US 2010O292090A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0292090 A1 Nakamura et al. (43) Pub. Date: Nov. 18, 2010 (54) PROGNOSTIC MARKERS AND Related U.S. Application Data THERAPEUTIC TARGETS FOR LUNG (60) Provisional application No. 60/840,376, filed on Aug. CANCER 25, 2006, provisional application No. 60/894,801, filed on Mar. 14, 2007. (75) Inventors: Yusuke Nakamura, Tokyo (JP); Publication Classification Yataro Daigo, Tokyo (JP); Shuichi Nakatsuru, Kanagawa (JP) (51) Int. Cl. C40B 30/04 (2006.01) CI2O I/68 (2006.01) Correspondence Address: GOIN 33/53 (2006.01) TOWNSEND AND TOWNSEND AND CREW, CI2O 1/02 (2006.01) LLP GOIN 33/00 (2006.01) TWO EMBARCADERO CENTER, EIGHTH (52) U.S. Cl. ............... 506/9: 435/6: 435/7.92; 435/7.94: FLOOR 435/29: 436/86 SAN FRANCISCO, CA 94111-3834 (US) (57) ABSTRACT The present invention provides a diagnostic marker to detect (73) Assignee: ONCOTHERAPY SCIENCE, ing lung cancer. In particular, the present invention provides INC., Kawasaki-shi (JP) lung cancer marker genes i.e. KIF4A, MAPJD, NPTX, or FGFR1OP. The present invention further provides methods (21) Appl. No.: 12/438,579 and kit for identifying compounds for treating lung cancer as well as methods for predicting a prognosis or diagnosis of lung cancer. In particular, the present invention provides (22) PCT Filed: Aug. 23, 2007 methods and kits for identifying inhibitors of the interaction between KIF4A/ZNF549, KIF4A/ZNF553, MAPJD/MYC, (86). PCT No.: PCT/UP2007/066825 or FGFR1OP/WRNIP1 which find utility in the treatment and prevention of lung. Alternatively, the present invention pro S371 (c)(1), vides MAPJD associated with HAT complex as therapeutic (2), (4) Date: Aug. 4, 2010 target. Patent Application Publication Nov. 18, 2010 Sheet 1 of 23 US 2010/0292090 A1 Fig. 1 Clinical SCLC K6 K24 K45 K46 K64 K76 K81 K95 onal sists ADC SCC ii is MAPUD NSCLC Normal lung NSCLC OEWS! saw how ww &lar a v. NPTX1 ADC SCC SCLC | | | | (/)|Q O |81||H-ION 01LZH-ION 6-Od 999?H-IONI ACTB Airway SC epithelia ADC |(_)02GH-ION ASCLCC SCLC 90/LH-ION 01LZH-ION |179H-ION Patent Application Publication Nov. 18, 2010 Sheet 2 of 23 US 2010/0292090 A1 Fig. Airway B NSCLC Cell line SCLC Cell line epithelia 3 KF4A & C st $2 cy C w SS. g. S SN SS isN N25 & 22 &a 3?m 3?m 3 (kDa) C - T of Y a ?h (/) (/) () B: anti-KIF4A IB: anti-ACTB NPTX1 N am --m- - - - - m a v - - - - - W - W -- FGFR1OP Clinical lung cancers - - - 15 16 17 (kDa) N T N T N T 250 1OO 50 FGFR1OP 37 inmammam ACTB Airway ... ...u. ... -- - - - - - --- epithelia Lung cancer cell lines cN Y S. S. g . S. S. N. S. LL SE cyp O O O- V - kDa is : C 9 ; ; 9 9 O 3 75 us sk a s xx 50 m m a name inter quipment oup m | FGFR1OP 37- - 37 Patent Application Publication Nov. 18, 2010 Sheet 3 of 23 US 2010/0292090 A1 Fig. 1 | |W NPTX1 Molleueuog| 1.35 |- Patent Application Publication Nov. 18, 2010 Sheet 4 of 23 US 2010/0292090 A1 Fig. 1 KIF4A Air DAPI KIF4A-Alexa488 MAPUD NPTX1 COS-7 NPTX1 A549 LC31 9 NCI-H520 SBC-5 SBC-5 hours after aphidicolin release 15 4 DAP Patent Application Publication Nov. 18, 2010 Sheet 5 of 23 US 2010/0292090 A1 Fig. 1 E KF4A MAPUD NPTX1 Lung SCLC FGFR1OP Patent Application Publication Nov. 18, 2010 Sheet 6 of 23 US 2010/0292090 A1 Fig. 2 A scLc ADC sco B KF4A 1 s 9.8 KLF4A negative 8-6 9 st d .4 .2 Normal Lung 5.2 - - - - - - - --- - -rrorror- O KIF4A positive (t O N - 127 P =0.0005 O 500 1 OOO1500 200025OO300035OO Postoperative days C E NPTX Normal FGFR1OP Strong Normal i. Lung Strong Weak * Y-8 NPTX1 Weak or absent .8 6. Weak,absent FGFR1OP Cas expression expression (N=208) .4 26 Strong expression FGFR1OP expression O N=139 (N=211) PC O.OOO1 0 500 100015002000250030003500 0 1000 20003000 40005000 6000 7000 Postoperative days Postoperative days Patent Application Publication Nov. 18, 2010 Sheet 7 of 23 US 2010/0292090 A1 Fig. 3 NPTX1 (U/ml) 7OO 600 300 200 1400 100 Patent Application Publication Nov. 18, 2010 Sheet 8 of 23 US 2010/0292090 A1 Fig. 4 A Control si-KIF4A C KFAA LUC SCR #1 #2 0.25 POOOO4 g 0.2 B S i 0.15 LUC .2 0.1 Y SCR O.05 D LUC SCR #1 #2 E COS-7 NH3T3 1 & S. 5 292 0.8 (kDa) l,a O2 a 8O O.6 250 sing Sadha- " : 8 150 e g 0.4 100 . COS-7 Y 0.2 O KIF4A(+) MOCK -- . KIF4A(+) MOCK KIF4A(+ MOCK PEO.OOO8 P=OOO37 G 80 140 r 70 - 120 3 60 100 S 50 - O 80 40 - g 30 - 60 w : 2O - 40 20 O O KIF4A(+) MOCK KIF4A(+) MOCK Patent Application Publication Nov. 18, 2010 Sheet 9 of 23 US 2010/0292090 A1 Fig. 5 A 9 5 S. MAPUD (O 5 MAPJD AcTB LUC si-1 SCR si-2 B LUC Si-MAPJD-2 3. 3 C LUC Si-MAPJD-2 ej8.7% 3.34.6% O y 2O FL, FL2, D mock MAPJD if A i B if A if B MAPJD ACTB xirs. MAPJD-A - X - MAPJD-B Patent Application Publication Nov. 18, 2010 Sheet 10 of 23 US 2010/0292090 A1 Fig. 6 NPTX1 A549 SBC5 Control anti-NPTX1 Control anti-NPTX1 SiRNA SiRNA SiRNA SiRNA LUC SCR- - - -si-2 - LUC SCR si-2 NPTX1ACTB RR NPTX1ACTB 0.500 2,000 CD g 2 0.400 1500 m marrimum c d 9 as O.300 3 g 1,000 --- m 0.200m CD > 2 t 0.500 '''''". "''''''''''''''''''" is 0.100 c Y Y O.000 0.000 SN p v i X i H I l 2 Patent Application Publication Nov. 18, 2010 Sheet 11 of 23 US 2010/0292090 A1 Fig. 7 A anti-FGFR1OP Control anti-FGFR1OP Control SiRNA SiRNA SiRNA SiRNA si-1 EGFPLUC SCR si-1 EGFPLUC SCR 3.0 32.5 2. O 2. O 5 1 O si-1 EGFP LUC SCR ? C 9 E (kDa) 1 5 250 1 O 100 75 50 mock 1 FGFR1OP 1 COS-7 COS-7 25 FGFR1OP/COS-7 WB: anti-c-Myc mock/COS-7 WB: anti-ACTB Patent Application Publication Nov. 18, 2010 Sheet 12 of 23 US 2010/0292090 A1 Fig. 7 p 0.0056 mock mock FGFR1OP P < 0.0001 ÇO o LO G mock CD © mock FGFR1OP Patent Application Publication Nov. 18, 2010 Sheet 13 of 23 US 2010/0292090 A1 Fig. 8 KFAA A P Normal anti-KIF4A (kDa) goat (gG pAb - - - - - - - - - - - 100 1 75 50 1. ZNF549 Zinc finger protein 549 2. ZNF553 Zinc finger protein 553 B DAP ZNF549-Alexa488 KIF4A-Alexa594 DAP - - ZNF553-Alexa488 KF4A-Alexa594 Merge Patent Application Publication Nov. 18, 2010 Sheet 14 of 23 US 2010/0292090 A1 Fig. 9 A WAPO mock SBNO TGFBRAP1 ROK1 RASGEF1A 1OOO 500 600 500 4OO 5 400 s 3OO & 3OO O s 200- 2OO 100. 1OO O 0 - O mock MAPJD mock MAPJD mock MAPJD mock MAPJD modk MAPJD 2, B C is a P: anti-MAPD Ab IP es 9e S. B anti-MYC. AO -- m -- -an P ... anti-MYC Ab SBNO1 E B: anti-MAPJD Abi TGFBRAP1 - IP: (-) : 3 RIOK1 - - B: anti-MYC Ab RASGEF1A - IBIP : anti-MAPJD( ) Abt. is " B23. D- - - - - - - - - - - - - - - - - - - - - - - - - - a r D x108 mock x104 SBNO1 x104 TGFBRAP1 x 106 ROK1 xg RASGEFA 5 III | - ON RSS "O : SS O>- 9 >-O 3 s : E a i i 2 a > n cl ?h-G -2 s CC C < f s S s s s Patent Application Publication Nov. 18, 2010 Sheet 15 of 23 US 2010/0292090 A1 Fig. 10 WAPO B protein MAPD MYC a swas sat w w a Y by (g) 0.51 2 0.51 2 Competitor (-) o c (coid probe) (-) g (-) g - O) --lsa s s : Patent Application Publication Nov. 18, 2010 Sheet 16 of 23 US 2010/0292090 A1 Fig. 11 A B MAPUD (D s 5 input ACH4 TRRAP MYC 2is sin sš So & So & So & So IP: () SBNo1 - - Acetylation Activation Repression Patent Application Publication Nov. 18, 2010 Sheet 17 of 23 US 2010/0292090 A1 Fig. 12 A P FGFR1OP P input O 2, () L : as M - 1 5O IgG heavy chain IB: anti-WRNIP WNRIP1 UV irradiation (10 J/m2) FGFR1 OP WRNIP1 IABL1 ACTB Patent Application Publication Nov. 18, 2010 Sheet 18 of 23 US 2010/0292090 A1 Fig. 12 FGFR1OP DAP merge with DAP UV (100 J/m2) (-) 60 min. E UV (100 J/m2) W (-) 5 min. 60 min. Patent Application Publication Nov. 18, 2010 Sheet 19 of 23 US 2010/0292090 A1 Fig. 12 F FGFR1OP DAP merge with DAP UV (100 J/m2) (-) 5 min. 60 min. Patent Application Publication Nov. 18, 2010 Sheet 20 of 23 US 2010/0292090 A1 Fig. 13 A FGFR1OP FGFR1OP (myc-IP) + + - WRNIP1 (myc-IP) + + recombinant ABL 1 - - recombinant ABL1 - -- -- (kDa) O O 9. (kDa) O O O 250 250 150 m # # 150 - iii. 100- 100 75 75 50 m 50 WB: phospho-Tyr (kDa) (kDa) O O. O. 250 150 m 100 m 75 50 m WB: c-myc(FGFR1OP) WB: C-myc(WRNIP1) c IP: myc Ab. B myc-tagged WRNIP1. * * Flag-tagged ABL1 - recombinant ABL1 * (kDa) WRNIP1 (myc-IP) + .