight, general health status, sex , diet , time of IDH1. As described below , a kit can also include buffers , administration , rate of excretion , drug combination , the solvents , and information related to the evaluation . severity and course of the disease , condition or symptoms, In one embodiment, the informational material can the patient's disposition to the disease , condition or symp- 50 include information about production of the compound , toms, and the judgment of the treating physician . molecular weight of the compound , concentration , date of Upon improvement of a patient's condition , a mainte expiration , batch or production site information , and so nance dose of a compound , composition or combination of forth . In one embodiment, the informationalmaterial relates this invention may be administered , if necessary . Subse to methods for administering the compound . quently , the dosage or frequency of administration , or both , 55 In one embodiment, the informational material can may be reduced , as a function of the symptoms, to a level at include instructions to administer a compound described which the improved condition is retained when the symp herein in a suitable manner to perform the methods toms have been alleviated to the desired level . Patients may , described herein , e.g. , in a suitable dose , dosage form , or however , require intermittent treatment on a long - term basis mode of administration ( e.g. , a dose , dosage form , or mode upon any recurrence of disease symptoms. 60 of administration described herein ). In another embodiment, Kits the informational material can include instructions to admin A compound described herein can be provided in a kit . ister a compound described herein to a suitable subject , e.g., In an embodiment the kit includes ( a ) a compound a human , e.g., a human having or at risk for a disorder described herein , e.g., a composition that includes a com described herein . pound described herein ( wherein , e.g. , the compound can be 65 The informational material of the kits is not limited in its an inhibitor described herein ) , and , optionally (b ) informa form . In many cases, the informational material , e.g. , tional material. The informational material can be descrip instructions , is provided in printed matter, e.g., a printed US 10,610,125 B2 61 62 text, drawing , and / or photograph , e.g., a label or printed Chemotherapy sheet . However, the informational material can also be In some embodiments , a compound or composition provided in other formats , such as Braille , computer read described herein , is administered with a chemotherapy . able material, video recording, or audio recording. In Chemotherapy is the treatment of cancer with drugs that can another embodiment, the informational material of the kit is 5 destroytoxic drugs cancer which cells affect. “ Chemotherapy rapidly dividing” usually cells refersin general to cyto, in contact information, e.g., a physical address , email address , contrast with targeted therapy. Chemotherapy drugs inter website , or telephone number, where a user of the kit can fere with cell division in various possible ways, e.g. ,with the obtain substantive information about a compound described duplication of DNA or the separation of newly formed herein and / or its use in the methods described herein . Of chromosomes. Most forms of chemotherapy target all rap course , the informational material can also be provided in 10 idly dividing cells and are not specific for cancer cells , any combination of formats . although some degree of specificity may come from the In addition to a compound described herein , the compo inability of many cancer cells to repair DNA damage , while normal cells generally can . sition of the kit can include other ingredients , such as a Examples of chemotherapeutic agents used in cancer solvent or buffer , a stabilizer , a preservative, a flavoring 15 therapy include , for example , antimetabolites ( e.g., folic agent ( e.g., a bitter antagonist or a sweetener) , a fragrance or acid , purine , and pyrimidine derivatives ) and alkylating other cosmetic ingredient , and / or a second agent for treating agents (e.g. , nitrogen mustards, nitrosoureas, platinum , alkyl a condition or disorder described herein . Alternatively , the sulfonates , hydrazines, triazenes, aziridines , spindle poison , other ingredients can be included in the kit, but in different cytotoxic agents , toposimeraerase inhibitors and others ) . Exem compositions or containers than a compound described 20 plary agents include Aclarubicin , Actinomycin , Alitretinon , herein . In such embodiments , the kit can include instructions Altretamine, Aminopterin , Aminolevulinic acid , Amrubicin , for admixing a compound described herein and the other Amsacrine , Anagrelide, Arsenic trioxide , Asparaginase , ingredients, or for using a compound described herein Atrasentan , Belotecan , Bexarotene, endamustine , Bleomy together with the other ingredients . cin , Bortezomib , Busulfan , Camptothecin , Capecitabine, A compound described herein can be provided in any 25 Carboplatin , Carboquone , Carmofur , Carmustine , Cele form , e.g., liquid , dried or lyophilized form . It is preferred coxib , Chlorambucil , Chlormethine, Cisplatin , Cladribine , that a compound described herein be substantially pure Clofarabine, Crisantaspase, Cyclophosphamide, Cytarabine , and /or sterile . When a compound described herein is pro Dacarbazine , Dactinomycin , Daunorubicin , Decitabine , vided in a liquid solution , the liquid solution preferably is an Demecolcine, Docetaxel , Doxorubicin , Efaproxiral, Eles aqueous solution , with a sterile aqueous solution being 30 clomol, Elsamitrucin , Enocitabine , Epirubicin , Estramus preferred . When a compound described herein is provided as tine, Etoglucid , Etoposide, Floxuridine, Fludarabine , Fluo a dried form , reconstitution generally is by the addition of a rouracil (5FU ) , Fotemustine , Gemcitabine, Gliadel implants , suitable solvent. The solvent, e.g. , sterile water or buffer, can Hydroxycarbamide, Hydroxyurea , Idarubicin , Ifosfamide , optionally be provided in the kit . Irinotecan , Irofulven , Ixabepilone, Larotaxel , Leucovorin , The kit can include one or more containers for the 35 Liposomal doxorubicin , Liposomal daunorubicin , composition containing a compound described herein . In Lonidamine , Lomustine, Lucanthone, Mannosulfan , Maso some embodiments , the kit contains separate containers, procol, Melphalan , Mercaptopurine, Mesna , Methotrexate, dividers or compartments for the composition and informa Methyl aminolevulinate , Mitobronitol, Mitoguazone, Mito tional material. For example, the composition can be con tane , Mitomycin , Mitoxantrone , Nedaplatin , Nimustine , tained in a bottle , vial , or syringe, and the informational 40 Oblimersen , Omacetaxine , Ortataxel , Oxaliplatin , Pacli material can be contained in a plastic sleeve or packet. In taxel, Pegaspargase , Pemetrexed , Pentostatin , Pirarubicin , other embodiments , the separate elements of the kit are Pixantrone, Plicamycin , Porfimer sodium , Prednimustine, contained within a single , undivided container. For example , Procarbazine, Raltitrexed , Ranimustine , Rubitecan , Sapac the composition is contained in a bottle , vial or syringe that itabine , Semustine , Sitimagene ceradenovec, Strataplatin , has attached thereto the informationalmaterial in the form of 45 Streptozocin , Talaporfin , Tegafur -uracil , Temoporfin , Temo a label. In some embodiments , the kit includes a plurality zolomide , Teniposide , Tesetaxel, Testolactone , Tetranitrate , ( e.g., a pack ) of individual containers , each containing one Thiotepa , Tiazofurine , Tioguanine , Tipifarnib , Topotecan , or more unit dosage forms ( e.g., a dosage form described Trabectedin , Triaziquone, Triethylenemelamine , Triplatin , herein ) of a compound described herein . For example , the Tretinoin , Treosulfan , Trofosfamide, Uramustine , Valrubi kit includes a plurality of syringes , ampules , foil packets , or 50 cin , Verteporfin , Vinblastine, Vincristine , Vindesine , Vinflu blister packs , each containing a single unit dose of a com nine , Vinorelbine, Vorinostat , Zorubicin , and other cytostatic pound described herein . The containers of the kits can be air or cytotoxic agents described herein . tight, waterproof (e.g. , impermeable to changes in moisture Because some drugs work better together than alone, two or evaporation ) , and /or light- tight. or more drugs are often given at the same time. Often , two The kit optionally includes a device suitable for admin- 55 or more chemotherapy agents are used as combination istration of the composition , e.g., a syringe , inhalant , pipette , chemotherapy . In some embodiments , the chemotherapy forceps, measured spoon , dropper ( e.g., eye dropper ), swab agents ( including combination chemotherapy ) can be used in ( e.g. , a cotton swab or wooden swab ) , or any such delivery combination with a compound described herein , e.g. , phen device . In an embodiment, the device is a medical implant formin . device, e.g., packaged for surgical insertion . 60 Targeted Therapy Combination Therapies In some embodiments , a compound or composition In some embodiments , a compound or composition described herein , is administered with a targeted therapy . described herein , is administered together with an additional Targeted therapy constitutes the use of agents specific for the cancer treatment. Exemplary cancer treatments include , for deregulated proteins of cancer cells. Small molecule tar example : surgery , chemotherapy, targeted therapies such as 65 geted therapy drugs are generally inhibitors of enzymatic antibody therapies , immunotherapy , and hormonal therapy . domains on mutated , overexpressed , or otherwise critical Examples of each of these treatments are provided below . proteins within the cancer cell . Prominent examples are the US 10,610,125 B2 63 64 tyrosine kinase inhibitors such as Axitinib , Bosutinib , Cedi Temozolomide is an orally active alkylating agent that is ranib , desatinib , erlotinib , imatinib , gefitinib , lapatinib , Les used for persons newly diagnosed with glioblastoma multi taurtinib , Nilotinib , Semaxanib , Sorafenib , Sunitinib , and forme . It was approved by the United States Food and Drug Vandetanib , and also cyclin -depdendent kinase inhibitors Administration (FDA ) in March 2005. Studies have shown such as Alvocidib and Seliciclib . Monoclonal antibody 5 that the drug was well tolerated and provided a survival therapy is another strategy in which the therapeutic agent is benefit . Adjuvant and concomitant temozolomide with an antibody which specifically binds to a protein on the radiation was associated with significant improvements in surface of the cancer cells . Examples include the anti -HER2 / median progression - free survival over radiation alone (6.9 vs 5 mo ) , overall survival ( 14.6 vs 12.1 mo ), and the neuin breast antibody cancer trastuzumab , and the anti (HERCEPTIN? -CD20 antibody ) typicallyrituximab used and 10 likelihood of being alive in 2 years ( 26 % vs 10 % ). Tositumomab typically used in a variety of B -cell malig Nitrosoureas : BCNU (carmustine )-polymer wafers (Glia nancies. Other exemplary antibodies include Cetuximab , del ) were approved by the FDA in 2002. Though Gliadel Panitumumab , Trastuzumab , Alemtuzumab , Bevacizumab , wafers are used by some for initial treatment, they have Edrecolomab, and Gemtuzumab . Exemplary fusion proteins 15 placeboshown only(13.8 a vs.modest 11.6 months increase ) in inthe median largest suchsurvival phase over III include Aflibercept and Denileukin diftitox . In some trial , and are associated with increased rates of CSF leak and embodiments , the targeted therapy can be used in combina increased intracranial pressure secondary to edema and mass tion with a compound described herein , e.g., a biguanide effect. such as metformin or phenformin , preferably phenformin . MGMT is a DNA repair enzyme that contributes to Targeted therapy can also involve small peptides as “ hom- 20 temozolomide resistance. Methylation of the MGMT pro ing devices” which can bind to cell surface receptors or moter, found in approximately 45 % of glioblastoma multi affected extracellular matrix surrounding the tumor. Radio formes , results in an epigenetic silencing of the gene , nuclides which are attached to these peptides ( e.g., RGDs ) decreasing the tumor cell's capacity for DNA repair and eventually kill the cancer cell if the nuclide decays in the increasing susceptibility to temozolomide . vicinity of the cell. An example of such therapy includes 25 When patients with and without MGMT promoter meth BEXXAR® . ylation were treated with temozolomide, the groups had Immunotherapy median survivals of 21.7 versus 12.7 months, and 2 - year In some embodiments , a compound or composition survival rates of 46 % versus 13.8 % , respectively . described herein , is administered with an immunotherapy . Though temozolomide is currently a first - line agent in the Cancer immunotherapy refers to a diverse set of therapeutic 30 treatment of glioblastoma multiforme, unfavorable MGMT strategies designed to induce the patient's own immune methylation status could help select patients appropriate for system to fight the tumor. Contemporary methods for gen future therapeutic investigations. erating an immune response against tumors include intra 06 -benzylguanine and other inhibitors of MGMT as well vesicular BCG immunotherapy for superficial bladder can as RNA interference -mediated silencing of MGMT offer cer, and use of interferons and other cytokines to induce an 35 promising avenues to increase the effectiveness of temozo immune response in renal cell carcinoma and melanoma lomide and other alkylating antineoplastics, and such agents patients . are under active study . Allogeneic hematopoietic stem cell transplantation can be Carmustine (BCNU ) and cis -platinum ( cisplatin ) have considered a form of immunotherapy, since the donor's been the primary chemotherapeutic agents used against immune cells will often attack the tumor in a graft- versus- 40 malignant gliomas . All agents in use have no greater than a tumor effect . In some embodiments , the immunotherapy 30-40 % response rate , and most fall into the range of agents can be used in combination with a compound or 10-20 % . composition described herein . Data from the University of California at San Francisco Hormonal Therapy indicate that, for the treatment of glioblastomas, surgery In some embodiments , a compound or composition 45 followed by radiation therapy leads to 1-, 3- , and 5- year described herein , is administered with a hormonal therapy . survival rates of 44 % , 6 % , and 0 % , respectively . By com The growth of some cancers can be inhibited by providing parison , surgery followed by radiation and chemotherapy or blocking certain hormones . Common examples of hor using nitrosourea -based regimens resulted in 1- , 3-, and mone- sensitive tumors include certain types of breast and 5 -year survival rates of 46 % , 18 % , and 18 % , respectively . prostate cancers . Removing or blocking estrogen or testos- 50 A major hindrance to the use of chemotherapeutic agents terone is often an important additional treatment. In certain for brain tumors is the fact that the blood -brain barrier cancers , administration of hormone agonists , such as pro (BBB ) effectively excludes many agents from the CNS . For gestogens may be therapeutically beneficial . In some this reason , novelmethods of intracranial drug delivery are embodiments , the hormonal therapy agents can be used in being developed to deliver higher concentrations of chemo combination with a compound or a composition described 55 therapeutic agents to the tumor cells while avoiding the herein . adverse systemic effects of these medications. In some embodiments , a compound or composition Pressure -driven infusion of chemotherapeutic agents described herein , is administered together with an additional through an intracranial catheter , also known as convection cancer treatment (e.g. , surgical removal) , in treating cancer enhanced delivery (CED ) , has the advantage of delivering in nervous system , e.g., cancer in central nervous system , 60 drugs along a pressure gradient rather than by simple e.g., brain tumor , e.g., glioma, e.g. , glioblastoma multiforme diffusion . CED has shown promising results in animal (GBM ) . models with agents including BCNU and topotecan . Several studies have suggested that more than 25 % of Initial attempts investigated the delivery of chemothera glioblastoma patients obtain a significant survival benefit peutic agents via an intraarterial route rather than intrave from adjuvant chemotherapy . Meta -analyses have suggested 65 nously . Unfortunately , no survival advantage was observed . that adjuvant chemotherapy results in a 6-10 % increase in Chemotherapy for recurrent glioblastoma multiforme pro 1 -year survival rate . vides modest, if any , benefit , and several classes of agents US 10,610,125 B2 65 66 are used . Carmustine wafers increased 6 -month survival nasal drainage or phlegm ), pleural fluid , pus, saliva , sebum , from 36 % to 56 % over placebo in one randomized study of semen , serum , sweat , tears, urine , vaginal secretion , or 222 patients , though there was a significant association vomit . between the treatment group and serious intracranial infec In some embodiments , a subject can be evaluated for tions . 5 neoactivity of an enzyme using magnetic resonance. For Genotyping of brain tumors may have applications in example , where themutant enzyme is IDH1 or IDH2 and the stratifying patients for clinical trials of various novel thera neoactivity is conversion of a -ketoglutarate to 2 -hydroxy pies. glutarate , the subject can be evaluated for the presence of The anti -angiogenic agent bevacizumab , when used with and / or an elevated amount of 2 - hydroxyglutarate , e.g., R - 2 irinotecan improved 6 -month survival in recurrent glioma 10 hydroxyglutarate relative to the amount of 2 -hydroxyglut patients to 46 % compared with 21 % in patients treated with arate , e.g., R - 2 -hydroxyglutarate present in a subject who temozolomide . This bevacizumab and irinotecan combina does nothave a mutation in IDH1 or IDH2 having the above tion for recurrent glioblastoma multiforme has been shown neoactivity . In some embodiments, neoactivity of IDH1 or to improve survival over bevacizumab alone. Anti- angio- 15 IDH2of a peak can becorresponding determined by to the 2- hydroxyglutaratepresence or elevated , e.g. amount, R -2 genic agents also decrease peritumoral edema, potentially hydroxyglutarate as determined by magnetic resonance. For reducing the necessary corticosteroid dose . example , a subject can be evaluated for the presence and / or Some glioblastomas responds to gefitinib or erlotinib strength of a signal at about 2.5 ppm to determine the ( tyrosine kinase inhibitors ). The simultaneous presence in presence and / or amount of 2 -hydroxyglutarate , e.g., R - 2 glioblastoma cells of mutant EGFR (EGFRviii ) and PTEN 20 hydroxyglutarate in the subject. This can be correlated to was associated with responsiveness to tyrosine kinase and /or predictive of a neoactivity described herein for the inhibitors , whereas increased p -akt predicts a decreased mutant enzyme IDH . Similarly , the presence , strength and /or effect. Other targets include PDGFR , VEGFR , mTOR , absence of a signal at about 2.5 ppm could be predictive of farnesyltransferase , and PI3K . a response to treatment and thereby used as a noninvasive Other possible therapy modalities include imatinib , gene 25 biomarker for clinical response . therapy, peptide and dendritic cell vaccines, synthetic chlo Neoactivity of a mutant enzyme such as IDH can also be rotoxins, and radiolabeled drugs and antibodies. evaluated using other techniques known to one skilled in the Patient Selection /Monitoring art. For example , the presence or amount of a labeled Described herein are methods of treating a cell prolifera substrate , cofactor, and /or reaction product can be measured tion -related disorder , e.g., cancer, in a subject and methods 30 reactionsuch as producta 13C .or The 14C neoactivity labeled substratecan be evaluated , cofactor by, andevalu/ or of identifying a subject for a treatment described herein . ating the forward reaction of the wild -type / non mutant Also described herein are methods of predicting a subject enzyme (such as the oxidative decarboxylation of ioscitrate who is at risk of developing cancer (e.g. , a cancer associate to a -ketoglutarate in a mutant IDH1 or IDH2 enzyme, with a mutation in an enzyme ( e.g. , an enzyme in the 35 specifically a mutant IDH1 enzyme) and /or the reaction metabolic pathway such as IDH1 and /or IDH2) ). The cancer corresponding to the neoactivity ( e.g. , the conversion of is generally characterized by the presence of a neoactivity, a -ketoglutarate to 2 -hydroxyglutarate , e.g., R - 2 -hydroxy such as a gain of function in one or more mutant enzymes glutarate in a mutant IDH1 or IDH2 enzyme, specifically a ( e.g. , an enzyme in the metabolic pathway leading to fatty mutant IDH1 enzyme) . acid biosynthesis, glycolysis , glutaminolysis , the pentose 40 Disorders phosphate shunt, the nucleotide biosynthetic pathway , or the The IDH - related methods disclosed herein , e.g., methods fatty acid biosynthetic pathway , e.g., IDH1 or IDH2 ). The of evaluating or treating subjects , are directed to subjects subject can be selected on the basis of the subject having a having a cell proliferation - related disorder characterized by mutant gene having a neoactivity , e.g., a neoactivity an IDH mutant, e.g., an IDH1 or IDH2, mutant having described herein . As used herein , “ select” means selecting in 45 neoactivity , e.g. , 2HG neoactivity . Examples of some of the whole or part on said basis . disorders below have been shown to be characterized by an In some embodiments , a subject is selected for treatment IDH1 or IDH2 mutation . Others can be analyzed , e.g. , by with a compound described herein based on a determination sequencing cell samples to determine the presence of a that the subject has a mutant enzyme described herein ( e.g., somatic mutation at amino acid 132 of IDH1 or at amino an enzyme in the metabolic pathway , e.g., a metabolic 50 acid 172 of IDH2 . Without being bound by theory it is pathway leading to fatty acid biosynthesis , glycolysis , glu expected that a portion of the tumors of given type of cancer taminolysis , the pentose phosphate shunt, the nucleotide will have an IDH , e.g., IDH1 or IDH2, mutant having 2HG biosynthetic pathway, or the fatty acid biosynthetic pathway, neoactivity. e.g., IDH1 or IDH2) . In some embodiments , the mutant The disclosed methods are useful in evaluating or treating enzyme has a neoactivity and the patient is selected on that 55 proliferative disorders, e.g. evaluating or treating solid basis . The neoactivity of the enzyme can be identified , for tumors , soft tissue tumors , and metastases thereof wherein example , by evaluating the subject or sample ( e.g. , tissue or the solid tumor, soft tissue tumor or metastases thereof is a bodily fluid ) therefrom , for the presence or amount of a cancer described herein . Exemplary solid tumors include substrate , cofactor and /or product of the enzyme. The pres malignancies ( e.g., sarcomas , adenocarcinomas, and carci ence and /or amount of substrate , cofactor and /or product can 60 nomas ) of the various organ systems, such as those of brain , correspond to the wild - type/ non -mutant activity or can cor lung , breast, lymphoid , gastrointestinal ( e.g., colon ), and respond to the neoactivity of the enzyme. Exemplary bodily genitourinary ( e.g. , renal, urothelial, or testicular tumors ) fluid that can be used to identify (e.g. , evaluate ) the neoac tracts , pharynx , prostate , and ovary. Exemplary adenocarci tivity of the enzyme include amniotic fluid surrounding a nomas include colorectal cancers , renal- cell carcinoma, liver fetus, aqueous humour , blood ( e.g., blood plasma) , Cere- 65 cancer, non - small cell carcinoma of the lung , and cancer of brospinal fluid , cerumen , chyme, Cowper's fluid , female the small intestine . The disclosed methods are also useful in ejaculate , interstitial fluid , lymph , breast milk , mucus (e.g. , evaluating or treating non - solid cancers . US 10,610,125 B2 67 68 The methods described herein can be used with any Hodgkin's , Adult ; Lymphoma, Non - Hodgkin's , Childhood ; cancer , for example those described by the National Cancer Lymphoma, Non -Hodgkin's During Pregnancy ; Lym Institute . A cancer can be evaluated to determine whether it phoma, Primary Central Nervous System ; Macroglobuline is using a method described herein . Exemplary cancers mia , Waldenstrom’s ; Male Breast Cancer ; MalignantMeso described by the National Cancer Institute include: Acute 5 thelioma, Adult; Malignant Mesothelioma, Childhood ; Lymphoblastic Leukemia , Adult ; Acute Lymphoblastic Leu Malignant Thymoma; Medulloblastoma, Childhood ; Mela kemia , Childhood ; Acute Myeloid Leukemia , Adult ; Adre noma; Melanoma, Intraocular; Merkel Cell Carcinoma ; nocortical Carcinoma; Adrenocortical Carcinoma, Child hood ; AIDS -Related Lymphoma; AIDS -Related Mesothelioma, Malignant; Metastatic Squamous Neck Can Malignancies ; Anal Cancer ; Astrocytoma, Childhood Cer- 10 cer with Occult Primary ; Multiple Endocrine Neoplasia ebellar ; Astrocytoma, Childhood Cerebral ; Bile Duct Can Syndrome, Childhood ;Multiple Myeloma/ Plasma CellNeo cer , Extrahepatic ; Bladder Cancer; Bladder Cancer, Child plasm ; Mycosis Fungoides ; Myelodysplastic Syndromes ; hood ; Bone Cancer, Osteosarcoma/ Malignant Fibrous Myelogenous Leukemia , Chronic ; Myeloid Leukemia , Histiocytoma; Brain Stem Glioma , Childhood ; Brain Tumor , Childhood Acute ; Myeloma, Multiple ; Myeloproliferative Adult; Brain Tumor, Brain Stem Glioma, Childhood ; Brain 15 Disorders, Chronic ; Nasal Cavity and Paranasal Sinus Can Tumor, Cerebellar Astrocytoma, Childhood ; Brain Tumor, cer ; Nasopharyngeal Cancer; Nasopharyngeal Cancer , Cerebral Astrocytoma/ Malignant Glioma, Childhood ; Brain Childhood ; Neuroblastoma ; Non -Hodgkin's Lymphoma, Tumor, Ependymoma, Childhood ; Brain Tumor, Medullo Adult; Non - Hodgkin's Lymphoma, Childhood ; Non - Hodg blastoma, Childhood ; Brain Tumor, Supratentorial Primitive kin's Lymphoma During Pregnancy ; Non -Small Cell Lung Neuroectodermal Tumors , Childhood ; Brain Tumor, Visual 20 Cancer ; Oral Cancer , Childhood ; Oral Cavity and Lip Can Pathway and Hypothalamic Glioma, Childhood ; Brain cer ; Oropharyngeal Cancer ; Osteosarcoma/Malignant Tumor, Childhood (Other ) ; Breast Cancer ; Breast Cancer Fibrous Histiocytoma of Bone ; Ovarian Cancer, Childhood ; and Pregnancy ; Breast Cancer , Childhood ; Breast Cancer , Ovarian Epithelial Cancer ; Ovarian Germ Cell Tumor; Male ; Bronchial Adenomas /Carcinoids , Childhood ; Carci Ovarian Low Malignant Potential Tumor; Pancreatic Can noid Tumor, Childhood ; Carcinoid Tumor, Gastrointestinal; 25 cer; Pancreatic Cancer, Childhood ; Pancreatic Cancer, Islet Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carci Cell ; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid noma of Unknown Primaiy; Central Nervous System Lym Cancer ; Penile Cancer ; Pheochromocytoma; Pineal and phoma, Primary ; Cerebellar Astrocytoma, Childhood ; Cere Supratentorial Primitive Neuroectodermal Tumors, Child bral Astrocytoma/ Malignant Glioma, Childhood ; Cervical hood ; Pituitary Tumor; Plasma Cell Neoplasm /Multiple Cancer ; Childhood Cancers ; Chronic Lymphocytic Leuke- 30 Myeloma ; Pleuropulmonary Blastoma; Pregnancy and mia ; Chronic Myelogenous Leukemia ; Chronic Myelopro Breast Cancer; Pregnancy and Hodgkin's Lymphoma; Preg liferative Disorders ; Clear Cell Sarcoma of Tendon Sheaths ; nancy and Non -Hodgkin's Lymphoma; Primary Central Colon Cancer ; Colorectal Cancer , Childhood ; Cutaneous Nervous System Lymphoma; Primary Liver Cancer , Adult ; T - Cell Lymphoma; Endometrial Cancer; Ependymoma, Primary Liver Cancer , Childhood ; Prostate Cancer; Rectal Childhood ; Epithelial Cancer, Ovarian ; Esophageal Cancer ; 35 Cancer ; Renal Cell (Kidney ) Cancer; Renal Cell Cancer , Esophageal Cancer , Childhood ; Ewing's Family of Tumors ; Childhood ; Renal Pelvis and Ureter , Transitional Cell Can Extracranial Germ Cell Tumor , Childhood ; Extragonadal cer ; Retinoblastoma; Rhabdomyosarcoma, Childhood ; Sali Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye vary Gland Cancer; Salivary Gland Cancer , Childhood ; Cancer, Intraocular Melanoma; Eye Cancer, Retinoblas Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's ; toma; Gallbladder Cancer , Gastric (Stomach ) Cancer; Gas- 40 Sarcoma (Osteosarcoma ) / Malignant Fibrous Histiocytoma tric ( Stomach ) Cancer , Childhood ; Gastrointestinal Carci of Bone ; Sarcoma, Rhabdomyosarcoma, Childhood ; Sar noid Tumor; Germ Cell Tumor, Extracranial , Childhood ; coma, Soft Tissue , Adult; Sarcoma, Soft Tissue , Childhood ; Germ Cell Tumor , Extragonadal; Germ Cell Tumor, Ovar Sezary Syndrome; Skin Cancer ; Skin Cancer , Childhood ; ian ; Gestational Trophoblastic Tumor; Glioma, Childhood Skin Cancer (Melanoma ) ; Skin Carcinoma, Merkel Cell ; Brain Stem ; Glioma , Childhood Visual Pathway and Hypo- 45 Small Cell Lung Cancer; Small Intestine Cancer ; Soft Tissue thalamic ; Hairy Cell Leukemia ; Head and Neck Cancer ; Sarcoma, Adult ; Soft Tissue Sarcoma, Childhood ; Hepatocellular ( Liver ) Cancer, Adult ( Primary ) ; Hepatocel Squamous Neck Cancer with Occult Primary , Metastatic ; lular (Liver ) Cancer, Childhood (Primary ) ; Hodgkin's Lym Stomach (Gastric ) Cancer ; Stomach (Gastric ) Cancer , phoma, Adult; Hodgkin's Lymphoma, Childhood ; Hodg Childhood ; Supratentorial Primitive Neuroectodermal kin's Lymphoma During Pregnancy; Hypopharyngeal 50 Tumors, Childhood ; T- Cell Lymphoma, Cutaneous ; Testicu Cancer ; Hypothalamic and Visual Pathway Glioma, Child lar Cancer ; Thymoma, Childhood ; Thymoma, Malignant ; hood ; Intraocular Melanoma; Islet Cell Carcinoma ( Endo Thyroid Cancer; Thyroid Cancer, Childhood ; Transitional crine Pancreas ); Kaposi's Sarcoma; Kidney Cancer ; Laryn Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic geal Cancer; Laryngeal Cancer, Childhood ; Leukemia , Tumor, Gestational; Unknown Primary Site , Cancer of, Acute Lymphoblastic , Adult; Leukemia , Acute Lymphoblas- 55 Childhood ; Unusual Cancers of Childhood ; Ureter and tic , Childhood ; Leukemia , Acute Myeloid , Adult; Leukemia , Renal Pelvis , Transitional Cell Cancer ; Urethral Cancer ; Acute Myeloid , Childhood ; Leukemia , Chronic Lympho Uterine Sarcoma; Vaginal Cancer ; Visual Pathway and cytic ; Leukemia , Chronic Myelogenous ; Leukemia , Hairy Hypothalamic Glioma, Childhood ; Vulvar Cancer ; Walden Cell ; Lip and Oral Cavity Cancer; Liver Cancer , Adult strom's Macro globulinemia ; and Wilms' Tumor. Metastases (Primary ); Liver Cancer , Childhood (Primary ) ; Lung Can- 60 of the aforementioned cancers can also be treated or pre cer, Non - Small Cell ;Lung Cancer, Small Cell; Lymphoblas vented in accordance with the methods described herein . tic Leukemia , Adult Acute ; Lymphoblastic Leukemia , The methods described herein are useful in treating cancer Childhood Acute ; Lymphocytic Leukemia , Chronic ; Lym in nervous system , e.g., brain tumor, e.g. , glioma, e.g. , phoma, AIDS - Related ; Lymphoma, Central Nervous System glioblastoma multiforme (GBM ) , e.g. , by inhibiting a neo ( Primary ) ; Lymphoma, Cutaneous T- Cell; Lymphoma, 65 activity of a mutant enzyme, e.g. , an enzyme in a metabolic Hodgkin's , Adult; Lymphoma, Hodgkin's , Childhood ; Lym pathway, e.g., a metabolic pathway leading to fatty acid phoma, Hodgkin's During Pregnancy ; Lymphoma, Non biosynthesis , glycolysis , glutaminolysis , the pentose phos US 10,610,125 B2 69 70 phate shunt, the nucleotide biosynthetic pathway, or the fatty and creating a C - terminal His8 tag (SEQ ID NO : 3 ) without acid biosynthetic pathway, e.g., IDH1 or IDH2. the N - terminal GST fusion . The original stop codon of IDH1 Gliomas , a type ofbrain tumors , can be classified as grade is change to serine , so the junction sequence in final IDH1 I to grade IV on the basis of histopathological and clinical protein is : Ser- Leu -Glu -His -His -His -His -His -His -His -His criteria established by the World Health Organization 5 Stop (SEQ ID NO : 4 ). (WHO ). WHO grade I gliomas are often considered benign . The C - terminal His tag strategy instead of N - terminal His Gliomas of WHO grade II or III are invasive, progress to tag strategy was chosen , because C - terminal tag might not higher- grade lesions. WHO grade IV tumors ( glioblastomas ) negatively impact IDH1 protein folding or activity . See , e.g., are themost invasive form . Exemplary brain tumors include , Xu X et al , J Biol Chem . 2004 Aug. 6 ; 279( 32 ): 33946-57 . e.g. , astrocytic tumor ( e.g. , pilocytic astrocytoma, sub- 10 The sequence for pET41a - IDH1 plasmid is confirmed by ependymal giant -cell astrocytoma, diffuse astrocytoma, DNA sequencing . FIG . 1 shows detailed sequence verifica pleomorphic xanthoastrocytoma, anaplastic astrocytoma, tion of pET41a - IDH1 and alignmentagainst published IDH1 astrocytoma, giant cell glioblastoma, glioblastoma, second CDS below . ary glioblastoma, primary adult glioblastoma , and primary 2. IDH1 Site Directed Mutagenesis to Create the IDHr132s pediatric glioblastoma) ; oligodendroglial tumor ( e.g., oligo- 15 and IDHr132h Mutants . dendroglioma, and anaplastic oligodendroglioma ); oligoas Site directed mutagenesis was performed to convert R132 trocytic tumor ( e.g., oligoastrocytoma , and anaplastic oli to S or H , DNA sequencing confirmed that G395 is mutated goastrocytoma) ; ependymoma ( e.g. , myxopapillary to A ( creating Arg-> His mutation in the IDH1 protein ) , and ependymoma, and anaplastic ependymoma) ; medulloblas C394 is mutated to A (creating Arg-> Ser in the IDH1 toma; primitive neuroectodermal tumor , schwannoma, men- 20 protein ) . Detailed method for site directed mutagenesis is ingioma, meatypical meningioma, anaplastic meningioma; described in the user manual for QuikChange® MultiSite and pituitary adenoma. Exemplary cancers are described in Directed Mutagenesis Kit ( Stratagene , cat # 200531) . FIG . 2 Acta Neuropathol (2008 ) 116 :597-602 and N Engl J Med . shows DNA sequence verification of such mutations. High 2009 Feb. 19 ; 360 ( 8 ) :765-73 , the contents of which are each lighted nucleotides were successfully changed in the muta incorporated herein by reference . 25 genesis : G395A mutation allows amino acid In embodiments the disorder is glioblastoma. Arg132 > His ; C394 > A mutation allows amino acid In an embodiment the disorder is prostate cancer, e.g., Arg132 -- Ser. stage T1 ( e.g., Tla , Tlb and Tic ) , T2 ( e.g. , T2a , T2b and 3. IDH1 Protein Expression and Purification . T2c ) , T3 ( e.g., T3a and T3b ) and T4 , on the TNM staging IDHwt, IDHR132S , and IDHR132H proteins were system . In embodiments the prostate cancer is grade G1, G2 , 30 expressed in the E. coli strain Rosetta and purified according G3 or G4 ( where a higher number indicates greater differ to the detailed procedure below . Active IDH1proteins are in ence from normal tissue ). Types of prostate cancer include, dimer form , and SEC column fraction / peak that correspond e.g. , prostate adenocarcinoma, small cell carcinoma, to the dimer form were collected for enzymology analysis squamous carcinoma, sarcomas, and transitional cell carci and cross comparison of catalytic activities of these proteins. noma. 35 A. Cell Culturing : Methods and compositions of the invention can be com Cells were grown in LB ( 20 ug/ ml Kanamycin ) at 37 ° C. bined with art- known treatment. Art -known treatment for with shaking until OD600 reaches 0.6 . The temperature was prostate cancer can include , e.g., active surveillance, surgery changed to 18 ° C. and protein was induced by adding IPTG (e.g. , radical prostatectomy, transurethral resection of the to final concentration of 1 mM . Cells were collected 12-16 prostate , orchiectomy, and cryosurgegry ), radiation therapy 40 hours after IPTG induction . including brachytherapy ( prostate brachytherapy ) and exter B. Buffer System : nal beam radiation therapy, High - Intensity Focused Ultra Lysis buffer : 20 mM Tris , pH7.4 , 0.1 % Triton X -100 , 500 sound ( HIFU ) , chemotherapy, cryosurgery , hormonal mM NaCl, 1 mM PMSF, 5 mM B -mercaptoethanol , 10 % therapy ( e.g., antiandrogens ( e.g., flutamide , bicalutamide, glycerol. nilutamide and cyproterone acetate , ketoconazole , amino- 45 Ni- Column Buffer A : 20 mM Tris , pH7.4 , 500 mM NaCl, glutethimide) , GnRH antagonists (e.g. , Abarelix )) , or a 5 mM B -mercaptoethanol , 10 % glycerol. combination thereof . Ni- column Buffer B : 20 mM Tris , pH7.4 , 500 mM NaCl, All references described herein are expressly incorporated 5 mM B -mercaptoethanol , 500 mM Imidazole , 10 % glycerol herein by reference . Gel filtration Buffer C : 200 mM NaCl, 50 mM Tris 7.5 , 5 50 mM B -mercaptoethanol , 2 mM MnSO4, 10 % glycerol . EXAMPLES C. Protein Purification Procedure 1. Cell pellet were resuspended in the lysis buffer ( 1 gram Example 1 IDH1 Cloning , Mutagenesis , Expression cel1/ 5-10 mlbuffer ). and Purification 2. Cells were broken by passing the cell through Micro 55 fludizer with at a pressure of 15,000 psi for 3 times . I. Wild Type IDH1 was Cloned into pET41a , Creating His8 3. Soluble protein was collected from supernatant after Tag at C - Terminus. centrifugation at 20,000 g (Beckman Avanti J - 26XP ) for 30 The IDH1 gene coding region (cDNA ) was purchased min at 4 ° C. from Invitrogen in PENTR221 vector (www.invitrogen 4. 5-10 mlof Ni- column was equilibrated by Buffer A until .com , Cat # B -068487_Ultimate_ORF ) . Oligo nucleotides 60 the A280 value reached baseline . The supernatant was were designed to PCR out the coding region of IDH1 with loaded onto a 5 -ml Ni- Sepharose column ( 2 ml/min ). The Ndel at the 5 ' end and Xhol at the 3 '. ( IDH1- f : TAATCATAT column was washed by 10-20 CV of washing buffer (90 % GTCCAAAAAAATCAGT (SEQ ID NO : 1 ) , IDH1- r : buffer A + 10 % buffer B ) until A280 reach the baseline ( 2 TAATCTCGAGTGAAAGTTTGGCCTGAGCTAGTT ml/min ). (SEQ ID NO : 2 ) ) . The PCR product is cloned into the 65 5. The protein was eluted by liner gradient of 10-100 % Ndel /Xhol cleaved PET41a vector. Ndel/ Xhol cleavage of buffer B (20 CV ) with the flow rate of 2 ml/min and the the vector pET41a releases the GST portion of the plasmid , sample fractions were collected as 2 ml/ tube . US 10,610,125 B2 71 72 6. The samples were analyzed on SDS -PAGE gel . C. The ICDH1R132H and R132S Mutants Display Reduced 7. The samples were collected and dialyzed against 200x Product Inhibition in the Oxidative Decarboxylation of Gel filtration buffer for 2 times ( 1 hour and > 4 hours ) . Isocitrate to a -ketoglutarate ( a - KG ) . 8. The samples were concentrated to 10 ml. A well- known regulatory mechanism for control of meta 9. 200 ml of S - 200 Gel - filtration column was equilibrated by 5 bolic enzymes is feedback inhibition , in which the product buffer C until the A280 value reached baseline . The samples of the reaction acts as a negative regulator for the generating were loaded onto Gel filtration column (0.5 ml/min ). enzyme. To examine whether the R132S or R132H mutants 10. The column was washed by 10 CV of buffer C , collect maintain this regulatory mechanism , the Ki for a -KG in the fractions as 2-4 ml/ tube . oxidative decarboxylation of ioscitrate to a -ketoglutarate 11. The samples were analyzed on SDS -PAGE gel and 10 was determined . Data is presented in FIGS . 13A - 13C and protein concentration was determined . summarized in Table 2. In all cases , a - KG acts as a D. Protein Purification Results competitive inhibitor of the isocitrate substrate . However , The results for purification of wild type IDH1 are shown R132H and R132S display a 20 - fold and 13 - fold increase in in FIGS . 3 , 4 , 5A and 5B . sensitivity to feedback inhibition as compared to the wild The results for purification of mutant IDH1R132S are 15 type enzyme. shown in FIGS . 6 , 7 , 8A and 8B . The results for purification of wild type IDH1R132H are TABLE 2 shown in FIGS . 9 , 10 , 11A and 11B . Enzyme Ki (UM ) Example 2 Enzymology Analysis of IDH1 Wild 20 Wt 612.2 Type and Mutants R132H 28.6 R132S 45.3 1. Analysis of IDH1 Wild - Type and Mutants R132H and R132S in the Oxidative Decarboxylation of Isocitrate to 25 D. The Effect of Mncl, in Oxidative Decarboxylation of a -ketoglutarate (C -KG ) . Isocitrate to a -ketoglutarate ( a -KG ). A. Methods MnCl2 can be substituted with MgCl, to examine if there To determine the catalytic efficiency of enzymes in the is any difference in oxidative decarboxylation of isocitrate to oxidative decarboxylation of isocitrate to a -ketoglutarate a -ketoglutarate ( C -KG ) . Vmax(a -KG and) direction Km for , isocitratereactions . wereIn these performed reactions , to the determine substrate 30 E. The Effect of R132 Mutations on the Inhibitory Effect of was varied while the cofactor was held constant at 500 M. Oxalomalate on IDH1 All reactions were performed in 150 mM NaCl, 20 mM The purpose of this example is to examine the suscepti Tris - Cl, pH 7.5 , 10 % glycerol, and 0.03 % (w / v ) BSA ). bility of IDH1R132S and IDH1R132H in oxidative decar Reaction progress was followed by spectroscopy at 340 nM boxylation of isocitrate to a -ketoglutarate ( a -KG ) to the monitoring the change in oxidation state of the cofactor. 35 knownformed IDH1to examine inhibitor if R132 oxalomalate mutations . Experiments circumvent were the inhiper Sufficient enzyme was added to give a linear change in bition by oxalomalate . absorbance for 10 minutes . Final concentrations: Tris 7.5 20 mM , NaCl 150 mM , B. ICDH1 R132H and ICDH1 R132S are Impaired for MnCl, 2 mM , Glycerol 10 % , BSA 0.03 % , NADP 0.5 mM , ConversionMichaelis of -Menten Isocitrate plots to fora -KG the . relationship of isocitrate 40 IDH1wt 1.5 ug/ ml , IDH1R132S 30 ug/ ml , IDH1R132H 60 concentration to reaction velocity are presented in FIGS. ug /ml , DL - isocitrate ( 5-650 M ). The results are summa 12A - 12C . Kinetic parameters are summarized in the Table 1 . rized in FIG . 17 and Table 3. The R132S mutation displays All data was fit to the Hill equation by least - squares regres approximately a two- fold increase in susceptibility to inhi sion analysis. bition by oxalomalate , while the R132H mutation is essen 45 tially unaffected . In all three cases , the same fully competi tive mode of inhibition with regards to isocitrate was TABLE 1 observed . Relative Vmax Hill Catalytic TABLE 3 Enzyme (umol / min /mg ) Km (UM ) Constant Vmax /Km Efficiency 50 Enzyme Wt 30.5 56.8 1.8 0.537 100 % Oxalomalate Ki (UM ) R132H 0.605 171.7 0.6 0.0035 0.35 % wt 955.4 R132S 95 > 1e6 0.479 9.5e7 < .001 % R132S 510 R132H 950.8 Both mutant enzymes display a reduced Hill coefficient 55 and an increase in Km for isocitrate , suggesting a loss of F. Forward Reactions ( Isocitrate to a -KG ) of Mutant co -operativity in substrate binding and /or reduced affinity Enzyme do not go to Completion . for substrate. R132H enzyme also displays a reduced Vmax , Forward reactions containing ICDH1 R132S or ICDH1 suggestive of a lower kcat. R132S displays an increase in R132H were assembled and reaction progress monitored by Vmax , suggesting an increase in kcat, although this comes 60 an increase in the OD340 of the reduced NADPH cofactor . at the expense of a 20,000 fold increase in Km so that the It was observed (FIG . 23) , that these reactions proceed in the overall effect on catalytic efficiency is a great decrease as forward direction for a period of time and then reverse compared to the wild - type enzyme. The relative catalytic direction and oxidize the cofactor reduced in the early stages efficiency, described as Vmax /Km , is dramatically lower for of the reaction , essentially to the starting concentration the mutants as compared to wild -type . The in vivo effect of 65 present at the initiation of the experiment. Addition of these mutations would be to decrease the flux conversion of further isocitrate re - initiated the forward reaction for a isocitrate to a -KG . period of time, but again did not induce the reaction to US 10,610,125 B2 73 74 proceed to completion . Rather, the system returned to initial The results are shown in FIG . 16 and Table 5. The R132S concentrations of NADPH . This experiment suggested that mutant demonstrated the ability to utilize NADH while the the mutant enzymes were performing a reverse reaction wild type and R132H show no measurable consumption of other than the conversion of a -KG to isocitrate . NADH in the presence of alpha- ketoglutarate . 2. Analysis of IDH1 Wild - Type and Mutants R132H and 5 R132S in the Reduction of a -ketoglutarate ( a - KG ) . A. Methods TABLE 5 To determine the catalytic efficiency of enzymes in the Consumption of NADH by R132S in the presence of reduction of a -ketoglutarate ( a -KG ) , reactions were per alpha -ketoglutarate formed determine Vmax and Km for a -KG . In these 10 reactions , substrate was varied while the cofactor was held R132S Mean SD constant at 500 M.All reactions were performed in 50 mM Rate (AA / sec ) 0.001117 0.001088 0.001103 2.05E - 05 potassium phosphate buffer , pH 6.5 , 10 % glycerol, 0.03 % Umol/ min /mg 0.718328 0.699678 0.709003 0.013187 ( w / v ) BSA , 5 mM MgCl2 , and 40 mM sodium hydrocar bonate . Reaction progress was followed by spectroscopy at 15 Summary 340 nM monitoring the change in oxidation state of the To understand how R132 mutations alter the enzymatic cofactor . Sufficient enzyme was added to give a linear properties of IDH1, wild - type and R132H mutant IDH1 change in absorbance for 10 minutes . proteins were produced and purified from E. coli. When B. The R132H and R132S Mutant Enzymes, but not the NADP + -dependent oxidative decarboxylation of isocitrate Wild - Type Enzyme, Support the Reduction of a -KG . 20 was measured using purified wild -type or R132H mutant To test the ability of the mutant and wild - type enzymes to IDH1 protein , it was confirmed that R132H mutation perform the reduction of a -KG , 40 ug/ ml of enzyme was impairs the ability of IDH1 to catalyze this reaction ( Yan , H. incubated under the conditions for the reduction of a -Ke et al. N Engl J Med 360 , 765-73 (2009 ) ; Zhao , S. et al . toglutarate ( a -KG ) as described above . Results are pre Science 324 , 261-5 ( 2009 ) ) , as evidentby the loss in binding sented in FIG . 14. The wild - type enzyme was unable to 25 affinity for both isocitrate and MgCl, along with a 1000 - fold consume NADPH , while R132S and R132H reduced a - KG decrease in catalytic turnover ( FIGS. 30A and 30C ) . In and consumed NADPH . contrast , when NADPH -dependent reduction of aKG was C. The Reduction of a - KG by the R132H and R132S assessed using either wild - type or R132H mutant IDH1 Mutants Occurs In Vitro at Physiologically Relevant Con protein , only R132H mutant could catalyze this reaction at centrations of a -KG . 30 a measurable rate (FIGS . 30 and 30C ) . Part of this increased To determine the kinetic parameters of the reduction of rate of aKG reduction results from an increase in binding a -KG performed by the mutant enzymes, a substrate titra affinity for both the cofactor NADPH and substrate aKG in tion experiment was performed , as presented in FIGS. the R132H mutant IDH1 ( FIG . 30C ). Taken together, these 15A - 15B . R132H maintained the Hill- type substrate inter data demonstrate that while the R132H mutation leads to a action as seen in the oxidative decarboxylation of isocitrate , 35 loss of enzymatic function for oxidative decarboxylation of but displayed positive substrate co -operative binding. isocitrate , this mutation also results in a gain of enzyme R132S showed a conversion to Michaelis -Menten kinetics function for the NADPH - dependent reduction of aKG . with the addition of uncompetitive substrate inhibition , as 2 : Analysis of Mutant IDH1 compared to wild - type enzyme in the oxidative decarboxy The R132H Mutant does not Result in the Conversion of lation of isocitrate . The enzymatic parameters of the mutant 40 a - KG to Isocitrate . enzyme are presented in Table 4. Since the wild - type Using standard experimental methods, an API2000 mass enzyme did not consume measurable NADPH in the experi spectrometer was configured for optimal detection of a - KG ment described above , a full kinetic workup was not per and isocitrate ( Table 6 ) . MRM transitions were selected and formed . tuned such that each analyte was monitored by a unique 45 transition . Then , an enzymatic reaction containing 1 mM TABLE 4 a - KG , 1 mM NADPH , and ICDH1R132H were assembled and run to completion as judged by the decrease to baseline Vmax Hill of the optical absorbance at 340 nM . A control reaction was Enzyme (umol /min /mg ) Km (MM ) Constant Ki (mm ) Vmax /Km performed in parallel from which the enzyme was omitted . R132H 1.3 0.965 1.8 1.35 50 Reactions were quenched 1 : 1 with methanol, extracted , and R132S 2.7 0.181 0.479 24.6 14.92 subjected to analysis by LC -MS / MS . FIG . 18A presents the control reaction indicating that The relative catalytic efficiency of reduction of a -KG is aKG was not consumed in the absence of enzyme, and no approximately ten - fold higher in the R132S mutant than in detectable isocitrate was present. FIG . 18B presents the the R132H mutant. The biological consequence is that the 55 reaction containing R132H enzyme, in which the a -KG has rate of metabolic flux should be greater in cells expressing been consumed , but no isocitrate was detected . FIG . 18C R132S as compared to R132H . presents a second analysis of the reaction containing enzyme D. Analysis of IDH1 Wild - Type and Mutants R132H and in which isocitrate has been spiked to a final concentration R132S in the Reduction of Alpha -Ketoglutarate with of 1 mM , demonstrating that had a -KG been converted to NADH . 60 isocitrate at any appreciable concentration greater than In order to evaluate the ability of the mutant enzymes to 0.01 % , the configured analytical system would have been utilize NADH in the reduction of alpha -ketoglutarate , the capable of detecting its presence in the reaction containing following experiment was conducted . Final concentrations: enzyme. The conclusion from this experiment is that while NaHCO3 40 mM , MgCl2 5 mM , Glycerol 10 % , K2HPO4 A -KG was consumed by R132H , isocitrate was not pro 50 mM , BSA 0.03 % , NADH 0.5 mM , IDH1 wt 5 ug /ml , 65 duced . This experiment indicates that one neoactivity of the R132S 30 ug /ml , R132H 60 ug/ ml , alpha -Ketoglutarate 5 R132H mutant is the reduction of a -KG to a compound mM . other than isocitrate . US 10,610,125 B2 75 76 TABLE 6 These are referred to by those with knowledge in the art as the R (or pro - R ) and S (or pro - S ) enantiomers , respec Instrument settings for MRM detection of compounds tively . In order to determine which form or both is produced Compound Q1 Q3 DP FP EP CEP CE CXP as a result of the ICDH1 neoactivity described above , the 5 relative amount of each chiral form in the reaction product a - KG 144.975 100.6 -6 -220 -10 -16 -10 -22 isocitrate 191.235 110.9 -11 -230 -4.5 -14 -16 -24 was determined in the procedure described below . a -hydro 147.085 128.7 -11 -280 -10 -22 -12 -24 Reduction of a -KG to 2 - HG was performed by xyglutarate ICDHR132H in the presence of NADPH as described above , and the reaction progress was monitored by a change The R132H Mutant Reduces a -KG to 2- Hydroxyglutaric 10 in extinction coefficient of the nucleotide cofactor at 340 Acid . nM ; once the reaction was judged to be complete , the Using standard experimental methods, an API2000 mass reaction was extracted with methanol and dried down com spectrometer was configured for optimal detection 2 -hy pletely in a stream of nitrogen gas . In parallel, samples of droxyglutarate ( Table 6 and FIG . 19 ). The reaction products chirally pure R - 2 -HG and a racemic mixture of R- and of the control and enzyme- containing reactions from above 15 were investigated for the presence of 2 -hydroxyglutaric S - 2 -HG ( produced by a purely chemical reduction of a - KG acid , FIG . 20. In the control reaction , no 2 -hydroxyglutaric to 2- HG ) were resuspended in ddH20 , similarly extracted acid was detected , while in reaction containing R132H , with methanol, and dried . 2 -hydroxyglutaric acid was detected . This data confirms that The reaction products or chiral standards were then resus one neoactivity of the R132H mutant is the reduction of 20 pended in a solution of dichloromethane: acetic acid ( 4 : 1 ) a -KG to 2 -hydroxyglutaric acid . containing 50 g /L DATAN and heated to 75º C. for 30 To determine whether R132H mutant protein directly minutes to promote the derivitization of 2 -HG in the scheme produced 2HG from aKG , the product of the mutant IDH1 described below : reaction was examined using negative ion mode triple quadrupole electrospray LC -MS . These experiments con- 25 firmed that 2HG was the direct product of NADPH -depen OH dent aKG reduction by the purified R132H mutant protein through comparison with a known metabolite standards + ( FIG . 31A ). Conversion of aKG to isocitrate was not HOOC COOH observed . M = 148 One can determine the enantiomeric specificity of the 30 reaction product through derivitization with DATAN ( di acetyl- L - tartaric acid ) and comparing the retention time to H3C CH3 that of known R and S standards . This method is described CH2Cl2 / HAC in Struys et al . Clin Chem 50 : 1391-1395 (2004 ) . The stereo 30 ' , 75 ° C . specific production of either the or S enantiomer of 35 alpha -hydroxyglutaric acid by ICDH1 R132H may modify the biological activity of other enzymes present in the cell. M = 216 The racemic production may also occur . For example , one can measure the inhibitory effect of -CH3 alpha- hydroxyglutaric acid on the enzymatic activity of 40 H3C enzymes which utilize a -KG as a substrate. In one embodi ment, alpha- hydroxyglutaric acid may be a substrate- or product- analogue inhibitor of wild - type ICDH1. In another HO embodiment alpha -hydroxyglutaric acid may be a substrate or product -analogue inhibitor ofHIF1 prolyl hydroxylase . In 45 the former case , inhibition of wild type ICDH1 by the * enzymatic product of R132H will reduce the circulating HOOC COOH levels of aKG in the cell. In the latter case , inhibition of M = 364 HIF1 prolyl hydroxylase will result in the stabilization of HIF1 and an induction of the hypoxic response cohort of cellular responses. 50 After cooling to room temperature , the derivitization ICDH R132H Reduces aKG to the R - Enantiomer of 2 -hy reactions were dried to completion and resuspended in droxyglutarate. ddH2O for analysis on an LC -MS / MS system . Analysis of There are two possible enantiomers of the ICDHR132H reaction products and chiral standards was performed on an reductive reaction product, converting alpha -ketoglutarate API2000 LC -MS / MS system using a 2x150 mM C18 col to 2 -hydroxyglutarate , with the chiral center being located at 55 umn with an isocratic flow of 200 ul /min of 90:10 ( ammo the alpha- carbon position . Exemplary products are depicted nium formate , pH 3.6 :methanol ) and monitoring the reten below . tion times of the 2 -HG -DATAN complex using XIC and the diagnostic MRM transition of 363/147 in the negative ion mode . 60 It should be noted that retention times in the experiments described below are approximate and accurate to within +/- 1 minute ; the highly reproducible peak seen at 4 minutes is an artefact of a column switching valve whose presence OHque OH has no result on the conclusions drawn from the experiment. R - 2 -hydroxyglutarate S - 2 -hydroxyglutarate 65 Injection of the racemic mixture gave two peaks of equal area at retention times of 8 and 10 minutes (FIG . 24A ), while injection of the R - 2 -HG standard resulted in a major peak of US 10,610,125 B2 77 78 > 95 % area at 10 minutes and a minor peak < 5 % area at 8 sodium salt at concentrations of either 750 ug /ml (CRL minutes (FIG . 24B ); indicating that the R -2 -HG standard is 2610 ) or 500 ug/ ml (HTB - 14 ) to select those cells in culture approximately 95 % R and 5 % S. Thus, this method allows that expressed the integrated DNA cassette expressing both us to separate the R and S - 2 -HG chiral forms and to the neomycin selectable marker and the ORF for human determine the relative amounts of each in a given sample . 5 wild type or R132H . Pooled populations of G418 resistant Coinjection of the racemic mixture and the R -2 - HG standard cells were generated and expression of either wild type resulted in two peaks at 8 and 10 minutes , with a larger peak at 10 minutes resulting from the addition of surplus pro - R IDH1 or R132 IDH1 was confirmed by standard Western form (the standard ) to a previously equal mixture of R- and blot analysis of cell lysates using commercial antibodies S - 2 - HG (FIG . 24C ) . These experiments allow us to assign 10 recognizing either human IDH1 antigen or the engineered the 8 minute peak to the S - 2 -HG form and the 10 minute carboxy - terminal MYC -DDK expression tag. These stable peak to the R - 2 -HG form . clonal pools were then utilized for metaobolite preparation Injection of the derivatized neoactivity enzyme reaction and analysis . product alone yields a single peak at 10 minutes , suggesting Procedure for Metabolite Preparation and Analysis . that the neoactivity reaction product is chirally pure R -2 -HG 15 Glioblastoma cell lines (CRL -2610 and HTB - 14 ) express (FIG . 24D ) . Coinjection of the neoactivity reaction product ing wildtype or mutant IDH - 1 protein were grown using with the R - 2 -HG standard results in a major peak of > 95 % standard mammalian tissue culture techniques on DMEM area at 10 minutes (FIG . 24E ) and a single minor peak of media containing 10 % FCS , 25 mM glucose , 4 mM gluta < 5 % area at 8 minutes (previously observed in injection of mine , and G418 antibiotic (CRL - 2610 at 750 ug /mL ; HTB the R -2 -HG standard alone ) confirming the chirality of the 20 14 at 500 ug /mL ) to insure ongoing selection to preserve the neoactivity product as R. Coinjection of a racemic mixture transfected mutant expression sequences. In preparation for and the neoactivity reaction product ( FIG . 24F ) results in a metabolite extraction experiments , cells were passaged into 60 % area peak at 10 minutes and a 40 % area peak at 8 10 cm round culture dishes at a density of 1x106 cells . minutes ; this deviation from the previously symmetrical Approximately 12 hours prior to metabolite extraction , the peak areas observed in the racemate sample being due to the 25 culture media was changed ( 8 mL per plate ) to DMEM excess presence of R - 2 -HG form contributed by the addition containing 10 % dialyzed FCS (10,000 mwco ) , 5 mM glu of the neoactivity reaction product . cose, 4 mM glutamine , and G -418 antibiotic as before; the These experiments allow us to conclude that the ICDH1 dialyzed FCS removes multiple small molecules form the neoactivity is a highly specific chiral reduction of a -KG to culture media and enables cell culture - specific assessment of R - 2 -HG . 30 metabolite levels . The media was again changed 2 hours Enzyme Properties of Other IDH1 Mutations prior to metabolite extraction . Metabolite extraction was To determine whether the altered enzyme properties accomplished by quickly aspirating the media from the resulting from R132H mutation were shared by other R132 culture dishes in a sterile hoc immediately placing the mutations found in human gliomas , recombinant R132C , dishes in a tray containing dry ice to cool them to -80 ° C., R132L and R132S mutant IDH1 proteins were generated 35 and as quickly as possible , adding 2.6 mL of 80 % MeOH / and the enzymatic properties assessed . Similar to R132H 20 % water , pre -chilled to -80 ° C. in a dry - ice/ acetone bath . mutant protein , R132C , R132L , and R132S mutations all These chilled ,methanol extracted cells were then physically result in a gain - of- function for NADPH -dependent reduc separated from the culture dish by scraping with a sterile tion of aKG (data not shown) . Thus, in addition to impaired polyethylene cell lifter (Corning # 3008 ), brought into sus oxidative decarboxylation of isocitrate , one common feature 40 pension and transferred to a 15 mL conical vial, then chilled shared among the IDH1 mutations found in human gliomas to -20 ° C. An additional 1.0 mL of 80 % MeOH /20 % water is the ability to catalyze direct NADPH -dependent reduction was applied to the chilled culture dish and the cell lifting of aKG . procedure repeated , to give a final extraction volume of 3.6 Identification of 2 -HG Production in Glioblastoma Cell mL . The extracts were centrifuged at 20,000xg for 30 Lines Containing the IDH - 1 R132H Mutant Protein . 45 minutes to sediment the cell debris , and 3.0 mL of the Generation of Genetic Engineered Glioblastoma Cell supernatants was transferred to a screw -cap freezer vial and Lines Expressing Wildtype or Mutant IDH - 1 Protein . stored at -80 ° C. until ready for analysis . A carboxy - terminal Myc- DDK -tagged open reading In preparation for analysis , the extracts were removed frame (ORF ) clone of human isocitrate dehydrogenase 1 from the freezer and dried on a nitrogen blower to remove ( IDH1; Ref . ID : NM_005896 ) cloned in vector pCMV6 was 50 methanol. The 100 % aqueous samples were analyzed by obtained from commercial vendor Origen Inc. Vector LCMS as follows. The extract ( 10 uL ) was injected onto a PCMV6 contains both kanamycin and neomycin resistance reverse -phase HPLC column (Synergi 150 mmx2 mm , Phe cassettes for selection in both bacterial and mammalian cell nomenex Inc. ) and eluted using a linear gradient of LCMS systems. Standard molecular biology mutagenesis tech grade methanol (Buffer B ) in Aq . 10 mM tributylamine , 15 niques were utilized to alter the DNA sequence at base pair 55 mM Acetic acid (Buffer A ), running from 3 % Buffer B to 364 of the ORF to introduce base pair change from guanine 95 % Buffer B over 45 minutes at 200 uL /min . Eluted to adenine resulting in a change in the amino acid code at metabolite ions were detected using a triple - quadrapole position 132 from argentine (wt ) to histidine (mutant ; or mass spectrometer , tuned to detect in negative mode with R132H ). Specific DNA sequence alteration was confirmed multiple - reaction -monitoring mode transition set (MRM's ) by standard methods for DNA sequence analysis. Parental 60 according to the molecular weights and fragmentation pat vector pCMV6 (no insert ), pCMV6 -wt IDH1 or PCMV6 terns for 38 known central metabolites, including 2 -hy R132H were transfected into immortalized human glioblas droxyglutarate (MRM parameters were optimized by prior toma cell lines ATCC® CRL - 2610 (LN - 18 ) or HTB - 14 infusion of known compound standards ). Data was pro ( U -87 ) in standard growth medium (DMEM ; Dulbecco's cessed using Analyst Software ( Applied Biosystems, Inc.) modified Eagles Medium containing 10 % fetal bovine 65 and metabolite signal intensities were converted into abso serum ) . Approximately 24 hrs after transfection , the cell lute concentrations using signal build - up curves from cultures were transitioned to DMEM containing G418 injected mixtures of metabolite standards at known concen US 10,610,125 B2 79 80 trations . Final metabolite concentrations were reported as 132 of human IDH1 protein from arginine (wild type ) to mean of at least three replicates, +/- standard deviation . histidine (mutant ) as has been previously reported . Results . Analyses reveal significantly higher levels of 2 -HG in TABLE 15 cells that express the IDH - 1 R132H mutant protein . As 5 Sequence variance at base pair 170 of the amplified shown in FIG . 26A , levels of 2 -HG in CRL - 2610 cell lines genomic DNA from human glioma samples . expressing the IDH - 1 R132H mutant protein are approxi mately 28 - fold higher than identical lines expressing the Sample Base IDH1 Amino wild - type protein . Similarly , levels of 2 - HG in HTB - 14 cell ID 170 Acid 132 Genotype 10 lines expressing the IDH - 1 R132H mutant protein are 1102 G arginine wild type approximately 38 -fold higher than identical lines expressing 1822 histidine mutant 496 G arginine wild type the wild - type protein , as shown in FIG . 26B . 1874 A histidine mutant Evaluation of 2 -Hydroxyglutarate (2 -HG ) Production in 816 histidine mutant Human Glioblastoma Tumors Containing Mutations in Iso 534 G arginine wild type citrate Dehydrogenase 1 ( IDH1) at Amino Acid 132 . 15 AP - 1 histidine mutant Heterozygous somatic mutations at nucleotide position AP - 2 histidine mutant 395 ( amino acid codon 132) in the transcript encoding isocitrate dehydrogenase 1 ( IDH1) can occur in brain Procedure for Metabolite Preparation and Analysis . tumors . Metabolite extraction was accomplished by adding a 10x Tissue Source : 20 volume ( m / v ratio ) of -80 C methanol:water mix ( 80 % : Human brain tumors were obtained during surgical resec 20 % ) to the brain tissue (approximately 100 mgs ) followed tion , flash frozen in liquid nitrogen and stored at -80 ° C. by 30 s homogenization at 4 C. These chilled , methanol Clinical classification of the tissue as gliomas was per extracted homogenized tissues were then centrifuged at formed using standard clinical pathology categorization and 14,000 rpm for 30 minutes to sediment the cellular and grading . 25 tissue debris and the cleared tissue supernatants were trans Genomic Sequence Analysis to Identify Brain Tumor ferred to a screw -cap freezer vial and stored at -80 ° C. For Samples Containing Either Wild Type Isocitrate Dehydro analysis , a 2x volume of tributylamine ( 10 mM ) acetic acid genase ( IDH1) or Mutations Altering Amino Acid 132 . ( 10 mM ) pH 5.5 was added to the samples and analyzed by Genomic DNA was isolated from 50-100 mgs of brain LCMS as follows. Sample extracts were filtered using a tumor tissue using standard methods. A polymerase chain 30 Millex - FG 0.20 micron disk and 10 uL were injected onto a reaction (PCR ) procedure was then performed on the iso reverse -phase HPLC column ( Synergi 150 mmx2 mm , Phe lated genomic DNA to amplify a 295 base pair fragment of nomenex Inc.) and eluted using a linear gradient LCMS the genomic DNA that contains both intron and 2nd exon grade methanol (50 % ) with 10 mM tributylamine and 10 sequences of human IDH1 (FIG . 27 ) . In FIG . 27 , intron mM acetic acid ) ramping to 80 % methanol: 10 mM tribu sequence is shown in lower case font; 2nd exon IDH1 DNA 35 tylamine : 10 mM acetic acid over 6 minutes at 200 uL /min sequence is shown in upper case font ; forward ( 5 ') and Eluted metabolite ions were detected using a triple - quadra reverse ( 3 ') primer sequences are shown in underlined font; pole mass spectrometer , tuned to detect in negative mode guanine nucleotide mutated in a subset of human glioma with multiple - reaction -monitoring mode transition set tumors is shown in bold underlined font. (MRM's ) according to the molecular weights and fragmen The amplified DNA fragment was then sequenced using 40 tation patterns for 8 known central metabolites, including standard protocols and sequence alignments were performed 2 -hydroxyglutarate (MRM parameters were optimized by to classify the sequences as either wild type or mutant at the prior infusion of known compound standards) . Data was guanine nucleotide at base pair 170 of the amplified PCR processed using Analyst Software ( Applied Biosystems, fragment. Tumors were identified that contained genomic Inc. ) and metabolite signal intensities were obtained by DNA having either two copies of guanine ( wild type ) or a 45 standard peak integration methods. mixed or monoalellic combination of one IDH1 allele con Results . taining guanine and the other an adenine (mutant ) sequence Analyses revealed dramatically higher levels of 2 -HG in at base pair 170 of the amplified product ( Table 15 ) . The cells tumor samples that express the IDH - 1 R132H mutant nucleotide change results in a change at amino acid position protein . Data is summarized in Table 16 and FIG . 28 . TABLE 16 Tumor Cells in Sam Tumor ple Primary Specimen Foci Geno Nucleotide 2HG KG Malate Fumarate Succinate Isocitrate ID Diagnosis Grade ( % ) type change Codon (Omole g ) (Omole / g ) (Omole / g ) ( mole / g ) ( mole/ g ) (Omole / g ) Glioblastoma, WHO n / a wild wild R132 0.18 0.161 1.182 0.923 1.075 0.041 residual/ recurrent grade type type IV 2 Glioblastoma WHO n / a wild wild R132 0.16 0.079 1.708 1.186 3.156 0.100 grade type type IV 3 Glioblastoma WHO n / a wild wild R132 0.13 0.028 0.140 0.170 0.891 0.017 grade type type IV US 10,610,125 B2 81 82 TABLE 16 - continued Tumor Cells in Sam Tumor ple Primary Specimen Foci Geno Nucleotide 2HG DKG Malate Fumarate Succinate Isocitrate ID Diagnosis Grade ( % ) type change Codon ( mole / g ) mole / g ) ( mole / g ) mole /g ) ( mole / g ) mole / g ) 4 Oligoastrocytoma WHO n / a wild wild R132 0.21 0.016 0.553 1.061 1.731 0.089 grade type type II 5 Glioblastoma WHO n / a mutant G364A R132H 16.97 0.085 1.091 0.807 1.357 0.058 grade IV 6 Glioblastoma WHO n / a mutant G364A R132H 19.42 0.023 0.462 0.590 1.966 0.073 grade IV 7 Glioblastoma WHO n / a mutant G364A R132H 31.56 0.068 0.758 0.503 2.019 0.093 grade IV 8 Oligodendroglioma, WHO 75 mutant G364A R132H 12.49 0.033 0.556 0.439 0.507 0.091 anaplastic grade III 9 Oligodendroglioma, WHO 90 mutant G364A R132H 4.59 0.029 1.377 1.060 1.077 0.574 anaplastic grade III 10 Oligoastrocytoma WHO n / a mutant G364A R132H 6.80 0.038 0.403 0.503 1.561 0.065 grade II 11 Glioblastoma WHO n / a wild wild R132 0.686 0.686 0.686 0.686 0.686 0.007 grade type type IV 12 Glioblastoma WHO n / a mutant G364A R132H 18.791 18.791 18.791 18.791 18.791 0.031 grade IV 13 Glioblastoma WHO n / a mutant G364A R132H 4.59 0.029 1.377 1.060 1.077 0.043 grade IV 14 Glioblastoma WHO n /a wild wild R132 0.199 0.046 0.180 0.170 0.221 0.014 grade type type IV 15 Glioblastoma WHO n / a mutant C363G R132G 13.827 0.030 0.905 0.599 1.335 0.046 grade IV 16 Glioblastoma WHO n / a mutant G364A R132H 28.364 0.068 0.535 0.488 2.105 0.054 grade IV 17 Glioblastoma WHO n / a mutant C363A R132S 9.364 0.029 1.038 0.693 2.151 0.121 grade IV 18 Glioblastoma WHO n / a wild wild R132 0.540 0.031 0.468 0.608 1.490 0.102 grade type type IV 19 Glioma, malignant, WHO 80 mutant G364A R132H 19.000 0.050 0.654 0.391 2.197 0.171 astrocytoma grade IV 20 Oligodendroglioma WHO 80 wild wild R132 0.045 0.037 1.576 0.998 1.420 0.018 grade type type III 21 Glioma, malignant , WHO 95 wild wild R132 0.064 0.034 0.711 0.710 2.105 0.165 astrocytoma grade type type IV 22 Glioblastoma WHO 70 wild wild R132 0.171 0.041 2.066 1.323 0.027 0.072 grade type type IV

55 To determine if 2HG production is characteristic of in all tumors that contained an R132 IDH1 mutation . All tumors harboring mutations in IDH1, metabolites were R132 mutant IDH1 tumors examined had between 5 and 35 extracted from human malignant gliomas that were either umol of 2HG per gram of tumor, while tumors with wild wild -type or mutant for IDH1. It has been suggested that type IDH1 had over 100 fold less 2HG . This increase in 2HG AKG levels are decreased in cells transfected with mutant 60 in R132 mutant tumors was statistically significant IDH1 ( Zhao , S. et al. Science 324 , 261-5 ( 2009 ) ). The (p < 0.0001 ). It was confirmed that (R ) -2HG was the isomer average aKG level from 12 tumor samples harboring vari present in tumor samples (data not shown ). Together these ous R132 mutations was slightly less than the average aKG data establish that the novel enzymatic activity associated level observed in 10 tumors which are wild - type for IDH1. with R132 mutations in IDH1 results in the production of This difference in aKG was not statistically significant, and 65 2HG in human brain tumors that harbor these mutations. a range of aKG levels was observed in both wild - type and 2HG is known to accumulate in the inherited metabolic mutant tumors . In contrast , increased 2HG levels were found disorder 2 -hydroxyglutaric aciduria . This disease is caused US 10,610,125 B2 83 84 by deficiency in the enzyme 2 -hydroxyglutarate dehydroge tive decarboxylation of isocitrate by the wild - type ICDH1, nase , which converts 2HG to AKG (Struys , E. A. et al . Am while the ( R ) -2 -hydroxyglutarate did not show any effect on J Hum Genet 76 , 358-60 (2005 )) . Patients with 2 -hydroxy the rate of the reaction . Since there are only two possible glutarate dehydrogenase deficiencies accumulate 2HG in the chiral products of the ICDH1 R132H mutant reduction of brain as assessed by MRI and CSF analysis , develop leu- 5 a - KG to 2 -HG , and the ( R ) -2 -HG did not show inhibition in koencephalopathy, and have an increased risk of developing this assay , it follows that the product of the mutant reaction brain tumors ( Aghili , M., Zahedi, F. & Rafiee , J Neurooncol is the ( S )-2 - HG form . This experiment is presented in FIG . 91 , 233-6 ( 2009 ) ; Kolker , S. , Mayatepek , E. & Hoffmann , G. 25 . F.Neuropediatrics 33, 225-31 ( 2002 ); Wajner ,M. , Latini, A., To determine the chirality of the 2HG produced , the Wyse , A. T. & Dutra -Filho , C. S. J Inherit Metab Dis 27 , 10 products of the R132H reaction was derivatized with 427-48 (2004 ) ) . Furthermore, elevated brain levels of 2HG diacetyl- L -tartaric anhydride, which allowed separating the result in increased ROS levels (Kolker , S. et al. Eur J ( S ) and (R ) enantiomers of 2HG by simple reverse- phase LC Neurosci 16 , 21-8 ( 2002 ); Latini, A. et al. Eur J Neurosci 17 , and detecting the products by tandem mass spectrometry 2017-22 ( 2003 ) ), potentially contributing to an increased (Struys , E. A., Jansen , E. E., Verhoeven , N.M. & Jakobs, C. risk of cancer. The ability of 2HG to act as an NMDA 15 Clin Chem 50 , 1391-5 (2004 )) (FIG . 31B ). The peaks receptor agonist may contribute to this effect (Kolker , S. et corresponding to the ( S ) and ( R ) isomers of 2HG were al. Eur J Neurosci 16 , 21-8 (2002 )) . 2HG may also be toxic confirmed using racemic and R ( - ) - 2HG standards . The to cells by competitively inhibiting glutamate and/ or aKG reaction product from R132H co - eluted with R ( - ) -2HG utilizing enzymes . These include transaminases which allow peak , demonstrating that the R ( - ) stereoisomer is the prod utilization of glutamate nitrogen for amino and nucleic acid 20 uct produced from aKG by R132H mutant IDH1. biosynthesis , and aKG -dependent prolyl hydroxylases such The observation that the reaction product of the mutant as those which regulate Hifla levels . Alterations in Hifla enzyme is capable of inhibiting a metabolic reaction known have been reported to result from mutant IDH1 protein to occur in cells suggests that this reaction product might expression (Zhao , S. et al. Science 324, 261-5 (2009 ) ). also inhibit other reactions which utilize a -KG , isocitrate , or Regardlessfunction ability of mechanism of cells to , produce it appears 2HG likely as athat result the ofgain R132 -of- 25 citrate as substrates or produce them as products in vivo or mutations in IDH1 contributes to tumorigenesis . Patients in vitro . with 2 -hydroxyglutarate dehydrogenase deficiency have a Example 3 Metabolomics Analysis of IDH1 Wild high risk of CNS malignancy (Aghili , M., Zahedi, F. & Type and Mutants Rafiee, E. J Neurooncol 91, 233-6 ( 2009 )) . The ability of 30 mutant IDH1 to directly act on aKG may explain the Metabolomics research can provide mechanistic basis for prevalence of IDH1 mutations in tumors from CNS tissue , why R132 mutations confer survival advantage for GBM which are unique in their high level of glutamate uptake and patients carrying such mutations. its ready conversion to AKG in the cytosol ( Tsacopoulos, M. 1. Metabolomics of GBM Tumor Cell Lines: Wild Type Vs J Physiol Paris 96 , 283-8 (2002 ) ) , thereby providing high 35 R132 Mutants levels of substrate for 2HG production . The apparent co Cell lines with R132 mutations can be identified and dominance of the activity of mutant IDH1 with that of the profiled . Experiments can be performed in proximal metabo wild -type enzyme is consistent with the genetics of the lite pool with a broad scope of metabolites . disease, in which only a single copy of the gene is mutated . 2. Oxalomalate Treatment of GBM Cell Lines As discussed above , the wild - type IDH1 could directly 40 Oxalomalate is a competitive inhibitor of IDH1. Change provide NADPH and aKG to themutant enzyme. These data of NADPH (metabolomics ) when IDH1 is inhibited by a also demonstrate that mutation of R132 to histidine , serine , small molecule can be examined . cysteine, glycine or leucine share a common ability to 3. Metabolomics of Primary GBM Tumors : Wild Type Vs catalyze the NADPH -dependent conversion of aKG to R132 Mutations 2HG . These findings help clarify why mutations at other 45 Primary tumors with R132 mutations can be identified . amino acid residues of IDH1, including other residues Experiments can be performed in proximal metabolite pool essential for catalytic activity , are not found . Finally , these with a broad scope of metabolites . findings have clinical implications in that they suggest that 4. Detection of 2- hydroxyglutarate in Cells that Overexpress 2HG production will identify patients with IDH1 mutant IDH1 132 Mutants brain tumors. This will be important for prognosis as 50 Overexpression of an IDH1 132 mutant in cells may cause patients with IDH1mutations live longer than patients with an elevated level of 2 -hydroxyglutarate and /or a reduced gliomas characterized by other mutations (Parsons , D. W. et level of alpha -ketoglutarate . One can perform a metabolo al . Science 321, 1807-12 (2008 ) ) . In addition , patients with mic experiment to demonstrate the consequence of this lower grade gliomas may benefit by the therapeutic inhibi tion of 2HG production . Inhibition of 2HG production by 55 mutation on the cellular metabolite pool. mutant IDH1 might slow or halt conversion of lower grade Example 4 Evaluation of IDH1 as a Cancer Target glioma into lethal secondary glioblastoma, changing the course of the disease . shRNAmir inducible knockdown can be performed to The Reaction Product of ICDH1 R132H Reduction of a -KG examine the cellular phenotype and metabolomics profiles . Inhibits the Oxidative Decarboxylation of Isocitrate by 60 HTS grade IDH1 enzymes are available . The IDH mutations Wild - Type ICDH1. described herein can be used for patient selection . A reaction containing the wild -type ICDHI, NADP , and a - KG was assembled ( under conditions as described above ) Example 5 siRNAS to which was added in a titration series either (R ) -2 -hy droxyglutarate or the reaction product of the ICDH1 65 IDH1 R1321H mutant reduction of a - KG to 2 -hydroxyglutarate . Exemplary siRNAs are presented in the following tables. The reaction product 2 -HG was shown to inhibit the oxida Art- known methods can be used to select other siRNAs. US 10,610,125 B2 85 86 siRNAs can be evaluated , e.g., by determining the ability of dehydrogenase by small interfering RNA enhances the sen an siRNA to silence an IDH , e.g., IDH1, e.g., in an in vitro sitivity of Hela cells toward stauropine, Lee et al ., 2009 , system , e.g., in cultured cells , e.g., HeLa cells or cultured glioma cells . siRNAs known in the art for silencing the Free Radical Research , 43 : 165-173 . target can also be used , see , e.g. , Silencing of cytosolic 5 The siRNAs in Tables 7 , 8 , 9 , 10 , 11 , 12 , 13 and 14 NADP + dependent isoccitrate dehydrogenase by small represent candidates spanning the IDH1 mRNA at nucleo interfering RNA enhances the sensitivity of Hela cells tide positions 628 and 629 according to the sequence at toward stauropine, Lee et al. , 2009 , Free Radical Research , GenBank Accession No. NM_005896.2 (SEQ ID NO : 9 , 43 : 165-173 . FIG . 22 ). The siRNAs in Table 7 (with the exception of entry 1356 ) 10 The RNAs in the tables can be modified , e.g., as described were generated using the siRNA selection tool available on herein . Modifications include chemical modifications to the worldwide web at jura.wi.mit.edu/bioc/siRNAext/ . enhance properties , e.g. , resistance to degradation , or the use ( Yuan et al. Nucl. Acids. Res . 2004 32 :W130 - W134 . ) Other of overhangs. For example , either one or both of the sense selection tools can be used as well. Entry 1356 was adapted and antisense strands in the tables can include an additional from Silencing of cytosolic NADP + dependent isoccitrate dinucleotide at the 3 ' end , e.g. , TT, UU , dTdT . TABLE 7 siRNAs targeting wildtype IDH1 Position on mRNA SEQ SEQ ( FIG . ID ID 21B ) sense ( 5 ' to 3 ' ) NO : antisense ( 5 ' to 3 ' ) NO : 13 GGUUUCUGCAGAGUCUACU 14 AGUAGACUCUGCAAAACC 15

118 CUCUUCGCCAGCAUAUCAU 16 AUGAUAUGCUGGCGAAGAG 17 140 GGCAGGCGAUAAACUACAU 18 AUGUAGUUUAUCGCCUGCC 19

145 GCGAUAAACUACAUUCAGU 20 ACUGAAUGUAGUUUAUCGC 21

199 GAAAUCUAUUCACUGUCAA 22 UUGACAGUGAAUAGAUUUC 23

257 GUUCUGUGGUAGAGAUGCA 24 UGCAUCUCUACCACAGAAC 25

272 GCAAGGAGAUGAAAUGACA 26 UGUCAUUUCAUCUCCUUGC 27

277 GGAGAUGAAAUGACACGAA 28 UUCGUGUCAUUUCAUCUCC 29

278 GAGAUGAAAUGACACGAAU 30 AUUCGUGUCAUUUCAUCUC 31

280 GAUGAAAUGACACGAAUCA 32 UGAUUCGUGUCAUUUCAUC 33 292 CGAAUCAUUUGGGAAUUGA 34 UCAAUUCCCAAAUGAUUCG 35 302 GGGAAUUGAUUAAAGAGAA 36 UUCUCUUUAAUCAAUUCCC 37 332 CCUACGUGGAAUUGGAUCU 38 AGAUCCAAUUCCACGUAGG 39

333 CUACGUGGAAUUGGAUCUA 40 UAGAUCCAAUUCCACGUAG 41

345 GGAUCUACAUAGCUAUGAU 42 AUCAUAGCUAUGUAGAUCC 43

356 GCUAUGAUUUAGGCAUAGA 44 UCUAUGCCUAAAUCAUAGC 45

408 GGAUGCUGCAGAAGCUAUA 46 UAUAGCUUCUGCAGCAUCC 47

416 CAGAAGCUAUAAAGAAGCA 48 UGCUUCUUUAUAGCUUCUG 49

418 GAAGCUAUAAAGAAGCAUA 50 UAUGCUUCUUUAUAGCUUC 51 432 GCAUAAUGUUGGCGUCAAA 52 UUUGACGCCAACAUUAUGC 53

467 CUGAUGAGAAGAGGGUUGA 54 UCAACCCUCUUCUCAUCAG 55

481 GUUGAGGAGUUCAAGUUGA 56 UCAACUUGAACUCCUCAAC 57

487 GAGUUCAAGUUGAAACAAA 58 UUUGUUUCAACUUGAACUC 59

495 GUUGAAACAAAUGUGGAAA 60 UUUCCACAUUUGUUUCAAC 61

502 CAAAUGUGGAAAUCACCAA 62 UUGGUGAUUUCCACAUUUG 63

517 CCAAAUGGCACCAUACGAA 64 UUCGUAUGGUGCCAUUUGG 65

528 CAUACGAAAUAUUCUGGGU 66 ACCCAGAAUAUUUCGUAG 67 US 10,610,125 B2 87 88 TABLE 7 - continued siRNAs targeting wildtype IDH1 Position on mRNA SEQ SEO ( FIG . ID ID 21B ) sense ( 5 ' to 3 ' ) NO : antisense ( 5 ' to 3 ' ) NO : 560 GAGAAGCCAUUAUCUGCAA 68 UUGCAGAUAAUGGCUUCUC 69

614 CUAUCAUCAUAGGUCGUCA 70 UGACGACCUAUGAUGAUAG 71

618 CAUCAUAGGUCGUCAUGCU 72 AGCAUGACGACCUAUGAUG 73

621 CAUAGGUCGUCAUGCUUAU 74 AUAAGCAUGACGACCUAUG 75 691 GAGAUAACCUACACACCAA 76 UUGGUGUGUAGGUUAUCUC 77 735 CCUGGUACAUAACUUUGAA 78 UUCAAAGUUAUGUACCAGG 79 747 CUUUGAAGAAGGUGGUGGU 80 ACCACCACCUUCUUCAAAG 81

775 GGGAUGUAUAAUCAAGAUA 82 UAUCUUGAUUAUACAUCCC 83

811 GCACACAGUUCCUUCCAAA 84 UUUGGAAGGAACUGUGUGC 85

818 GUUCCUUCCAAAUGGCUCU 86 AGAGCCAUUUGGAAGGAAC 87

844 GGUUGGCCUUUGUAUCUGA 88 UCAGAUACAAAGGCCAACC 89

851 CUUUGUAUCUGAGCACCAA 90 UUGGUGCUCAGAUACAAAG 91

882 GAAGAAAUAUGAUGGGCGU 92 ACGCCCAUCAUAUUUCUUC 93

942 GUCCCAGUUUGAAGCUCAA 94 UUGAGCUUCAAACUGGGAC 95

968 GGUAUGAGCAUAGGCUCAU 96 AUGAGCCUAUGCUCAUACC 97

998 GGCCCAAGCUAUGAAAUCA 98 UGAUUUCAUAGCUUGGGCC 99 1001 CCCAAGCUAUGAAAUCAGA 100 UCUGAUUUCAUAGCUUGGG 101

1127 CAGAUGGCAAGACAGUAGA 102 UCUACUGUCUUGCCAUCUG 103

1133 GCAAGACAGUAGAAGCAGA 104 UCUGCUUCUACUGUCUUGC 105 1184 GCAUGUACCAGAAAGGACA 106 UGUCCUUUCUGGUACAUGC 107 1214 CCAAUCCCAUUGCUUCCAU 108 AUGGAAGCAAUGGGAUUGG 109 1257 CCACAGAGCAAAGCUUGAU 110 AUCAAGCUUUGCUCUGUGG 111

1258 CACAGAGCAAAGCUUGAUA 112 UAUCAAGCUUUGCUCUGUG 113

1262 GAGCAAAGCUUGAUAACAA 114 UUGUUAUCAAGCUUUGCUC 115 1285 GAGCUUGCCUUCUUUGCAA 116 UUGCAAAGAAGGCAAGCUC 117 1296 CUUUGCAAAUGCUUUGGAA 118 UUCCAAAGCAUUUGCAAAG 119

1301 CAAAUGCUUUGGAAGAAGU 120 ACUUCUUCCAAAGCAUUUG 121

1307 CUUUGGAAGAAGUCUCUAU 122 AUAGAGACUUCUUCCAAAG 123 1312 GAAGAAGUCUCUAUUGAGA 124 UCUCAAUAGAGACUUCUUC 125 1315 GAAGUCUCUAUUGAGACAA 126 UUGUCUCAAUAGAGACUUC 127

1356 GGACUUGGCUGCUUGCAUU 128 AAUGCAAGCAGCCAAGUCC 129

1359 CUUGGCUGCUUGCAUUAAA 130 UUUAAUGCAAGCAGCCAAG 131

1371 CAUUAAAGGUUUACCCAAU 132 AUUGGGUAAACCUUUAAUG 133

1385 CCAAUGUGCAACGUUCUGA 134 UCAGAACGUUGCACAUUGG 135

1390 GUGCAACGUUCUGACUACU 136 AGUAGUCAGAACGUUGCAC 137

1396 CGUUCUGACUACUUGAAUA 138 UAUUCAAGUAGUCAGAACG 139

1415 CAUUUGAGUUCAUGGAUAA 140 UUAUCCAUGAACUCAAAUG 141 US 10,610,125 B2 89 90 TABLE 7 - continued siRNAs targeting wildtype IDH1 Position on mRNA SEQ SEO ( FIG . ID ID 21B ) sense ( 5 ' to 3 ' ) NO : antisense ( 5 ) to 3 ' ) NO :

1422 GUUCAUGGAUAAACUUGGA 142 UCCAAGUUUAUCCAUGAAC 143 1425 CAUGGAUAAACUUGGAGAA 144 UUCUCCAAGUUUAUCCAUG 145 1455 CAAACUAGCUCAGGCCAAA 146 UUUGGCCUGAGCUAGUUUG 147 1487 CCUGAGCUAAGAAGGAUAA 148 UUAUCCUUCUUAGCUCAGG 149 1493 CUAAGAAGGAUAAUUGUCU 150 AGACAAUUAUCCUUCUUAG 151

1544 CUGUGUUACACUCAAGGAU 152 AUCCUUGAGUGUAACACAG 153 1546 GUGUUACACUCAAGGAUAA 154 UUAUCCUUGAGUGUAACAC 155

1552 CACUCAAGGAUAAAGGCAA 156 UUGCCUUUAUCCUUGAGUG 157

1581 GUAAUUUGUUUAGAAGCCA 158 UGGCUUCUAAACAAAUUAC 159

1646 GUUAUUGCCACCUUUGUGA 160 UCACAAAGGUGGCAAAAC 161

1711 CAGCCUAGGAAUUCGGUUA 162 UAACCGAAUUCCUAGGCUG 163

1713 GCCUAGGAAUUCGGUUAGU 164 ACUAACCGAAUUCCUAGGC 165

1714 CCUAGGAAUUCGGUUAGUA 166 UACUAACCGAAUUCCUAGG 167

1718 GGAAUUCGGUUAGUACUCA 168 UGAGUACUAACCGAAUUCC 169

1719 GAAUUCGGUUAGUACUCAU 170 AUGAGUACUAACCGAAUUC 171

1725 GGUUAGUACUCAUUUGUAU 172 AUACAAAUGAGUACUAACC 173

1730 GUACUCAUUUGUAUUCACU 174 AGUGAAUACAAAUGAGUAC 175

1804 GGUAAAUGAUAGCCACAGU 176 ACUGUGGCUAUCAUUUACC 177

1805 GUAAAUGAUAGCCACAGUA 178 UACUGUGGCUAUCAUUUAC 179 1816 CCACAGUAUUGCUCCCUAA 180 UUAGGGAGCAAUACUGUGG 181 1892 GGGAAGUUCUGGUGUCAUA 182 UAUGACACCAGAACUUCCC 183 1897 GUUCUGGUGUCAUAGAUAU 184 AUAUCUAUGACACCAGAAC 185

1934 GCUGUGCAUUAAACUUGCA 186 UGCAAGUUUAAUGCACAGC 187

1937 GUGCAUUAAACUUGCACAU 188 AUGUGCAAGUUUAAUGCAC 189

1939 GCAUUAAACUUGCACAUGA 190 UCAUGUGCAAGUUUAAUGC 191

1953 CAUGACUGGAACGAAGUAU 192 AUACUUCGUUCCAGUCAUG 193

1960 GGAACGAAGUAUGAGUGCA 194 UGCACUCAUACUUCGUUCC 195

1961 GAACGAAGUAUGAGUGCAA 196 UUGCACUCAUACUUCGUUC 197 1972 GAGUGCAACUCAAAUGUGU 198 ACACAUUUGAGUUGCACUC 199 1976 GCAACUCAAAUGUGUUGAA 200 UUCAACACAUUUGAGUUGC 201

1982 CAAAUGUGUUGAAGAUACU 202 AGUAUCUUCAACACAUUUG 203

1987 GUGUUGAAGAUACUGCAGU 204 ACUGCAGUAUCUUCAACAC 205

1989 GUUGAAGAUACUGCAGUCA 206 UGACUGCAGUAUCUUCAAC 207

2020 CCUUGCUGAAUGUUUCCAA 208 UUGGAAACAUUCAGCAAGG 209

2021 CUUGCUGAAUGUUUCCAAU 210 AUUGGAAACAUUCAGCAAG 211

2024 GCUGAAUGUUUCCAAUAGA 212 UCUAUUGGAAACAUUCAGC 213