USOO924.9466B2
(12) United States Patent (10) Patent No.: US 9.249,466 B2 Yu et al. (45) Date of Patent: Feb. 2, 2016
(54) METHODS AND COMPOSITIONS FOR WO WO-2007/017047 A1 2/2007 TREATING CANCER WO WO-2007/047754 A2 4/2007 WO WO-2008.005457 A2 1, 2008 (75) Inventors: Qiang Yu, Singapore (SG); Jing Tan, OTHER PUBLICATIONS Singapore (SG) Peifer et al.New anti-cancer role for PDK1 inhibitors: preventing (73) Assignee: Agency for Science, Technology and resistance to tamoxifen, 2009, Biochem J. 417.e5-e7. Research, Singapore (SG) Feldman et al., Novel Small Molecule Inhibitors of 3-Phosphoinositide-dependent Kinase-1. The Journal of Biological (*) Notice: Subject to any disclaimer, the term of this Chemistry, 2005, pp. 19867-19874, vol. 280, No. 20. patent is extended or adjusted under 35 Falasca et al. A novel inhibitor of the Pl3k/Akt pathway based on the U.S.C. 154(b) by 0 days. structure of inositol 1,3,4,5,6-pentakisphosphate, British Journal of Cancer, 2010, pp. 104-114, vol. 102. (21) Appl. No.: 13/520,472 Peifer et al., New anti-cancer role for PDK1 inhibitors: preventing resistance to tamoxifen, Biochem. J., 2009, pp. e5-e7, vol. 417. (22) PCT Filed: Jan. 10, 2011 Weisberg et al., Potentiation of antileukemic therapies by the dual P13k/PDK-1 inhibitor BAG956: effects on BCR-ABL- and mutant (86). PCT No.: PCT/SG2O11AOOOO11 FLT3-expressing cells, Blood, 2008, pp. 3723-3734, vol. 111, No. 7. Jing Tan, et al., B55 B-Associated PP2A Complex Controls PDK1 S371 (c)(1), Directed Myc Signaling and Modulates Rapamycin Sensitivity in (2), (4) Date: Jul. 3, 2012 Colorectal Cancer, Cancer Cell, 2010, 459-471, vol. 18. International Search Report for PCT/SG2011/000011, dated Apr. 8, (87) PCT Pub. No.: WO2011/084108 2011, 5 pages. PCT Pub. Date: Jul. 14, 2011 Written Opinion for for PCT/SG2011/000011, dated Apr. 8, 2011, 11 pageS. (65) Prior Publication Data Tan, J. and Yu, Q, PDK1-driven Myc signaling regulates cellular response to mTOR inhibitors, Cell Cycle, 10(7): 1019-1020 (2011). US 2013/O123328A1 May 16, 2013 Yuan, R.R. et al., Targeting tumorigenesis: development and use ofm TOR inhibitors in cancer therapy, Journal of Hematology & Oncol (30) Foreign Application Priority Data ogy, 2(45): 1-12 (2009). Jan. 8, 2010 (SG) ...... 201OOO114-7 * cited by examiner Mar. 4, 2010 (SG) ...... 2O1 OO1629-3 Primary Examiner — Mark Shibuya (51) Int. Cl. Assistant Examiner — Kate Poliakova CI2N IS/II (2006.01) CI2O I/68 (2006.01) (74) Attorney, Agent, or Firm — Choate, Hall & Stewart A6 IK 45/06 (2006.01) LLP; Charles E. Lyon; Nishat A. Shaikh GOIN33/574 (2006.01) A6 IK3I/436 (2006.01) (57) ABSTRACT A6 IK3I/506 (2006.01) We describe a method of determining whether a cancer cell is A6 IK3I/713 (2006.01) likely to be resistant to treatment by an mTOR inhibitor. The CI2O I/42 (2006.01) method may comprise detecting PPP2R2B (GenBank Acces GOIN33/68 (2006.01) sion Number: NM 18167) in or of the cell. It may, alterna (52) U.S. Cl. tively, or in addition, comprise detecting PDK1 (GenBank CPC ...... CI2O I/6886 (2013.01); A61 K31/436 Accession Number: NM 002613), in or of the cell. The (2013.01); A61 K3I/506 (2013.01); A61 K method may comprise detecting methylation of the PPP2R2B 3 1/713 (2013.01); A61K 45/06 (2013.01); promoter in or of the cell. It may comprise detecting the CI2O 1/42 (2013.01); G0IN33/57407 expression and/or activity of PPP2R2B in or of the cell. It may (2013.01); G0IN33/6893 (2013.01) comprise detecting PDK1 mediated Myc phosphorylation (58) Field of Classification Search activity. Methods of choosing a treatment for an individual None Suffering from or Suspected to be suffering from a cancer, See application file for complete search history. determining whether an individual Suffering from or Sus (56) References Cited pected to be suffering from a cancer will respond to treatment by an mTOR inhibitor, increasing the sensitivity of a cancer U.S. PATENT DOCUMENTS cell to treatment by an mTOR inhibitor, for treating or pre venting cancer in an individual Suffering or Suspected to be 2004/O186065 A1 9/2004 Ionescu et al. suffering from cancer are also provided. We further provide 2005/0216961 A1 9/2005 Delaney for a combination of an inhibitor of PDK1 expression and/or FOREIGN PATENT DOCUMENTS activity and an mTOR inhibitor for use in a method of treat ment or prevention of cancer. GB 2488O28 A 8, 2012 WO WO-2005/007846 A1 1, 2005 18 Claims, 85 Drawing Sheets U.S. Patent Feb. 2, 2016 Sheet 1 of 85 US 9.249,466 B2
FIGURE A PPP2R2B -N 12.5 CN O) O 5 10.0 C/D C/D 9 7.5 *p-0.001 CD CC Z n1, 5.0 E $S. 2.5 S. O.O N(24) T(24) U.S. Patent Feb. 2, 2016 Sheet 2 of 85 US 9.249,466 B2
*:::::83. is
U.S. Patent Feb. 2, 2016 Sheet 3 Of 85 US 9.249,466 B2
::::::8: {
& 8:
iii.2:32:3
g; ; ; 3.8
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8:::::::
Coion cancer Cell : : x8 8 5 x C 3.
s p---2; 3
pff-p2A
popf SC
{A-Off
U.S. Patent Feb. 2, 2016 Sheet 6 of 85 US 9.249,466 B2
:::::::::::
*::::::::: U.S. Patent Feb. 2, 2016 Sheet 7 Of 85 US 9.249,466 B2
{{8:8: {
{ - O Xs O Y O O <
PPP2R2B
GAPD U.S. Patent Feb. 2, 2016 Sheet 8 of 85 US 9.249,466 B2
*::::::::::
HCT116 DKO
PPP2R2B U.S. Patent US 9.249,466 B2
FIGURE 1
CpG island BGS
U.S. Patent Feb. 2, 2016 Sheet 10 Of 85 US 9.249,466 B2
FIGURE 2A DLD1-PPP2R2B DOX: Anti-myc ... a-PPP2R2B-myc(B55) Anti-B55 Alias Endogenous B55o.
U.S. Patent Feb. 2, 2016 Sheet 11 of 85 US 9.249,466 B2
*:::::::::: SA-B-gal
U.S. Patent Feb. 2, 2016 Sheet 12 of 85 US 9.249,466 B2
FIGURE 2C
50
4 O
1 O
O Vector PPP2R2B U.S. Patent Feb. 2, 2016 Sheet 13 of 85 US 9.249,466 B2
8: 388. 3:
C8 2: 3:3 - - - - *** **w
U.S. Patent Feb. 2, 2016 Sheet 14 of 85 US 9.249,466 B2
FIGURE 2E
1000 rer Vector+PBS Vector--Dox
900t-- PPP2R2B+PBS 8 800 PPP2R2B+Dox
O 1 3 5 6 9 10 13 15 16 17 20 2123 Days of treatment U.S. Patent Feb. 2, 2016 Sheet 15 Of 85 US 9.249,466 B2
FIGURE 2F 1.2 O
O. 2 U.S. Patent Feb. 2, 2016 Sheet 16 of 85 US 9.249,466 B2
FIGURE 2G SiNC
-- rr - - -- a------
SiPPP2R2BiSP SiPPP2R - E. . . ;-- G -
. sp h " a , u ise r i ------...- ... .".
U.S. Patent Feb. 2, 2016 Sheet 18 of 85 US 9.249,466 B2
FIGURE 3B
U.S. Patent Feb. 2, 2016 Sheet 19 Of 85 US 9.249,466 B2
PPP2R2B-mye
p-S6(S235,236)
payo Myc
B-Actin FIGURE 3C U.S. Patent Feb. 2, 2016 Sheet 20 Of 85 US 9.249,466 B2
IP: PPP2R2Binyo .C.
Wector Fpp2R2B Wecief PPP2R2B
0X. . g0 A. x
PP2R A
PP2AC
FIGURE 3D U.S. Patent Feb. 2, 2016 Sheet 21 of 85 US 9.249,466 B2
FIGURE 3E
- 41D CtrlDOX 2 3 CC S n 2 CD 2 (V 1 n1
O Vector PPP2R2B
U.S. Patent Feb. 2, 2016 Sheet 23 Of 85 US 9.249,466 B2
FIGURE 3G
HCT 116 31000 SiNC O redesiMyc y 750-v-sis6K
O E D C 5 O U.S. Patent Feb. 2, 2016 Sheet 24 of 85 US 9.249,466 B2
FIGURE 4A DLD1-PPP2R2B 1OOO-- Ctrl a DOx Rapamycin
500
250 U.S. Patent Feb. 2, 2016 Sheet 25 Of 85 US 9.249,466 B2
FIGURE 4B
DLD1-Vector DLD1-PPP2R2B
U.S. Patent Feb. 2, 2016 Sheet 26 of 85 US 9.249,466 B2
DLD1-PPP2R2B 75 s S. 50 CVS C Ol d o 25 CD
O Ctrl Dox Rapa R+D U.S. Patent Feb. 2, 2016 Sheet 27 Of 85 US 9.249,466 B2
FIGURE 4D
4 O O 300 200 1OO
1 3 5 7 9 11 14 16 18 Days of treatment U.S. Patent Feb. 2, 2016 Sheet 28 Of 85 US 9.249,466 B2
FIGURE 4E VectOr PPP2R2B Rapa: - - + + - - + + DOX: - + - + - + - + PPP2R2B-myc p-S6K(T421/S424) is sets as U.S. Patent Feb. 2, 2016 Sheet 29 Of 85 US 9.249,466 B2
Myc
p-AKTS473)
AK
FIGURE 4F U.S. Patent Feb. 2, 2016 Sheet 30 of 85 US 9.249,466 B2
FIGURE 4G Y .C. Y (Y
U.S. Patent Feb. 2, 2016 Sheet 31 of 85 US 9.249,466 B2
FIGURE 5A DLD1 (serum starved)
4. 8 24 (h) U.S. Patent Feb. 2, 2016 Sheet 32 Of 85 US 9.249,466 B2
P-403. p-Mye
MyC
p-AKI (S473)
AKT FIGURE 5B U.S. Patent Feb. 2, 2016 Sheet 33 of 85 US 9.249,466 B2
FIGURE SC
U.S. Patent Feb. 2, 2016 Sheet 34 of 85 US 9.249,466 B2
FIGURE SD
p-AKT(S473) p-S6K(T389) is
U.S. Patent Feb. 2, 2016 Sheet 35 of 85 US 9.249,466 B2
FIGURE SE
U.S. Patent Feb. 2, 2016 Sheet 36 of 85 US 9.249,466 B2
FIGURE SF
its seat U.S. Patent Feb. 2, 2016 Sheet 37 Of 85 US 9.249,466 B2
FIGURE 6A 293T
P: O-HA Input PPP2R2B-Myc: ------HA-PDK1: - - - - + + ol-MyC
O-PDK1
P: O-MyC Input PPP2R2B-Myc: - - - -
HA-PDK1: + - -- O-HA U.S. Patent Feb. 2, 2016 Sheet 38 of 85 US 9.249,466 B2
FIGURE 6B DLD1-PPP2R2B P: O-MyC Input
U.S. Patent Feb. 2, 2016 Sheet 39 Of 85 US 9.249,466 B2
FIGURE 6C
Membrane DOX: - -- --
U.S. Patent Feb. 2, 2016 Sheet 41 of 85 US 9.249,466 B2
*::::::::: { Normal (p-PDK1) U.S. Patent Feb. 2, 2016 Sheet 42 of 85 US 9.249,466 B2
FIGURE 7A
HCT 116 50 & 5 40 o 30 - q . 20 or isa 10 O) O siRNA. NC Myc PDK1 AKT1 PIK3CA U.S. Patent Feb. 2, 2016 Sheet 43 of 85 US 9.249,466 B2
FIGURE 7B SW48O -N75 s O CD 5 O 50 C - C - S. (r.y is3 25 - O) O ShNC shPDK1 U.S. Patent Feb. 2, 2016 Sheet 44 of 85 US 9.249,466 B2
FIGURE 7C
DD1 SW48O 50
<+coCN– 2O
10 OOC-DO cS V 6 U.S. Patent Feb. 2, 2016 Sheet 45 of 85 US 9.249,466 B2
FIGURE 7D DLD1 SW480
7 5 O Ct r --BX912 -- Rapa 500-BX912+Rapa
250
O U.S. Patent Feb. 2, 2016 Sheet 46 of 85 US 9.249,466 B2
FIGURE 7E
Rapa Rapa--BX912 Rapa Rapa--BX912 U.S. Patent Feb. 2, 2016 Sheet 47 of 85 US 9.249,466 B2
*::::::
*8883:08 U.S. Patent Feb. 2, 2016 Sheet 48 of 85 US 9.249,466 B2
FIGURE 8
Normal PPP2R2B is Tumor
Bladder Esophagus 2.5 20 . . os |EFLIESLI i. I 0.5 -10 2 15 2.0 U.S. Patent Feb. 2, 2016 Sheet 49 Of 85 US 9.249,466 B2
:::::::::::: A S-8 ---assasssssssssssssssssssssssssss
s U.S. Patent Feb. 2, 2016 Sheet 50 Of 85 US 9.249,466 B2
& 3:38. pMN pMN-PPP2R2B
s s U.S. Patent Feb. 2, 2016 Sheet 51. Of 85 US 9.249,466 B2
FIGURE 10A
U.S. Patent Feb. 2, 2016 Sheet 52 Of 85 US 9.249,466 B2
FIGURE 10B NIH/3T3raS QQ CN QC CN Q Q Q
U.S. Patent Feb. 2, 2016 Sheet 53 Of 85 US 9.249,466 B2
FIGURE 11A
U.S. Patent Feb. 2, 2016 Sheet 54 of 85 US 9.249,466 B2
FIGURE 11B
CD IP: PPP2R2B-myc 9 -
DOX: ------
PP2AA subla as U.S. Patent Feb. 2, 2016 Sheet 55 of 85 US 9.249,466 B2
FIGURE 12
DLD1-Vector DLD1-PPP2R2B Ctrl Ctrl DOX 3 8 8 8 o 8 3 P c s > is N N o C) is 3 3 3
0 200 400 600 800 1000 Bobo 6, 200, 460 600 800 000 O 200 400 600 800 1000 DNA contents DNA contents DNA contents c & s 8 5 s g ce E 5 5 s 8 3 3 e o Y rt w q ed C O 200 400 600, 800 1000 O 200 400 600 800 tooo DNA contents DNA contents DNA contents DNA contents U.S. Patent Feb. 2, 2016 Sheet 56 of 85 US 9.249,466 B2
FIGURE 13A
U.S. Patent Feb. 2, 2016 Sheet 57 Of 85 US 9.249,466 B2
FIGURE 13B
12 E. - HCT116
100 SS 80 6 s d 40 O 2O O Rapa (dayS) U.S. Patent Feb. 2, 2016 Sheet 58 Of 85 US 9.249,466 B2
FIGURE 13C
U.S. Patent Feb. 2, 2016 Sheet 59 of 85 US 9.249,466 B2
FIGURE 13D 100
8 O
6 O
4 O
2 O
HT29 HCT116 SW480 HT25 RKO SW403 U.S. Patent Feb. 2, 2016 Sheet 60 of 85 US 9.249,466 B2
FIGURE 14A
Renal Liver Lymphoma Ovarian
O.5 O.O -0.5 - 1.0 : U.S. Patent Feb. 2, 2016 Sheet 61 of 85 US 9.249,466 B2
FIGURE 14B PPP2R2B expression
còcoCNCNJCN) FIGURE 14C U.S. Patent Feb. 2, 2016 Sheet 63 of 85 US 9.249,466 B2 FIGURE 14D 120 Rapamycin 10nM treated for 5 days 1OO 8O 60 40 20 U.S. Patent Feb. 2, 2016 Sheet 64 of 85 US 9.249,466 B2 FIGURE 15A DAPI PPP2R2B-myc Merge 5 s X rare arra s' '. Y s 's P. X Y, " . . . X c S. A . s U.S. Patent Feb. 2, 2016 Sheet 65 of 85 US 9.249,466 B2 FIGURE 1SB DAPI P-PDK1 Merge U.S. Patent Feb. 2, 2016 Sheet 66 of 85 US 9.249,466 B2 *::::::::::: DAP p-Myc Merge U.S. Patent Feb. 2, 2016 Sheet 67 of 85 US 9.249,466 B2 FIGURE 16A PDK1 inhibitor BX912 PIK3CA inhibitor PK90 -: .N. s ...... sp. U.S. Patent Feb. 2, 2016 Sheet 68 of 85 US 9.249,466 B2 FIGURE 16B 3 DMSO (s BX912 PK90 s S G2/M: s 19.5% S. s S. . . . . s 200400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 DNA contents DNA Contents DNA Contents s Rapa-BX912 Rapa-PK90 s s 3 S. 9. S. s s s ed 200 400. 600 800 1000 200 400 soo 800 1000 O 200 400 600 800 1000 DNA contents DNA contents DNA contents d DMSO s BX912 s PIK90 st- s 3 3 3 s R S 9. s S. grid, Sr.'Essa's, 5"::"... DNA contents DNA contents DNA contents Rapa Rapa-BX912 Rapa--PIK90 200 400 600 8 200 400 600 800 1000 DNA contents DNA contents DNA contents U.S. Patent Feb. 2, 2016 Sheet 69 Of 85 US 9.249,466 B2 FIGURE 17 B 2536 -0-hek tervpmn --hek tervpdk1 hek terv myc sh pten 0.65 1.25mm 2.5m Sinn conc U.S. Patent Feb. 2, 2016 Sheet 70 of 85 US 9.249,466 B2 FIGURE 17 (CONTINUED) GW 843682)K --hek terv pmn o -D-hek tervpdk O hek terv myc Co o U 0.0 0.01um 0.05um 0.1Um 2.5um COC U.S. Patent Feb. 2, 2016 Sheet 71 Of 85 US 9.249,466 B2 FIGURE 18 HEK-TERV a 75 S 8 3 50 SS D 25 g O pMN PDK1 PIK3CA-E545 B2536 (10nM, 4days) RWPE1 57 O5 2 5 pMN PDK PIK3CA-E545 B2536 (10nM, 4days) U.S. Patent Feb. 2, 2016 Sheet 72 Of 85 US 9.249,466 B2 K.R. 50 4. ge 30 O K K3CAES5 FIGURE 19 U.S. Patent Feb. 2, 2016 Sheet 73 Of 85 US 9.249,466 B2 FIGURE 20A HEK-TERV A 2 Na () S X Ol n > U.S. Patent Feb. 2, 2016 Sheet 74. Of 85 US 9.249,466 B2 FIGURE 20B HMEC RWPE B ATCC1 ATCC2 RWPE1 RWPE2 n U.S. Patent Feb. 2, 2016 Sheet 75 Of 85 US 9.249,466 B2 FIGURE 21 MyC - + - + PLK1 - - + + p-MyC Myc Actinium is a U.S. Patent Feb. 2, 2016 Sheet 76 of 85 US 9.249,466 B2 FIGURE 22 HEK-PDK1 U.S. Patent Feb. 2, 2016 Sheet 77 of 85 US 9.249,466 B2 FIGURE 23 B 2536: - -- - - BEZ235: p-Myc(S62) p-AKT(S473), 3rasaacsitatasaakaasa.e : see . As se: res left it takes A Cti suggestinese:stees:Sis be • . . " ... • :- '...... "... a- if U.S. Patent Feb. 2, 2016 Sheet 78 of 85 US 9.249,466 B2 FIGURE 24 SW480 750 -s- DMSO -- B2536 -- BEZ 235 -- B-BEZ 750 - DMSO -- B2536 S. --- BEZ 235 9 -- B-BEZ 3, 500 5 O E s E 250 5 O U.S. Patent Feb. 2, 2016 Sheet 79 of 85 US 9.249,466 B2 FIGURE 25 SW480 48h 40 N 30 SS 5d 2O o CV) 10 is . O. l I. DMSO B2356 BEZ235 B-BEZ DLD1 48h 50 40 s NF 30 p O D 20 CO 10 O DMSO B2356 BEZ235 B+BEZ U.S. Patent Feb. 2, 2016 Sheet 80 Of 85 US 9.249,466 B2 FIGURE 26 U.S. Patent Feb. 2, 2016 Sheet 81 of 85 US 9.249,466 B2 FIGURE 27 HEKPDK1 U.S. Patent Feb. 2, 2016 Sheet 82 of 85 US 9.249,466 B2 FIGURE 28 Control B2536 U.S. Patent Feb. 2, 2016 Sheet 83 of 85 US 9.249,466 B2 *:::: 88, Control S-K- six U.S. Patent Feb. 2, 2016 Sheet 84 of 85 US 9.249,466 B2 FIGURE 30 SOX2 1OO LN28B 5 150 75 9. 2 Os i OO GDX 50 O.s G o O) t 2 as 25 s 50 4. 4. O--Ea- O w PMN PDK1 MYC shipTEN PMN PK1 MYC shon FOXA2 ALDH1A2 10.0 2OO 5 5 o2 7.5 150 O. Ol s 5.0 0X 100 2 g w vs naas 2.5 2s 50 O.O . O PMN PDK1 MYC shipTEN PMN PDK1 MYC shon U.S. Patent Feb. 2, 2016 Sheet 85 of 85 US 9.249,466 B2 FIGURE 31 HEK-TERV 400-s-PDK1(1x103) (Y) --MYC(1x10) --MYC(1x10) 23 OO OO 10 O 1 O 13 16 19 22 Time (days) US 9.249,466 B2 1. 2 METHODS AND COMPOSITIONS FOR phosphorylation through PI3K and mTORC2 (O'Reilly et al., TREATING CANCER 2006; Sarbaisov et al., 2006), further understanding the resis tance mechanism and identifying the biomarkers that help to This application is a U.S. National Stage of International predict the therapeutic response have become important top Application No. PCT/SG11/00011, filed Jan. 1, 2011, which 5 ics (Mao et al., 2008; Scott et al., 2009: Thomas et al., 2006). claims the benefit of SG 201000114-7, filed Jan. 8, 2010 and SG 201001629-3. filed Mar. 4, 2010, the entire content of SUMMARY each of which is hereby incorporated herein by reference. Here, we describe a B subunit of the PP2A holoenzyme, SEQUENCE LISTING 10 PPP2R2B, encoding B55 B, epigenetically inactivated by pro moter DNA hypermethylation in human colorectal cancer The instant application contains a Sequence Listing which (CRC). has been submitted electronically in ASCII format and is We show that PP2A-PPP2R2B complex functions as a hereby incorporated by reference in its entirety. Said ASCII tumor suppressor by mitigating PDK1-directed Myc phos copy, created on Nov. 4, 2015, is named 2008187 15 phorylation. Loss of this specific PP2A complex in CRC 0034 SL.txt and is 26,017 bytes in size. resulted in strong Myc phosphorylation in response to FIELD mTORC1 inhibitor rapamycin, which confers rapamycin resistance. The present invention relates to the fields of medicine, cell Intriguingly, unlike rapamycin-induced AKT phosphory biology, molecular biology and genetics. This invention lation, rapamycin-induced Myc phosphorylation does not relates to the field of medicine. In particular, it relates to require P13KCA, but is PDK1-dependent. treatment and diagnosis of diseases such as cancer, as well as We therefore identify a new tumor suppressor mechanism compositions for Such use. controlling PDK1-Myc signaling and show that a defect in 25 this regulation leads to resistance to mTOR-based cancer BACKGROUND therapy. According to a 1 aspect of the present invention, we pro Protein phosphatase 2A (PP2A) functions as a multimetric vide a method of determining whethera cancer cell is likely to enzyme that contains the catalytic C Subunit, a scaffolding be resistant to treatment by an mTOR inhibitor, the method A-Subunit and one of a large array of regulatory B-Subunits. 30 comprising detecting: (i) PPP2R2B (GenBank Accession Eukaryotic cells contain over 200 biochemical distinct PP2A Number: NM 181678) or (ii) PDK1 (GenBank Accession complexes derived from differential combinations of A, B, C Number: NM 002613), or both, in or of the cell. and other subunits. The regulatory Subunits are expressed in a The method may comprise detecting methylation of the tissue-specific manner, leading to the presence of different PPP2R2B promoter in or of the cell. The method may com PP2A complexes in different mammalian tissues (Virshup 35 and Shenolikar, 2009). Moreover, it is these regulators, rather prise detecting hypermethylation of the PPP2R2B promoter. than the catalytic subunit that provide the substrate specificity The methylation or hypermethylation may be compared to a and to catalyze distinct dephosphorylation events to impose cancer cell that is not mTOR inhibitor resistant. Detection of different functional outcomes. hypermethylation may indicate that the cancer cell is likely to Although a tumor suppressor role of PP2A has been shown 40 be resistant to treatment by an mTOR inhibitor. in a variety of immortalized human cell types (Chen et al., The method may comprise detecting expression or activity 2004; Eichhornet al., 2009; Janssens and Goris, 2001; Ran of PPP2R2B or PDK1, or both, in the cell. The PDK1 activity garajan et al., 2004; Sablina et al., 2007; Zhao et al., 2003), the may comprise PDK1 mediated Myc phosphorylation activity. genetic and/or the epigenetic evidence pointing to a prevalent The method may be such that a decreased expression and/ inactivation of PP2A in human malignancy have not been 45 or activity of PPP2R2B compared to a cancer cell that is not reported. Somatic mutations in the A subunit of the PP2A mTOR inhibitor resistant, indicates that the cancer cell is complex, which can result in the loss of B subunit binding likely to be resistant to treatment by an mTOR inhibitor. The (Ruediger et al., 2001), were found only in up to 15% of some method may be such that an increased expression and/or human cancers (Calin et al., 2000; Ruediger et al., 2001; activity of PDK1 compared to a cancer cell that is not mTOR Takagi et al., 2000; Tamaki et al., 2004; Wang et al., 1998: 50 inhibitor resistant, indicates that the cancer cell is likely to be Westermarck and Hahn, 2008), and the reduced expression of resistant to treatment by an mTOR inhibitor. PP2A subunit B56Y3 has been reported only in some cancer There is provided, according to a 2" aspect of the present cell lines (Chen et al., 2004; Zhao et al., 2003). In general, the invention, a method of choosing a treatment for an individual genetic or epigenetic changes of PP2A complexes in human Suffering from or Suspected to be suffering from a cancer, the cancer remains to be defined, as is its impact on cancer sig 55 method comprising: (a) providing a cell of the patient; (b) naling or therapeutic responses to targeted therapy. detecting mTOR inhibitor resistance of the cell by method as One of the PP2A regulated cancer signaling pathways is set out above; and (c) where the cell is determined not to be the mTOR pathway, a key component of PI3K pathway that mTOR inhibitor resistant, choosing an mTOR inhibitor as a many cancer cells are addicted to for growth advantage treatment for the individual, and where the cell is determined (Guertin and Sabatini, 2007; Sabatini, 2006). Although small 60 to be mTOR inhibitor resistant, choosing a different treatment molecule mTORC1 inhibitors, such as rapamycin and its for the individual. analogues have shown promising rationals for their use in We provide, according to a 3" aspect of the present inven cancer therapy and have been approved for clinical applica tion, a method of determining whetheran individual Suffering tion (Guertin and Sabatini, 2007; Hudes et al., 2007), these from or Suspected to be suffering from a cancer will respond inhibitors have had only limited successes and the clinical 65 to treatment by an mTOR inhibitor. The method may com outcome is unpredictable. Whilea known mechanism of rapa prise performing a method as set out above on a cell of the mycin resistance is linked to its feedback activation of AKT individual. The method may comprise, where the cell is deter US 9.249,466 B2 3 4 mined not to be mTOR inhibitor resistant, determining that ogy: DNA Structure Part A. Synthesis and Physical Analysis the individual is likely to respond to treatment by an mTOR of DNA Methods in Enzymology, Academic Press; Using inhibitor. Antibodies: A Laboratory Manual: Portable Protocol NO. I As a 4" aspect of the present invention, there is provided a by Edward Harlow, David Lane, Ed Harlow (1999, Cold method of increasing the sensitivity of a cancer cell to treat Spring Harbor Laboratory Press, ISBN 0-87969-544-7); ment by an mTOR inhibitor, the method comprising increas Antibodies: A Laboratory Manual by Ed Harlow (Editor), ing expression and/or activity of PPP2R2B, or decreasing David Lane (Editor) (1988, Cold Spring Harbor Laboratory expression and/or activity of PDK1, or both, in or of the cell. Press, ISBN 0-87969-314-2), 1855. Handbook of Drug We provide, according to a 5" aspect of the present inven Screening, edited by Ramakrishna Seethala, Prabhavathi B. tion, a method of treating or preventing cancer in an indi 10 vidual Suffering or Suspected to be suffering from cancer, the Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN method comprising increasing expression and/or activity of 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, PPP2R2B, or decreasing expression and/or activity of PDK1, Reagents, and Other Reference Tools for Use at the Bench, or both, in or of the individual. Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring The method may be such that expression of PPP2R2B is 15 Harbor Laboratory, ISBN 0-87969-630-3. Each of these gen increased by decreasing methylation of the PPP2R2B pro eral texts is herein incorporated by reference. moter. Methylation of the PPP2R2B promoter may be achieved by exposing the cell to, or administering to a patient, BRIEF DESCRIPTION OF THE FIGURES a demethylating agent Such as 5-aza-dC or AZacitidine. The method may comprise exposing the cell to, or admin FIGS. 1A-1I. Loss of PPP2R2B expression by promoter istering to a patient, a PDK1 inhibitor such as BX912 (CAS DNA hypermethylation in CRC Accession Number: 702674-56-4) or BX795 (CAS Acces FIG.1.A. Differential expression of PPP2R2B in 24 pairs of sion Number: 702675-74-9). patient-derived CRC and matched normal colon mucosa as The present invention, in a 6" aspect, provides a method of determined by Illumina array analysis: PPP2R2B-mRNA treating or preventing cancer in an individual Suffering or 25 expression is presented relative to log 2. ** p-0.001. Suspected to be suffering from cancer, the method comprising FIG. 1B. Hierarchical clustering of PP2A subunits expres administering an inhibitor of PDK1 expression and/or activ sion in 24 pairs of colorectal tumors (T) and matched normal ity together with an mTOR inhibitor. mucosa (N). In a 7" aspect of the present invention, there is provided a FIG. 1C. RT-PCR analysis of PPP2R2B expression from combination of an inhibitor of PDK1 expression and/or activ 30 eight randomly selected pairs of human CRC (T) and ity and an mTOR inhibitor for use in a method of treatment or matched mucosa (N). B-actin was used as a PCR control. prevention of cancer. FIG. 1D RT-PCR analysis of PPP2R2B, PPP2R2A and According to an 8” aspect of the present invention, we PPP2R5C inapanel of CRC cell lines compared to the normal provide use of a combination of an inhibitor of PDK1 expres colon tissue. sion and/or activity and an mTOR inhibitor in the manufac 35 FIG. 1E. Methylation specific PCR (MSP) analysis of ture of a medicament for the treatment or prevention of can PPP2R2B promoter in CRC cell lines. C. FIG. 1F. MSP analysis of PPP2R2B promoter in eight We provide, according to a 9' aspect of the invention, use tumor and normal controls of PPP2R2B or PDK1, or both, as a biomarker for sensitivity FIG. 1G. RT-PCR analysis of PPP2R2B in HCT116 and of a cancer cell to an mTOR inhibitor or for colorectal cancer 40 DKO or DLD1 cells treated with or without 5-AzaC (5 M) (CRC). Such as rapamycin resistance colorectal cancer. for 3 days. The mTOR inhibitor may comprise rapamycin or a deriva FIG.1H. MSP analysis of PPP2R2B promoter in HCT116 tive thereof. The PDK1 inhibitor may comprise a shRNA, and DKO cells. BX912 or BX795 (or any combination of these). FIG. 1I. Methylation analysis of PPP2R2B promoter by There is provided, in accordance with a 10" aspect of the 45 bisulfite-sequencing analysis (BGS). The region analyzed is present invention, PDK1 or an antagonist thereof for use in a indicated. The arrow indicates the transcriptional start site. method of treating colorectal cancer (CRC). Open circles represent unmethylated CpGs; closed circles The cancer may be selected from the group consisting of denote methylated CpGs. colorectal cancer (CRC), bladder cancer, brain cancer and FIGS. 2A-2G. Gain- or loss-of-function analysis of oesophageal cancer, preferably colorectal cancer (CRC). 50 PPP2R2B as a tumor suppressor The practice of the present invention will employ, unless FIGS. 2A. Immunoblot analysis of DLD1-PPP2R2B cells otherwise indicated, conventional techniques of chemistry, on endogenous B55C. and ectopic B55 B-Myc, using anti-Myc molecular biology, microbiology, recombinant DNA and tag or anti-B55 subunit antibody. Cells were treated with or immunology, which are within the capabilities of a person of without 1.0 g/ml Doxycycline (Dox) for 3 days. ordinary skill in the art. Such techniques are explained in the 55 FIG. 2B. SA-B-gal assay of DLD1-PPP2R2B or vector literature. See, for example, J. Sambrook, E. F. Fritsch, and T. control cells treated with or without Dox for 96 hr. Scale Maniatis, 1989, Molecular Cloning: A Laboratory Manual, bars=100 uM. Second Edition, Books 1-3, Cold Spring Harbor Laboratory FIG.2C. BrdU incorporation of DLD1-PPP2R2B or vector Press; Ausubel, F. M. et al. (1995 and periodic supplements: control cells treated with or without Dox for 96 h. Shown is Current Protocols in Molecular Biology, ch. 9, 13, and 16, 60 mean+S.D. of three independent experiments. ** p-0.001. John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and FIG. 2D. Anchorage-independent growth assessed by soft A. Kahn, 1996, DNA Isolation and Sequencing. Essential agar assay in DLD1-PPP2R2B or HCT116-PPP2R2B cells, Techniques, John Wiley & Sons; J. M. Polak and James O'D. treated with or without Dox for 12 days. McGee, 1990. In Situ Hybridization: Principles and Prac FIG. 2E. Xenograft tumor growth of DLD1-PPP2R2B or tice: Oxford University Press: M. J. Gait (Editor), 1984, Oli 65 control cells in nude mice treated with Dox at 100 mg/kg daily gonucleotide Synthesis. A Practical Approach, Irl Press: D. as described in Experimental Procedures. Error bars repre M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymol sents S.D. US 9.249,466 B2 5 6 FIG. 2F and FIG. 2G. HEK-TERV cells were transfected FIG. 5C. Immunoblot analysis of DLD1 cells for Myc, with a PPP2R2B Smart pool siRNA (SP) or an independent AKT, PI3K, or PDK1. Cells were transfected with siRNAs PPP2R2B siRNA and the PPP2R2B mRNA was assessed by targeting the indicated genes or a negative control siRNA for Taqman assay (FIG. 2F) and the colony formation capacity 48 h, followed by 10 nM rapamycin treatment for 24hr. was assessed by soft-agar assay (FIG. 2G). Shown on the left FIG.5D. Immunoblot analysis of PDK1, Myc and AKT in are representative images of three independent experiments. DLD1 cells transfected with two different PDK1 siRNAS or a SV40 small antigen (ST) expressing HEK-TERV cells were negative control siRNA and then treated with 10 nM rapamy used as positive control. cin for 24hr. FIGS. 3A-3G. Restoration of PPP2R2B-PP2A complex FIG.5E. Immunoblot analysis of PDK1, Myc and AKT in results in inhibition of p70S6K and Myc phosphorylation 10 SW480 cells infected with a retroviral PDK1 shRNA. FIG. 3A. Immunoblot analysis of HCT116-PPP2R2B or FIG. 5F. Immunoblot analysis of Myc, AKT and S6K in the vector control cells for indicated proteins in the presence DLD1 cells treated with PDK1 inhibitor BX912 (2.5uM), or or absence of Dox for 48 hr. PIK3CA inhibitor PIK90 (5 uM), rapamycin (10 nM) or FIG. 3B and FIG. 3C. Immunoblot analysis of DLD1 indicated combinations for 48 hr. PPP2R2B or the vector control cells treated with or without 15 FIGS. 6A-6E. PPP2R2B binds to PDK1 and inhibits its Dox for the indicated length of times. activity that is upregulated in CRC. FIG. 3D. Co-immunoprecipitation of PP2A scaffolding A FIG. 6A. Co-immunoprecipitation assay in 293T cells subunit and catalytic C subunit with PPP2R2B. DLD1 transfected with PPP2R2B-Myc, PDK1-HA, or both. PPP2R2B or vector control cells were treated with Dox and FIG. 6B. Co-immunoprecipitation assays in DLD1 PPP2R2B was immunoprecipitated with anti-Myc tag anti PPP2R2B cells. Cells were treated with or without Dox for 24 body. WCL, whole cell lysates. h and PPP2R2B-Myc or PDK1 were pulled down and sub FIG.3E. Serine/threonine phosphatase activity for DLD1 jected to immunoblot analysis. PPP2R2B or vector control cells. Protein phosphatase activ FIG. 6C. Immunoblotting analysis of the membrane frac ity of the immunoprecipitates of PPP2R2B-Myc was mea tions on PDK1 prepared from DLD1-PPP2R2B cells treated Sured in triplicates from three independent experiments. 25 with or without Dox for 48 hr. FIG. 3F. Immunoblot analysis of DLD1-PPP2R2B cells FIG. 6D. Immunofluorescence for PDK1-HA in DLD1 for Myc and p70S6K, treated PPP2R1A siRNA or a negative PPP2R2B with or without serum and Dox treatment. control siRNA, with or without Dox treatment. FIG. 6E. Immunohistochemical (IHC) analysis of phos FIG. 3G. Growth curve of HCT 116 and DLD1 cells treated phorylated PDK1 in human colon and normal appendix tis with Myc or p70S6K siRNA or a negative control siRNA for 30 Sue. Dark brown color represents positive signal of phospho indicated days. PDK1 at S241 and blue color represents the nuclear staining. FIGS. 4A-4G. PPP2R2B re-expression in CRC sensitizes Magnification for scanning IHC is 40x. rapamycin both in vitro and in Vivo, and overrides rapamycin FIGS. 7A-7F. Inhibition of PDK1-Myc signaling over induced Myc phosphorylation. comes rapamycin resistance. FIG. 4A. Proliferation of DLD1-PPP2R2B cells treated 35 FIG. 7A. HCT 116 cells were transfected with siRNAs with 10 ng/ml Dox (Dox) or 10 nM rapamycin or both (R--D) targeting Myc, PDK1, AKT1 or PIK3CA for 48 h, and then for indicated days. treated with 100 nM rapamycin for 5 days. The graph bars FIG. 4B. Dense foci formation on a monolayer of DLD1 show the rapamycin-induced growth inhibition relative to PPP2R2B or DLD1 vector control cells treated with 10 nM non-treated cells. rapamycin, with or without 10 ng/ml Dox treatment for 14 40 FIG. 7B. Rapamycin-induced growth inhibition in SW480 days. cells expressing PDK1 shRNA or a negative control shRNA. FIG. 4C. Cell cycle G2/M arrest in DLD1-PPP2R2B or FIG. 7C. G2/M phase arrest in SW480 and DLD1 cells vector control cells treated with Dox or rapamycin or both for induced by rapamycin (10 nM), BX912 (2.5 uM), PIK90 (5 48 h. uM), single or in combinations, assessed by PI staining and FIG. 4D. Xenograft tumor growth of DLD1-PPP2R2B 45 FACS analysis. Data are presented as meaniSD of the per cells in nude mice treated with Dox at 100 mg/kg, or rapa centages of cells arrested in G2/M. mycin at 4 mg/kg or both, every other day as described in FIG. 7D. Cell viability of DLD1 and SW480 cells treated Experimental Procedures. Error bars represent SEM. *** with BX912 (2.5 LM), rapamycin (10 nM), or both for indi p<0.01. cated days. FIG. 4E. DLD1-PPP2R2B or DLD1 vector control cells 50 FIG.7E. Dense foci formation for 14 days on a monolayer were treated with 10 nM rapamycin in the presence or of DLD1 and SW480 cells treated as FIG. 7D. absence of Dox for 48 hr. The immunoblot analysis shows FIG. 7F. A model indicating a role of PPP2R2B-regulated that rapamycin induces Myc phosphorylation, which is abro PDK1-Myc pathway in modulating rapamycin response. gated upon PPP2R2B expression. Loss of PPP2R2B expression in CRC results in induction of FIG. 4F. Immunoblot analysis of indicated proteins in 55 PDK1-dependent Myc phosphorylation by rapamycin, con DLD1 cells treated with 10 nM rapamycin for the indicated ferring rapamycin resistance. times FIG. 8. PPP2R2B is significantly suppressed in multiple FIG. 4G. Immunoblot analysis of Myc in DLD1 cells human cancers. ONCOMINE data analysis showing the nor treated with shRNAs targeting mTOR, raptor or rictor. malized expression of PPP2R2B in malignant and corre FIGS. 5A-5F. Rapamycin induced Myc phosphorylation 60 sponding normal tissues of bladder (Dyrskjot et al., 2004; requires DK1, but not PIK3CA-AKT. Sanchez-Carbayo et al., 2006), brain (Rickman et al., 2001; FIG. 5A. Immunoblot analysis of DLD1 cells for AKT and Shai et al., 2003; Sun et al., 2006), esophagus (Wang et al., Myc. DLD1 cells were serum starved for 48 h and followed 2006) and colon (Ki et al., 2007). Representative data are by treatment with 10 nM rapamycin for the indicated times. shown across multiple independently published microarray FIG.5B. Immunoblot analysis of DLD1 cells for AKT and 65 Studies from the ONCOMINE website. Myc in response to a dual mTORC1 and P110C inhibitor FIGS. 9A-9B. Effects of PPP2R2B re-expression on colon PI-103 (0.5 M) for the indicated times. cancer cell growth. DLD1, HT29 and SW480 cells were US 9.249,466 B2 7 8 infected with pMN-GFP (control vector) or pMN-GFP with anti-Myc (9E10) and DAPI to detect the localization of PPP2R2B retrovirus and cells were sorted with GFP Selec PPP2R2B-Myc in DLD1 cells. Scale bars=10 im. tion. The morphologies of DLD1 and SW480 cells induced FIG. 15B. DLD1 cells stained with anti-p-PDK1(S241) to by PPP2R2B re-expression are shown in FIG. 9A and the detect the localization. Scale bars=10 im. anchorage-independent growth of SW480 and HT29 is 5 FIG.15C. DLD1 cells treated with Rapamycin for 48 hand shown in FIG.9B. Scale bars=100 um. stained with anti-p-Myc (T58/S62). FIGS. 10A-10B. Ectopic expression of PPP2R2B inhibits FIGS. 16A-16B. Pharmacological effects of PDK1 inhibi the growth of NIH/3T3 cells transformed with Ras. tor BX912 and shPDK1 in colon cancer cells. FIG. 10A. Anchorage-independent growth following ret FIG. 16A. Morphologies of SW480 cells under indicated roviral packaged pMN-PPP2R2B in NIH/3T3-Ras cells is 10 treatmentS. shown. Colony formation in Soft agar was measured. FIG. 16B. FACS analysis of DLD1 and SW480 cells FIG. 10B. Immunoblotting studies of Myc and S6K pro treated with 2.5 M BX912 or 2.5 uM PIKCA inhibitor teins in NIH/3T3-Ras cells infected with PPP2R2B or control PIK90 with or without rapamycin for 48 h. Cells were stained Vector. with PI and cell cycle was measured by FACS analysis. Per 15 centages of cells arrested in G2/M were indicated. FIGS. 11A-11B. S6K interacts with PPP2R2B-associated FIG. 17. PLK1 inhibitors BI2536 and GW843682X more PP2A complex. selectively decrease the cell viability of PDK1 or Myc-trans FIG. 11 A. DLD1-PPP2R2B tet-on cells were treated with formed cells, compared to control or PTEN stable knock 1.0 ug/ml Dox for 8 h and cellular extracts were subjected to down induced transformation. The cell viability assay was immunoprecipitation with S6K antibody and interaction of carried out in the presence of PLK1 inhibitors for 4 days. S6K with PPP2R2B and PP2A C subunit were studied by FIG. 18. The PLK1 inhibitor BI2536 preferentially inhibits immunoblotting of the immunoprecipitates (IP). the proliferation of PDK1 transformed cells, but has less FIG. 11B. DLD1-PPP2R2B tet-on cells were treated as effect on control cells (PMN) and PI3K transformed cells FIG. 11A and cell lysate were immunoprecipitated with Myc (E545) in both HEK-TERV and RWPE1 systems tagged antibody and the interaction of PPP2R2B with S6K 25 FIG. 19. Apoptotic assay in HEK-TERV cells treated with and PP2A A subunit were studied by Western blotting of the PLK1 inhibitor BI2536 10 nM for 24 h. immunoprecipitates. FIGS. 20A-20B. PDK1 induces PLK1 and Myc phospho FIG. 12. PPP2R2B and rapamycin synergistically induce rylation in multiple epithelial cells. (FIG. 20A). Immunoblot G2/M cell cycle arrest. FACS analysis of DLD1-PPP2R2B or analysis of Myc and PLK1 in HEK-TERV cells transformed vector control cells treated with Dox or rapamycin or both for 30 with PDK1, Myc or shPTEN (FIG.20B). Immunoblot analy 48 h. The percentages of cells arrested in G2/M are indicated. sis of Myc and PLK1 in human mammary epithelial cells FIGS. 13A-13D. Rapamycin-induced Myc phosphoryla (HMEC) and prostate epithelial cells (RWPE). tion is associated with PPP2R2B expression and rapamycin FIG. 21. PLK1 overexpression induces Myc phosphoryla sensitivity in colon cancer cells. tion and accumulates Myc protein in 293T cell. FIG. 13A. Immunoblot analysis of Myc, AKT and p70S6K 35 FIG. 22. PLK1 knockdown by siRNA suppresses Myc in HCT116 and DKO cells treated 10 nM rapamycin for the phosphorylation in HEK-PDK1 cells. indicated times. FIG. 23. BEZ 23.5-induced Myc phosphorylation required FIG. 13B. Rapamycin induced growth inhibition in PLK1. Immunoblot analysis of Myc and AKT phosphoryla HCT 116 and DKO cells. tion in DLD1 cells treated with PLK1 inhibitor BI2536 (10 FIG. 13C. Immunoblot analysis of Myc, AKT, or p70S6K 40 nM), mTOR inhibitor BEZ 235 (100 nM) or indicated com in a series of CRC cell lines treated with rapamycin for 48 hr. bination for 48 hr FIG. 13D. Rapamycin-induced growth inhibition in CRC FIG. 24. Cell viability of SW480 and DLD1 cells treated cell lines. Cell viability measured after 5 days treatment with with BI2536 (10 nM), BEZ 235 (10 nM) or both for indicated 10 nM rapamycin. The graph bar represents the rapamycin days. induced growth inhibition (%) compared to untreated cells. 45 FIG.25. Apoptotic assay in SW480 and DLD1 cells treated FIGS. 14A-14D. Rapamycin does not induce Myc phos with BI2536 (10 nM), BEZ 235 (100 nM) or both for 48 hr. phorylation and is more sensitive in cancer cells expressing FIG. 26. Monolayer colony formation assay in DLD1 cells PPP2R2B. treated with BEZ 235 or BI2436 or combination for 12 days. FIG. 14A. ONCOMINE database showing the expression FIG. 27. PDK1 transformed cells generated a greater num of PPP2R2B in renal (Boer et al., 2001), liver (Chen et al., 50 ber of tumor spheres as compared with pMN control cells, 2002), lymphoma (Alizadeh et al., 2000) and ovarian cancer PIK3CA-E545K or shPTEN-transformed cells. (Hendrix et al., 2006). FIG. 28. The PLK1 inhibitor BI2536 blocks the tumor FIG. 14B. Tagman assay of PPP2R2B mRNA in indicated sphere formation in HEK-PDK1 cells. The cells were treated cancer cell lines and normal colon mucosa. ACT values were with or without 5 nM BI2536 for 7 days. shown. 55 FIG. 29. PLK1 knockdown by siRNAs inhibit tumor FIG. 14C. Immunblotting analysis of Myc and AKT phos sphere formation in HEK-PDK1 cells. phorylation in a panel of PPP2R2B-expressing cancer cell FIG. 30. qRT-PCR analysis was performed for stem cell lines. associated genes in HEK-TERV cells. FIG. 14D. Rapamycin-induced growth inhibition in FIG. 31. Tumor growth curve (meant-SD) in nude mice 3 PPP2R2B-expressing cancer cell lines. Cell viability was 60 weeks after injection of 1000 HEK-PDK1 cells as compared measured after 5 days treatment with 10 nM rapamycin. The with 1x10 or 1x10" HEK-Myc cells. graph bar represents the rapamycin-induced growth inhibi tion (%) compared to untreated cells. DETAILED DESCRIPTION FIGS. 15A-15C. Subcellular localizations of PPP2R2B, PDK1, and Myc in DLD1 cells. 65 Detection of mTOR Inhibitor Resistant Cancers FIG. 15A. DLD1-PPP2R2B cells were treated with Dox As shown in Example 17, colorectal cancer (CRC) cells for 48 h to induce PPP2R2B expression and cells were stained display epigenetic loss of PPP2R2B (GenBank Accession US 9.249,466 B2 10 Number: NM 181678) activity and expression. PPP2R2B PDK1 antagonist or PDK1 inhibitor. PDK1 inhibitors and may therefore be used as a marker for colorectal cancer. antagonists may be identified as described in further detail Example 13 and FIG.8 also show similar loss of PPP2R2B in below, and may include for example PDK1 inhibitors such as other cancers such as bladder cancer, oesophageal cancer and BX912 (CAS Accession Number: 702674-56-4) and BX795 brain cancer. 5 (CAS Accession Number: 702675-74-9). Other PDK1 inhibi We therefore provide for a method of detecting a cancer tors are known in the art and are described in, for example, R cell or a patient suffering or likely to suffer from such a I Feldman et al. Novel Small Molecule Inhibitors of 3-Phos cancer, such as colorectal cancer, bladder cancer, oesoph phoinositide-dependent Kinase-1. J. Biol. Chem..., 2005, 280, ageal cancer or brain cancer, the method comprising detect 20, 19867-19874 and C Peifer, D. R. Alessi, Small-Molecule ing decreased expression and/or activity of PPP2R2B in or of 10 Inhibitors of PDK1. ChemMedChem. 2008, 3 (10). a cell, or in or of a cell of a patient. We therefore provide for the use of a PDK1 inhibitor or We demonstrate that such loss results in resistance to antagonist in the treatment of cancers such as colorectal can mTOR inhibitor treatment, such as treatment with rapamycin cer, bladder cancer, oesophageal cancer and brain cancer, as (or a derivative of rapamycin). Wetherefore provide for use of well as mTOR inhibitor resistant forms thereof (e.g., rapamy PPP2R2B as a tumour suppressor. We further provide for 15 cin resistant or rapamycin derivative resistant forms of Such methods of detecting cancers which are resistant to treatment cancers). Treatment may further comprise administration of with mTOR inhibitor, including mTOR inhibitor resistant PDK1 inhibitors or antagonists in combination with an colorectal cancer, mTOR inhibitor resistant bladder cancer, mTOR inhibitor such as rapamycin or a derivative thereof. mTOR inhibitor resistant oesophageal cancer and mTOR Accordingly, we provide for a combination of (or compo inhibitor resistant brain cancer, the methods comprising sition comprising) an inhibitor or antagonist of PDK1 and an detecting reduced expression and/or activity of PPP2R2B, as mTOR inhibitor, such as for use in treatment of a cancer described above. The mTOR inhibitor may comprise in par including colorectal cancer, bladder cancer, oesophageal can ticular rapamycin or a derivative thereof. cer and brain cancer (as well as mTOR inhibitor resistant Up-regulation of expression of PPP2R2B activity in such forms, including rapamycin or rapamycin derivative resistant cells, in combination with rapamycin treatment, inhibits 25 forms, of such cancers). The mTOR inhibitor may include growth of the cells. Cancers such as colorectal cancer, bladder rapamycin or a derivative thereof and the PDK1 inhibitor or cancer, oesophageal cancer and brain cancer, including antagonist may comprise BX912 (CAS Accession Number: mTOR inhibitor resistant forms, may therefore be treated by 702674-56-4) or BX795 (CAS Accession Number: 702675 up-regulating the activity and/or expression of PPP2R2B, 74-9). optionally in combination with treatment with mTOR inhibi 30 We further provide for the use of PDK1 and PLK1 for tors such as rapamycin or a derivative thereof, and we there cancers such as cancer stem cells or Myc-driven tumors. fore provide for such methods in this document. Expression Treatment of Cancer and/or activity of PPP2R2B may be achieved for example by The methods and compositions described here suitably use of demethylating agents such as cytidine analogues, enable an improvement in a measurable criterion in an indi including 5-aza-dC and AZacitidine (Issa J P Kantarian H. 35 vidual to whom the treatment is applied, compared to one Azacitidine. Nat Rev Drug Discov 2005; May Suppl:S6-7). who has not received the treatment. Accordingly, we provide for a combination of (or compo For this purpose, a number of criteria may be designated, sition comprising) a demethylating agent and an mTOR which reflect the progress of cancer or the well-being of the inhibitor, such as for use in treatment of a cancer including patient. Useful criteria may include tumour size, tumour colorectal cancer, bladder cancer, oesophageal cancer and 40 dimension, largest dimension of tumour, tumour number, brain cancer (as well as mTOR inhibitor resistant forms, presence of tumour markers (such as alpha-feto protein), including rapamycin or rapamycin derivative resistant forms, degree or number of metastates, etc. of such cancers). The mTOR inhibitor may include rapamy Thus, as an example, a treated individual may show a cin or a derivative thereof and the demethylating agent may decrease in tumour size or number as measured by an appro include 5-aza-dC or AZacitidine. 45 priate assay or test. A treated individual may for example We show in Example 19 that rapamycin induced Myc show a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, phosphorylation (leading to mTOR inhibitor resistance) is 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%; 90%, 100% or mediated by PDK1 kinase. Detection of PDK1 may therefore more decrease in tumour size of a particular tumour, or be used as a proxy for detection of PPP2R2B (as described decrease in tumour number, or both, compared to an indi above) for detecting cancers such as colorectal cancer, blad 50 vidual who has not been treated. der cancer, oesophageal cancer and brain cancer, as well as In some embodiments, the effect of the treatment is suit mTOR inhibitor resistant forms thereof (e.g., rapamycin ably quantified using standard tests, such as the international resistant or rapamycin derivative resistant forms of Such can criteria proposed by the Response Evaluation Criteria in Solid cers). We therefore provide for methods of detecting such Tumours (RECIST) Committee, as described in detail in cancers, by detection of PDK1 activity and/or expression, 55 Therasse, P. S. G. Arbuck, et al. (2000). “New guidelines to optionally together with detection of PPP2R2B activity and/ evaluate the response to treatment in Solid tumors. European or expression. Organization for Research and Treatment of Cancer, National We further provide for use of PDK1 as a therapeutic target Cancer Institute of the United States, National Cancer Insti for cancers including colorectal cancer, bladder cancer, tute of Canada.”J Natl Cancer Inst 92(3): 205-16. oesophageal cancer and brain cancer. Accordingly, we pro 60 In other embodiments, the effect of the treatment may be vide for the treatment of cancers such as colorectal cancer, quantified by following the administration and testing proto bladder cancer, oesophageal cancer and brain cancer, as well cols described in the Clinical Trial (Examples E1 to E8). as mTOR inhibitor resistant forms thereof (e.g., rapamycin Thus, assessment of the effect of the treatment may be carried resistant or rapamycin derivative resistant forms of Such can out using one or more of the protocols, preferably all, as set cers) by down-regulation of PDK1 activity and/or expression. 65 out in Example E8: Measurement of Effect. Where this is the PDK1 activity and expression may be down-regulated by use case, the treatment may result in a Partial Response (PR) or a a modulator of PDK1 activity and/or expression, such as a Complete Response (CR). US 9.249,466 B2 11 12 Although the controls described above have been Preferably, the term “homologue' is intended to cover iden described as individuals who have not received treatment, in tity with respect to structure and/or function such that the Some cases, a more Suitable control may be the patient him resultant nucleotide sequence encodes a polypeptide which self, prior to receiving treatment. has mTOR activity. With respect to sequence identity (i.e. For the purposes of this document, the term “cancer can similarity), preferably there is at least 70%, more preferably comprise any one or more of the following: acute lympho at least 75%, more preferably at least 85%, more preferably at cytic leukemia (ALL), acute myeloid leukemia (AML). least 90% sequence identity. More preferably there is at least adrenocortical cancer, anal cancer, bladder cancer, blood can 95%, more preferably at least 98%, sequence identity. These cer, bone cancer, brain tumor, breast cancer, cancer of the terms also encompass allelic variations of the sequences. female genital system, cancer of the male genital system, 10 The following description of mTOR, referred to as FRAP central nervous system lymphoma, cervical cancer, child is provided from the Online Mendelian Inheritance in Man hood rhabdomyosarcoma, childhood sarcoma, chronic lym website (http://www.ncbi.nlm.nih.gov/entrez/dispomim phocytic leukemia (CLL), chronic myeloid leukemia (CML), .cgi?id=601231) colon and rectal cancer, colon cancer, endometrial cancer, FKBP12-rapamycin associated protein (FRAP) is one of a endometrial sarcoma, esophageal cancer, eye cancer, gall 15 family of proteins involved in cell cycle progression, DNA bladder cancer, gastric cancer, gastrointestinal tract cancer, recombination, and DNA damage detection. In rat, it is a hairy cell leukemia, head and neck cancer, hepatocellular 245-kD protein (symbolized RAFT1) with significant homol cancer, Hodgkin’s disease, hypopharyngeal cancer, Kaposi's ogy to the Saccharomyces cerevisiae protein TOR1 and has sarcoma, kidney cancer, laryngeal cancer, leukemia, leuke been shown to associate with the immunophilin FKBP12 mia, liver cancer, lung cancer, malignant fibrous histiocy (186945) in a rapamycin-dependent fashion (Sabatini et al., toma, malignant thymoma, melanoma, mesothelioma, mul 1994). Brown et al. (1994) noted that the FKBP12-rapamycin tiple myeloma, myeloma, nasal cavity and paranasal sinus complex was known to inhibit progression through the G1 cancer, nasopharyngeal cancer, nervous system cancer, neu cell cycle stage by interfering with mitogenic signaling path roblastoma, non-Hodgkin’s lymphoma, oral cavity cancer, ways involved in G1 progression in several cell types, as well oropharyngeal cancer, osteosarcoma, ovarian cancer, pancre 25 as in yeast. The authors stated that the binding of FRAP to atic cancer, parathyroid cancer, penile cancer, pharyngeal FKBP12-rapamycin correlated with the ability of these cancer, pituitary tumor, plasma cell neoplasm, primary CNS ligands to inhibit cell cycle progression. lymphoma, prostate cancer, rectal cancer, respiratory system, Rapamycin is an efficacious anticancer agent against Solid retinoblastoma, Salivary gland cancer, skin cancer, Small tumors. In a hypoxic environment, the increase in mass of intestine cancer, Soft tissue sarcoma, Stomach cancer, stom 30 Solid tumors is dependent on the recruitment of mitogens and ach cancer, testicular cancer, thyroid cancer, urinary system nutrients. When nutrient concentrations change, particularly cancer, uterine sarcoma, Vaginal cancer, vascular system, those of essential amino acids, the mammalian target of rapa Waldenstrom's macroglobulinemia and Wilms’ tumor. mycin (mTOR/FRAP) functions in regulatory pathways that mTOR control ribosome biogenesis and cell growth. In bacteria, Where the term mTOR is used in this document, it should 35 ribosome biogenesis is independently regulated by amino be taken to refer a polypeptide sequence having the accession acids and ATP. Dennis et al. (2001) demonstrated that the number NM 004958.2, P42345 or NP 004949, more par human mTOR pathway is influenced by the intracellular con ticularly NM 004958.2. centration of ATP, independent of the abundance of amino Preferably, mTOR refers to a human sequence. Thus, par acids, and that mTOR/FRAP itself is an ATP sensor. ticular homologues encompassed by this term include human 40 Castedo et al. (2001) delineated the apoptotic pathway homologues, for example, accession numbers resulting from human immunodeficiency virus(HIV)-1 enve NM 004958.2, NP 004949, Hs.509145. However, the term lope glycoprotein (EnV)-induced syncytia formation in vitro also covers alternative peptides homologous to mTOR. Such and in vivo. Immunohistochemical analysis demonstrated the as polypeptides derived from other species, including other presence of phosphorylated ser15 of p53 (191170) as well as mammalian species. For example, mouse homologues of 45 the preapoptotic marker tissue transglutaminase (TGM2; mTOR having accession number NM 020009. 1, 190196) in syncytium in the apical light Zone (T-cell area) of NP 064393, Mm 0.21158, Q9JLN9, AAF73196 and lymph nodes, as well as in peripheral blood mononuclear AF152838 are included. Bovine and rat homologues of cells, from HIV-1-positive but not HIV-1-negative donors. mTOR are also known (accession numbers NM 174319 and The presence of these markers correlated with viral load NM 019906 respectively). 50 (HIV-1 RNA levels). Quantitative immunoblot analysis mTOR is also known as FKBP12-Rapamycin Complex showed that phosphorylation of ser15 of p53 in response to Associated Protein 1, FRAP1, FK506-Binding Protein HIV-1 Env is mediated by FRAP and not by other phosphati 12-Rapamycin Complex-Associated Protein 1, FRAP dylinositol kinase-related kinases, and it is accompanied by FRAP2, Mammalian Target of Rapamycin and RAFT1. downregulation of protein phosphatase 2A (see 176915). The Preferably, mTOR includes fragments, homologues, vari 55 phosphorylation is significantly inhibited by rapamycin. ants and derivatives of such a nucleotide sequence. The terms Immunofluorescence microscopy indicated that FRAP is “variant”, “homologue', 'derivative' or “fragment” as used enriched in syncytial nuclei and that the nuclear accumulation here include any Substitution of variation of modification of precedes the phosphorylation of ser15 of p53. Castedo et al. replacement of, deletion of or addition of one (or more) (2001) concluded that HIV-1 Env-induced syncytium forma nucleic acids from or to the sequence of a mTOR nucleotide 60 tion leads to apoptosis via a pathway that involves phospho sequence. Unless the context admits otherwise, references to rylation of ser15 of p53 by FRAP, followed by activation of “mTOR’ include references to such variants, homologues, BAX (600040), mitochondrial membrane permeabilization, derivatives and fragments of mTOR. These are described in release of cytochrome C, and caspase activation. more detail below. Fang et al. (2001) identified phosphatidic acid as a critical Preferably, the resultant nucleotide sequence encodes a 65 component of mTOR signaling. In their study, mitogenic polypeptide having mTOR activity, preferably having at least stimulation of mammalian cells led to a phospholipase D-de the same activity of the human mTOR referred to above. pendent accumulation of cellular phosphatidic acid, which US 9.249,466 B2 13 14 was required for activation of mTOR downstream effectors. tomas. Given the role of PIK-related kinase proteins in DNA Phosphatidic acid directly interacted with the domain in repair, recombination, and cell cycle checkpoints, the authors mTOR that is targeted by rapamycin, and this interaction was suggested that the possible role of FRAP in solid tumors with positively correlated with mTOR’s ability to activate down deletions at 1p36 should be investigated. Onyango et al. stream effectors. The involvement of phosphatidic acid in (1998) established the order of genes in the 1p36 region, mTOR signaling reveals an important function of this lipid in telomere to centromere, as CDC2L1 (176873)-PTPRX2 signal transduction and protein synthesis, as well as a direct (604008)-ENO1 (172430)-PGD (172200)-XBX1 (604007)- link between mTOR and mitogens. Fang et al. (2001) con FRAP2 (FRAP1)-CD30 (153243). cluded that their study suggested a potential mechanism for mTOR is described in detail in Beugnet, et al. J. Biol. the in vivo actions of the immunosuppressant rapamycin. 10 Kim et al. (2002) and Hara et al. (2002) reported that Chem. 278 (42), 40717-40722 (2003); Kristof, et al., J. Biol. mTOR binds with RAPTOR (607130), an evolutionarily con Chem. 278 (36), 33637-33644 (2003); Chen, Y., et al., Onco served protein with at least 2 roles in the mTOR pathway. Kim gene 22(25),3937-3942 (2003); Garami, et al., Mol. Cell. 11 et al. (2002) showed that RAPTOR has a positive role in (6), 1457-1466 (2003); Nojima, et al., J. Biol. Chem. 278 nutrient-stimulated signaling to the downstream effector 15 (18), 15461-15464 (2003); Kimura, et al., Genes Cells 8 (1), S6K1 (601684), maintenance of cell size, and mTOR protein 65-79 (2003); McMahon, et al., Mol. Cell. Biol. 22 (21), expression. The association of RAPTOR with mTOR also 7428-7438 (2002); Tee, et al., Proc. Natl. Acad. Sci. U.S.A. negatively regulates mTOR kinase activity. Conditions that 99 (21), 13571-13576 (2002); Hudson, et al., Mol. Cell. Biol. repress the pathway, such as nutrient deprivation and mito 22 (20), 7004-7014 (2002); Choi, et al., EMBO Rep. 3 (10), chondrial uncoupling, stabilize the mTOR-RAPTOR asso 988-994 (2002); Inoki, et al., Nat. Cell Biol. 4 (9), 648-657 ciation and inhibit mTOR kinase activity. Kim et al. (2002) (2002); Zhang, et al., J. Biol. Chem. 277 (31), 28.127-28134 proposed that RAPTOR is a component of the mTOR path (2002); Castedo, et al., EMBO.J. 21 (15), 4070-4080 (2002); way that, through its association with mTOR, regulates cell Hara, et al., Cell 110(2), 177-189 (2002); Kim, et al., Cell 110 size in response to nutrient levels. (2), 163-175 (2002); Fingar, et al., Genes Dev. 16(12), 1472 Hara et al. (2002) showed that the binding of RAPTOR to 25 1487 (2002); Reynolds, et al., J. Biol. Chem. 277 (20), 17657 mTOR is necessary for the mTOR-catalyzed phosphorylation 17662 (2002); Fang, et al., Science 294 (5548), 1942-1945 of 4EBP1 (602223) in vitro and that it strongly enhances the (2001); Dennis, et al., Science 294 (5544), 1102-1105 (2001): mTOR kinase activity toward p70-alpha (S6K1). Rapamycin Onyango, et al., Genomics 50 (2), 187-198 (1998); Lench, et oramino acid withdrawal increased, whereas insulin strongly al., Hum. Genet. 99 (4), 547-549 (1997); Choi, et al., Science inhibited, the recovery of 4EBP1 and RAPTOR on 7-methyl 30 GTPsepharose. Partial inhibition of RAPTOR expression by 273 (5272), 239-242 (1996); Moore, et al., Genomics 33 (2), RNA interference reduced mTOR-catalyzed 4EBP1 phos 331-332 (1996); Chen, et al., Proc. Natl. Acad. Sci. U.S.A. 92 phorylation in vitro. RNA interference of C. elegans Raptor (11), 4947-4951 (1995); Chiu et al., Proc. Natl. Acad. Sci. yielded an array of phenotypes that closely resembled those U.S.A. 91 (26), 12574-12578 (1994); Brown, et al., Nature produced by inactivation of CE-Tor. Thus, the authors con 35 369 (6483), 756-758 (1994). cluded that RAPTOR is an essential scaffold for the mTOR Inhibitor of mTOR Activity catalyzed phosphorylation of 4EBP1 and mediates TOR The methods and compositions described here rely, in action in vivo. Some embodiments, on blocking, reducing, or decreasing the Vellai et al. (2003) demonstrated that TOR deficiency in C. activity of mTOR protein. Such inhibition of mTOR activity elegans more than doubles its natural life span. The absence 40 may be used in conjunction to treat cancer or prevent cell or of Let363/TOR activity caused developmental arrest at the L3 tissue growth or proliferation according to the methods and larval stage. At 25.5 degrees C., the mean life span of Let363 compositions described here. mutants was 25 days compared with a life span of 10 days in While any means of doing so may be used, in general, the wildtype worms. methods and compositions described here employ modula Huntington disease (HD; 143100) is an inherited neurode 45 tors of mTOR activity or expression. Agents which are generative disorder caused by a polyglutamine tract expan capable of decreasing the activity of mTOR protein are sion in which expanded polyglutamine proteins accumulate referred to as inhibitors or antagonists of that activity. For the abnormally in intracellular aggregates. Ravikumar et al. purpose of this document, the terms “inhibitor' and “antago (2004) showed that mammalian target of rapamycin (mTOR) nist may be regarded as synonymous, where the context is sequestered in polyglutamine aggregates in cell models, 50 requires. transgenic mice, and human brains. Sequestration of mTOR In preferred embodiments, antagonists of mTOR activity impairs its kinase activity and induces autophagy, a key clear have the ability to decrease a relevant activity of mTOR, for ance pathway for mutant huntingtin fragments. This protects example, kinase activity, by 10%, 20%, 30%, 40%, 50%, against polyglutamine toxicity, as the specific mTOR inhibi 60%, 70%, 80%, 90%. 95% or more. Preferably, mTOR tor rapamycin attenuates huntingtin accumulation and cell 55 activity is assayed as described below in the section "Assays death in cell models of HD, and inhibition of autophagy has for mTOR Activity”. converse effects. Furthermore, rapamycin protects against The term “antagonist', as used in the art, is generally taken neurodegeneration in a fly model of HD, and the rapamycin to refer to a compound which binds to an enzyme and inhibits analog CCI-779 improved performance on 4 different behav the activity of the enzyme. The term as used here, however, is ioral tasks and decreased aggregate formation in a mouse 60 intended to refer broadly to any agent which inhibits the model of HD. The data provided proof of principle for the activity of a molecule, not necessarily by binding to it. potential of inducing autophagy to treat HD. Accordingly, it includes agents which affect the expression of Moore et al. (1996) assigned the FRAP gene to 1p36 by an mTOR protein, or the biosynthesis of a regulatory mol fluorescence in situ hybridization (FISH). Lench et al. (1997) ecule, or the expression of modulators of the activity of mapped the FRAP gene to 1p36.2 by FISH following radia 65 mTOR. The specific activity which is inhibited may be any tion-hybrid mapping to that general region. Chromosome activity which is exhibited by, or characteristic of the enzyme 1p36.2 is the region most consistently deleted in neuroblas or molecule, for example, any activity of mTOR as the case US 9.249,466 B2 15 16 may be, for example, a kinase activity. The kinase activity acid and a carbohydrate. An agent may be in solution or in may comprise the ability to phosphorylate one or either of Suspension (e.g., in crystalline, colloidal or other particulate S6K1 and/or 4E-BP1. form). The agent may be in the form of a monomer, dimer, The antagonist may bind to and compete for one or more oligomer, etc, or otherwise in a complex. sites on the relevant molecule preferably, the catalytic site of 5 The terms “modulator”, “antagonist' and "agent” are also the enzyme. Preferably, such binding blocks the interaction intended to include, a protein, polypeptide or peptide includ between the molecule and another entity (for example, the ing, but not limited to, a structural protein, an enzyme, a interaction between a enzyme and its substrate). However, the cytokine (such as an interferon and/or an interleukin) an antagonist need not necessarily bind directly to a catalytic antibiotic, a polyclonal or monoclonal antibody, or an effec site, and may bind for example to an adjacent site, another 10 tive part thereof, such as an Fv fragment, which antibody or protein (for example, a protein which is complexed with the part thereofmay be natural, synthetic or humanised, a peptide enzyme) or other entity on or in the cell, so long as its binding hormone, a receptor, a signalling molecule or other protein; a reduces the activity of the enzyme or molecule. nucleic acid, as defined below, including, but not limited to, Where antagonists of a enzyme such as mTOR are con an, oligonucleotide or modified oligonucleotide, an antisense cerned, an antagonist may include a substrate of the enzyme, 15 oligonucleotide or modified antisense oligonucleotide, or a fragment of this which is capable of binding to the cDNA, genomic DNA, an artificial or natural chromosome enzyme. In addition, whole or fragments of a Substrate gen (e.g. a yeast artificial chromosome) or a part thereof, RNA, erated natively or by peptide synthesis may be used to com including mRNA, tRNA, rRNA or a ribozyme, or a peptide pete with the substrate for binding sites on the enzyme. Alter nucleic acid (PNA); a virus or virus-like particles; a nucle natively, or in addition, an immunoglobulin (for example, a otide or ribonucleotide or synthetic analogue thereof, which monoclonal or polyclonal antibody) capable of binding to the may be modified or unmodified; an amino acid or analogue enzyme may be used. The antagonist may also include a thereof, which may be modified or unmodified; a non-peptide peptide or other small molecule which is capable of interfer (e.g., Steroid) hormone; a proteoglycan; a lipid; or a carbohy ing with the binding interaction. Other examples of antago drate. Small molecules, including inorganic and organic nists are set forth in greater detail below, and will also be 25 chemicals, which bind to and occupy the active site of the apparent to the skilled person. polypeptide thereby making the catalytic site inaccessible to Non-functional homologues of a mTOR may also be tested Substrate Such that normal biological activity is prevented, are for inhibition of mTOR activity as they may compete with the also included. Examples of small molecules include but are wild type protein for binding to other components of the cell not limited to Small peptides or peptide-like molecules. machinery whilst being incapable of the normal functions of 30 In a particular embodiment, the technique of RNA inter the protein. Alternatively, they may block the function of the ference (RNAi) may be used to abolish or knockout or reduce protein bound to the cell machinery. Such non-functional gene activity, for example, mTOR activity. The overall strat homologues may include naturally occurring mutants and egy is to prepare double stranded RNA (dsRNA) specific to modified sequences or fragments thereof. each gene of interest and to transfect this into a cell of interest Alternatively, instead of preventing the association of the 35 to inhibit the expression of the particular gene. components directly, the Substance may suppress the biologi The following protocol may be used: a sample of PCR cally available amount of a mTOR. This may be by inhibiting product is analysed by horizontal gel electrophoresis and the expression of the component, for example at the level of DNA purified using a Qiagen QiaGuick PCR purification kit. transcription, transcript stability, translation or post-transla 1 Jug of DNA is used as the template in the preparation of gene tional stability. An example of such a substance would be 40 specific single stranded RNA using the Ambion T7 Megas antisense RNA or double-stranded interfering RNA cript kit. Single stranded RNA is produced from both strands sequences which suppresses the amount of mRNA biosyn of the template and is purified and immediately annealed by thesis. heating to 90 degrees C. for 15 mins followed by gradual Blocking the activity of an inhibitor of the mTOR protein cooling to room temperature overnight. A sample of the may therefore also be achieved by reducing the level of 45 dsRNA is analysed by horizontal gel electrophoresis, and expression of the protein or an inhibitor in the cell. For introduced into the relevant cell by conventional means. example, the cell may be treated with antisense compounds, Antagonists of mTOR Activity for example oligonucleotides having sequences specific to Any agent which is capable of reducing mTOR activity or the mTOR mRNA. The level of expression of pathogenic expression, as described above, may be used as an antagonist forms of adhesion proteins may also be regulated this way. 50 of mTOR for the purposes of reducing its activity. In general, agonists, antagonists of mTOR may comprise Butanol agents such as an atom or molecule, wherein a molecule may 1-Butanol is an inhibitor of mTOR activity, as described in be inorganic or organic, a biological effector molecule and/or Kam and Exton, FASEB J. 2004 February; 18(2):311-9 and a nucleic acid encoding an agent such as a biological effector Fang et al., Science 294: 1942-1945. Butanol may therefore molecule, a protein, a polypeptide, a peptide, a nucleic acid, a 55 be used in the methods and compositions described here as an peptide nucleic acid (PNA), a virus, a virus-like particle, a agent capable of reducing mTOR activity. nucleotide, a ribonucleotide, a synthetic analogue of a nucle Anti-Peptide mTOR Antibodies otide, a synthetic analogue of a ribonucleotide, a modified Anti-peptide antibodies may be produced against mTOR nucleotide, a modified ribonucleotide, an amino acid, an peptide sequences. The sequences chosen may be based on amino acid analogue, a modified amino acid, a modified 60 the mouse sequences as follow from the following mTOR amino acid analogue, a steroid, a proteoglycan, a lipid, a fatty reference sequence: 1 milgtgpavat a saatssnvs vlcqfasglk srneetraka akelghyvtm elirems gees 61 trfyddlnhh if elvis ssda nerkggilai asligveggn strigrfany lirnllipssop US 9.249,466 B2 19 20 Thus, preferred anti-peptide antibodies may be raised from Rapamycin is also known as Sirolimus. any one or more of the following sequences: amino acids Methods for the preparation of rapamycin are disclosed in 22-139; amino acids 647-907; amino acids 937-1140; amino Sehgal et al., U.S. Pat. Nos. 3,929,992, and 3,993,749. In acids 1382-1982; amino acids 2019-2112, or amino acids 2181-2549. addition, monoacyl and diacyl derivatives of rapamycin and Corresponding sequences from human mTOR may be cho methods for their preparation are disclosed by Rakhit, U.S. Sen for use in eliciting anti-peptide antibodies from immu Pat. No. 4,316,885. Furthermore, Stella et al., U.S. Pat. No. nised animals. Antibodies may be produced by injection into 4,650,803 disclose water soluble prodrugs of rapamycin, i.e., rabbits, and other conventional means, as described in for rapamycin derivatives including the following rapamycin example, Harlow and Lane (Supra). prodrugs: glycinate prodrugs, propionate prodrugs and the Antibodies are checked by Elisa assay and by Western 10 pyrrolidinobutyrate prodrugs. blotting, and used for immunostaining as described in the The methods and compositions described here include the Examples. use of natural and synthetic rapamycin, genetically engi Rapamycin neered rapamycin and all derivatives and prodrugs of rapa In Some embodiments, an agent capable of reducing mycin, such as described in the aforementioned U.S. Pat. mTOR activity comprises rapamycin. As the term is used in 15 Nos. 3,929,992: 3,993,749; 4,316,885; and 4,650,803, the this document, “rapamycin includes the specific compound contents of which are hereby incorporated by reference. rapamycin (also known as Sirolimus, C5H7NO, which is Rapamycin is a 31-membered macrollide lactone, described below) as well as any of its derivatives. Such CH7NO, with a molecular mass of 913.6Da. In solution, derivatives are described in detail and include rapamycin Sirolimus forms two conformational trans-, cis-isomers with prodrugs, rapamycin dialdehydes, structural analogues of O a ratio of 4:1 (chloroform) due to hindered rotationaround the rapamycin (rapalogs), etc. pipecolic acid amide bond. It is sparingly soluble in water, Rapamycin, including its derivatives, etc., is therefore pro aliphatic hydrocarbons and diethyl ether, whereas it is soluble vided as a specific antagonist of mTOR activity. in alcohols, halogenated hydrocarbons and dimethyl sulfox Rapamycin and its derivatives may be employed at con ide. Rapamycin is unstable in solution and degrades in plasma centrations over 1 nM, for example, 10 nM, 20 nM, 30 nM, 40 25 and low-, and neutral-pH buffers at 37 degrees C. with half nM, 50 nM, 100 nM, 500 nM, 10 um, 100 um, or more. In life of <10 h. the structures of the degradation products have Some embodiments, rapamycin and its derivatives are used at recently been characterized. Rapamycin is a macrocyclic about 50 nM. Rapamycin and its derivatives may be admin triene antibiotic produced by Streptomyces hygroscopicus, istered to human individuals at dosages of for example between about 1 mg/day and 10 mg/day. which was found to have antifungal activity, particularly Rapamycin (Sirolimus) 30 against Candida albicans, both in vitro and in vivo C. Vezina Rapamycin (C5H7NO. molecular mass 914.172 et al., J. Antibiot. 28, 721 (1975); S, N. Sehgal et al., J. g/mol.) is an antifungal antibiotic which is extractable from a Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, streptomycete, e.g., Streptomyces hygroscopicus. 539 (1978); U.S. Pat. No. 3,929,992; and U.S. Pat. No. 3,993, Rapamycin has an IUPAC name of (3S,6R,7E.9R,10R, 749). 12R,14S,15E,17E, 19E.21S,23S.26R,27R,34aS)-9,10,12,13, 35 Rapamycin alone (U.S. Pat. No. 4.885,171) or in combi 14.21.22.23.24.25,26.27.32.33.34.34a-hexadecahydro-9.27 nation with picibanil (U.S. Pat. No. 4,401,653) has been dihydroxy-3-(1R)-2-(1S,3R,4R)-4-hydroxy-3- shown to have antitumor activity. R. Martel et al. Can. J. methoxycyclohexyl-1-methylethyl-10,21-dimethoxy-6,8. Physiol. Pharmacol. 55, 48 (1977) disclosed that rapamycin 12, 14.20.26-hexamethyl-23.27-epoxy-3H-pyrido.2.1-c. 1, is effective in the experimental allergic encephalomyelitis 40 model, a model for multiple sclerosis; in the adjuvant arthritis 4-oxaazacyclohentriacontine-1,5,11,28.29(4H,6H,31H)- model, a model for rheumatoid arthritis; and effectively pentone. inhibited the formation of IgE-like antibodies. Rapamycin is identified by its CAS number 53123-88-9, The immunosuppressive effects of rapamycin have been ATC code L04AA10, PubChem 6436030, Drug Bank disclosed in FASEB 3, 3411 (1989). Cyclosporin A and APRD00178. The structural formula of rapamycin is shown 45 FK-506, other macrocyclic molecules, also have been shown below: to be effective as immunosuppressive agents, therefore useful HO, in preventing transplant rejection (FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and R.Y. Caine et al., Lancet 1 183 (1978). Although it shares structural homology with the 50 immunosuppressant tacrolimus and binds to the same intra cellular binding protein in lymphocytes, rapamycin inhibits S6p70-kinase and therefore has a mechanism of immunosup pressive action distinct from that of tacrolimus. Rapamycin was found to prolong graft Survival of different transplants in 55 several species alone or in combination with other immuno Supressants. In animal models its spectrum of toxic effects is different from that of cyclosporin or FK-506, comprising impairment of glucose homeostasis, stomach, ulceration; weight loss and thrombocytopenia, although no nephrotoxic 60 ity has been detected. Rapamycin Derivatives Rapamycin derivatives include rapamycin prodrugs, rapa mycin dialdehydes, structural analogues of rapamycin (rapa logs), etc., and are described in detail below. 65 Specific derivatives of rapamycin which may be used in the methods and compositions described here include RAD001 (Everolimus) and CCI-779 (Wyeth). US 9.249,466 B2 21 22 RAD001 (Everolimus) RAD001 (CHNO molecular mass 958.224 g/mol) is CH a derivative of rapamycin. RAD001 is identified by its CAS number 159351-69-6, ATC code L04AA18 and PubChem 5 OH 6442177. The structural formula of RAD001 is shown below: OH 10 15 25 CCI 779 is also known as Temsirolimus and is manufac tured by Wyeth. Temsirolimus binds to the cytosolic protein, FKBP, which subsequently inhibits mTOR (mammalian tar 30 get of rapamycin). In animal models of human cancers, temsirolimus has been found to inhibit the growth of a diverse range of cancer types RAD001 is also known as Everolimus and is manufactured even when an intermittent dosing schedule was used. The by Novartis AG. It is currently used as an immunosuppressant compound also appears to have potential for the blockade of to prevent rejection of organ transplants. 35 inflammatory responses associated with autoimmune and rheumatic diseases by inhibiting T-cell proliferation. RAD001 is described in detail in O'Reilly TM, Wood JM, CCI 779 is a water soluble ester (prodrug) of rapamycin Littlewood-Evans A, et al. Differential anti-vascular effects that releases rapamycin in vivo. It is believed to be more of mTOR or VEGFR pathway inhibition: a rational basis for tolerable than rapamycin when used clinically and is cur combining RAD001 and PTK787/ZK222584. Presented at: 40 rently being studied for use in oncology patients in Phase II 96th Annual Meeting of the American Association for Cancer and III trials (including brain tumors). Research. Anaheim, Calif.; Apr. 16-20, 2005. Abstract 3038. CCI 779 is described in detail in Nat. Genet. 2004:36:585 RAD001 is also described in 105. Van Oosterom A T, 95 and J Clin Oncol. 2004; 22:2336-47. Reference should Dumez, H. Desai J. et al. Combination signal transduction also be made to KYu, L Toral-Barza, C Discafani, W G inhibition: a phase I/II trial of the oral mTOR-inhibitor 45 Zhang, J Skotnicki, P Frost, and JJ Gibbons (2001). mTOR, everolimus (E, RAD001) and imatinib mesylate (IM) in a novel target in breast cancer: the effect of CCI-779, an patients (pts) with gastrointestinal stromal tumor (GIST) mTOR inhibitor, in preclinical models of breast cancer. Endo refractory to IM abstract. Proc Am Soc Clin Oncol. 2004: crine-Related Cancer 8 (3)249-258 and Josep Maria Peralba, 23:195. Abstract 3002. Linda deGraffenried, William Friedrichs, Letitia Fulcher, 50 Viktor Grunwald, Geoffrey Weiss and Manuel Hidalgo CCI 779 (Temsirolimus) (2003. Pharmacodynamic Evaluation of CCI-779, an Inhibi CCI 779 (cell cycle inhibitor-779, CH, NO, molecular tor of mTOR, in Cancer Patients. Clinical Cancer Research weight 1030.3) is an ester analogue of Rapamycin. Vol. 9, 2887-2892. CCI 779 is also known as rapamcyin-28-N,N-dimethlyg Rapamycin Prodrugs 55 The mTOR inhibitor, particularly rapamycin, may be pro lycinate methanesulfonate salt, rapamycin, 42-3-hydroxy-2- vided in the form of a prodrug. A specific example of a (hydroxymethyl)-2-methylpropanoate, (3S,6R,7E,9R,10R, rapamycin prodrug is CCI 779, described above. 12R,14S,15E,17E, 19E.21S,23S.26R,27R,34aS)-9,10,12,13, The term “prodrug” as used in this application refers to a 14.21.22.23.24.25,26.27.32.33.34.34a-hexadecahydro-9.27 precursor or derivative form of a pharmaceutically active dihydroxy-3-(1R)-2-(1S,3R,4R)-4-hydroxy-3- 60 Substance that is less cytotoxic to tumor cells compared to the methoxycyclohexyl-1-methylethyl-10,21-dimethoxy-6,8. parent drug and is capable of being enzymatically activated or 12, 14.20.26-hexamethyl-23.27-epoxy-3H-pyrido.2.1-c. 1, converted into the more active parent form. See, e.g., Wilman, 4-oxaazacyclohentriacontine-1.5.11.28.29(4H,6H,31H)- “Prodrugs in Cancer Chemotherapy” Biochemical Society pentone 4'-2.2-bis(hydroxymethyl)propionate and Transactions, 14, pp. 375 382,615th Meeting Belfast (1986) rapamycin 42-2.2-bis(hydroxymethyl)propionate. 65 and Stella et al., “Prodrugs: A Chemical Approach to Targeted CCI 779 is identified by its CAS registry number 162635 Drug Delivery.” Directed Drug Delivery, Borchardt et al., 04-3. The structural formula of CCI 779 is shown below: (ed.), pp. 247 267, Humana Press (1985). The prodrugs US 9.249,466 B2 23 24 described here include, but are not limited to, phosphate these may be employed in the methods and compositions containing prodrugs, thiophosphate-containing prodrugs, described here. Sulfate-containing prodrugs, peptide-containing prodrugs, For example, the extensive literature on analogs, D-amino acid-modified prodrugs, glycosylated homologs, derivatives and other compounds related structur prodrugs, beta.-lactam-containing prodrugs, optionally Sub ally to rapamycin (“rapalogs’) include among others variants stituted phenoxyacetamide-containing prodrugs or option of rapamycin having one or more of the following modifica ally substituted phenylacetamide-containing prodrugs, tions relative to rapamycin: demethylation, elimination or 5-fluorocytosine and other 5-fluorouridine prodrugs which replacement of the methoxy at C7, C42 and/or C29; elimina can be converted into the more active cytotoxic free drug. Examples of drugs that can be derivatized into a prodrug form tion, derivatization or replacement of the hydroxy at C13, for use in the methods and compositions described here 10 C43 and/or C28; reduction, elimination or derivatization of include, but are not limited to, those chemotherapeutic agents the ketone at C14, C24 and/or C30; replacement of the described above. 6-membered pipecolate ring with a 5-membered prolyl ring; Rapamycin Dialdebydes and alternative Substitution on the cyclohexyl ring or replace Rapamycin prodrugs such as rapamycin dialdehydes ment of the cyclohexyl ring with a substituted cyclopentyl described in U.S. Pat. No. 6,680,330 (Zhu, et al) may be 15 ring. In nearly all cases, potent immunosuppressive activity is employed in the methods and compositions described here. reported to accompany antifungal activity of the rapalogs. Mono- and diacylated derivatives of rapamycin (esterified Additional historical information is presented in the back at the 28 and 43 positions) have been shown to be useful as ground sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and antifungal agents (U.S. Pat. No. 4.316,885) and used to make 5,362,718. water soluble prodrugs of rapamycin (U.S. Pat. No. 4,650, Rapalogs 803). Recently, the numbering convention for rapamycin has "Rapalogs' as that term is used herein denotes a class of been changed; therefore according to Chemical Abstracts compounds comprising the various analogs, homologs and nomenclature, the esters described above would be at the 31 derivatives of rapamycin and other compounds related struc and 42-positions. Carboxylic acid esters (PCT application turally to rapamycin. “Rapalogs' include compounds other No. WO92/05179), carbamates (U.S. Pat. No. 5,118,678), 25 than rapamycin (or those rapamycin derivatives modified in amide esters (U.S. Pat. No. 5,118,678), (U.S. Pat. No. 5,118, comparison to rapamycin only with respect to Saturation of 678) fluorinated esters (U.S. Pat. No. 5,100,883), acetals one or more of the carbon-carbon double bonds at the 1, 2, 3, (U.S. Pat. No. 5,151,413), silyl ethers (U.S. Pat. No. 5,120, 4 or 5, 6 positions) which comprise the substructure shown in 842), bicyclic derivatives (U.S. Pat. No. 5,120,725), rapamy Formula I, bearing any number of a variety of Substituents, cin dimers (U.S. Pat. No. 5,120,727) and O-aryl, O-alkyl, 30 and optionally unsaturated at one or more carbon-carbon O-alkyenyl and O-alkynyl derivatives (U.S. Pat. No. 5.258, bonds unless specified to the contrary herein. 389) have been described. Rapalogs include, among others, variants of rapamycin Rapamycin is metabolized by cytochrome P-4503A to at having one or more of the following modifications relative to least six metabolites. During incubation with human liver and rapamycin: demethylation, elimination or replacement of the Small intestinal microsomes, sirolimus was hydroxylated and 35 methoxy at C7, C42 and/or C29; elimination, derivatization demethylated and the structure of 39-O-demethyl sirolimus or replacement of the hydroxy at C13, C43 and/or C28; reduc was identified. In bile of sirolimus-treated rats >16 hydroxy tion, elimination or derivatization of the ketone at C14, C24 lated and demethylated metabolites were detected. and/or C30; replacement of the 6-membered pipecolate ring In rapamycin, demethylation of methoxy group at C-7 with a 5-membered prolyl ring; and elimination, derivatiza Carbon will lead to the change in the conformation of the 40 tion or replacement of one or more Substituents of the cyclo Rapamycin due to the interaction of the released C-7 hexyl ring or replacement of the cyclohexyl ring with a Sub hydroxyl group with the neighbouring pyran ring system stituted or unsubstituted cyclopentyl ring. Rapalogs, as that which is in equilibrium with the open form of the ring system. term is used herein, do not include rapamycin itself, and The C-7 hydroxyl group will also interact with the triene preferably do not contain an oxygen bridge between C1 and system and possibly alter the immunosupressive activity of 45 C30. Illustrative examples of rapalogs are disclosed in the rapamycin. This accounts for the degradation of rapamycin documents listed in Table I. Examples of rapalogs modified at molecule and its altered activity. C7 are shown in Table II. TABLE I WO97105O2 WO94182O7 WO93O468O U.S. Pat. No. 5,527,907 U.S. Pat. No. 5,225,403 WO9641807 WO94.10843 WO9214.737 U.S. Pat. No. 5,484,799 U.S. Pat. No. 5,221,625 WO963S423 WO9409010 WO9205179 U.S. Pat. No. 5,457,194 U.S. Pat. No. 5,210,030 WO96O3430 WO94O4S4O U.S. Pat. No. 5,604,234 U.S. Pat. No. 5,457,182 U.S. Pat. No. 5,208,241 WO960O282 WO94O2485 U.S. Pat. No. 5,597,715 U.S. Pat. No. 5,362,735 U.S. Pat. No. 5,200,411 WO9516691 WO94O2137 U.S. Pat. No. 5,583,139 U.S. Pat. No. 5,324,644 U.S. Pat. No. 5, 198421 WO9515328 WO94O2136 U.S. Pat. No. 5,563,172 U.S. Pat. No. 5,318,895 U.S. Pat. No. 5,147,877 WO9507468 WO9325533 U.S. Pat. No. 5,561.228 U.S. Pat. No. 5,310,903 U.S. Pat. No. 5,140,018 WO9504738 WO931.8043 U.S. Pat. No. 5,561,137 U.S. Pat. No. 5,310,901 U.S. Pat. No. 5,116,756 WO9504060 WO9313663 U.S. Pat. No. 5,541,193 U.S. Pat. No. 5,258,389 U.S. Pat. No. 5,109,112 WO942SO22 WO931.1130 U.S. Pat. No. 5,541,189 U.S. Pat. No. 5,252,732 U.S. Pat. No. 5,093,338 WO9421644 WO931.0122 U.S. Pat. No. 5,534,632 U.S. Pat. No. 5,247,076 U.S. Pat. No. 5,091,389 Structural Analogues of Rapamycin (Rapalogs) Anti-Peptide mTOR Antibodies A large number of structural variants of rapamycin have Anti-peptide antibodies may be produced against mTOR been reported, typically arising as alternative fermentation 65 peptide sequences: The sequences chosen may be based on products or from synthetic efforts to improve the compounds the mouse sequences as follow from the following mTOR therapeutic index as an immunosuppressive agent. Each of reference sequence: US 9.249,466 B2 27 28 - Continued 2461 velgepahkik agttvipesih sfigdglivkp ealinkkaidi inrvrdkltg rolfshddtld 2521 vptovellik catshenlcq cyligwcpfw Thus, preferred anti-peptide antibodies may be raised from and brain cancer, as well as mTOR inhibitor resistant forms any one or more of the following sequences: amino acids thereof (e.g., rapamycin resistant or rapamycin derivative 22-139; amino acids 647-907; amino acids 937-1140; amino resistant forms of Such cancers). acids 1382-1982; amino acids 2019-2112, or amino acids 10 The level of PDK1 may be detected in a cell of an indi 2181-2549. vidual with cancer, in a cancer or non-cancer cell, and the Corresponding sequences from human mTOR may be cho sensitivity of the cancer to treatments such as mTOR inhibi Sen for use in eliciting anti-peptide antibodies from immu tors assessed. A high level of PDK1 amount, expression or nised animals. Antibodies may be produced by injection into activity compared with a normal cell indicates an mTOR rabbits, and other conventional means, as described in for 15 inhibitor resistant form (e.g., rapamycin resistant or rapamy example, Harlow and Lane (Supra). cin derivative resistant form) of cancers such as colorectal Antibodies are checked by Elisa assay and by Western cancer, bladder cancer, oesophageal cancer and brain cancer. blotting, and used for immunostaining as described in the Alternative therapies may therefore be chosen, including use Examples. of PDK1 inhibitors and antagonists. Downregulation of PDK1 A cancer is defined as being “treated if a condition asso ciated with the disease is significantly inhibited (i.e., by 50% The methods and compositions described here involve in or more) relative to controls. The inhibition may be by at least part down-regulating the activity and/or expression of PDK1. 75% relative to controls, such as by 90%, by 95% or 100% In general terms, our methods involve manipulation of relative to controls. The condition may comprise cell prolif cancer cells, by modulating (such as down-regulating) the eration, or it may comprise cell cycle time, cell number, cell expression, amount or activity of PDK1 in the cell. A step of migration, cell invasiveness, etc. By the term “treatment we detecting modulated PDK1 expression, amount or activity in mean to also include prophylaxis or alleviation of cancer. a cell may be conducted before or after the manipulation step. PDK1 polypeptide represents a target for inhibition of its The detection step may detect up-regulated or down-regu function for therapy, particularly in tumour cells and other lated PDK1 expression, amount or activity. Any of the meth proliferative cells. ods of modulating or down-regulating PDK1, as described in The term proliferative disorder is used herein in a broad detail elsewhere in this document, may be used. sense to include any disorder that requires control of the cell For example, PDK1 activity and/or expression may be cycle. In particular, a proliferative disorder includes malig down-regulated by use of PDK1 inhibitors, including small nant and pre-neoplastic disorders. The methods and compo molecule inhibitors of PDK1. An example of a PDK1 inhibi 35 sitions described here are especially useful in relation to tor is BX795 (CAS No. 702675-74-9). treatment or diagnosis of adenocarcinomas Such as: Small cell lung cancer, and cancer of the kidney, uterus, prostrate, blad der, ovary, colon and breast. For example, malignancies which may be treatable include acute and chronic leukemias, 40 lymphomas, myelomas, sarcomas Such as Fibrosarcoma, myxosarcoma, liposarcoma, lymphangioendotheliosarcoma, angiosarcoma, endotheliosarcoma, chondrosarcoma, osteo genic sarcoma, chordoma, lymphangiosarcoma, Synovioma, mesothelioma, leimyosarcoma, rhabdomyosarcoma, colon 45 carcinoma, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, squamous cell carcinoma, basal cell carci noma, adenocarcinoma, Sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarci nomas, cystadenocarcinoma, medullary carcinoma, bron 50 chogenic carcinoma, choriocarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma seminoma, embryonal carci Other PDK1 inhibitors which may be suitable for use in noma, cervical cancer, testicular tumour, lung carcinoma, down-regulating the activity and/or expression of PDK1 are Small cell lung carcinoma, bladder carcinoma, epithelial car described in RI Feldman et al. Novel Small Molecule Inhibi cinoma, glioma, astrocytoma, ependymoma, pinealoma, tors of 3-Phosphoinositide-dependent Kinase-1. J. Biol. 55 hemangioblastoma, acoustic neuoma, medulloblastoma, Chem., 2005, 280, 20, 19867-19874 and C Peifer, D R. craniopharyngioma, oligodendroglioma, menangioma, Alessi, Small-Molecule Inhibitors of PDK1. ChemMed melanoma, neutroblastoma and retinoblastoma. Chem. 2008, 3 (10). One possible approach for therapy of such disorders is to The method may comprise exposing the cell to an siRNA or express anti-sense constructs directed against PDK1 poly shRNA or an anti-PDK1 antibody capable of specifically 60 nucleotides as described here, and administering them to binding to PDK1. PDK1 may be modulated by targeting a tumour cells, to inhibit gene function and prevent the tumour PDK1 target site selected from any suitable site in the PDK1 cell from growing or progressing. Sequence. Anti-sense constructs may be used to inhibit gene function According to our methods, the cancer cell becomes non to prevent growth or progression in a proliferative cell. Anti cancerous or sensitive to mTOR inhibitors as a result of the 65 sense constructs, i.e., nucleic acid, Such as RNA, constructs manipulation. The cancer may in particular comprise cancers complementary to the sense nucleic acid or mRNA, are Such as colorectal cancer, bladder cancer, oesophageal cancer described in detail in U.S. Pat. No. 6,100,090 (Monia et al.),