PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland

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1 Award Number: W81XWH TITLE: A Gene Expressin Prfile f BRCAness That Predicts fr Respnsiveness t Platinum and PARP Inhibitrs PRINCIPAL INVESTIGATOR: Panagitis Knstantinpuls CONTRACTING ORGANIZATION: Dana Farber Cancer Institute Bstn, MA, REPORT DATE: Octber 2015 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland DISTRIBUTION STATEMENT: Apprved fr Public Release; The views, pinins and/r findings cntained in this reprt are thse f the authr(s) and shuld nt be cnstrued as an fficial Department f the Army psitin, plicy r decisin unless s designated by ther dcumentatin. 1

2 REPORT DOCUMENTATION PAGE Frm Apprved OMB N Public reprting burden fr this cllectin f infrmatin is estimated t average 1 hur per respnse, including the time fr reviewing instructins, searching existing data surces, gathering and maintaining the data needed, and cmpleting and reviewing this cllectin f infrmatin. Send cmments regarding this burden estimate r any ther aspect f this cllectin f infrmatin, including suggestins fr reducing this burden t Department f Defense, Washingtn Headquarters Services, Directrate fr Infrmatin Operatins and Reprts ( ), 1215 Jeffersn Davis Highway, Suite 1204, Arlingtn, VA Respndents shuld be aware that ntwithstanding any ther prvisin f law, n persn shall be subject t any penalty fr failing t cmply with a cllectin f infrmatin if it des nt display a currently valid OMB cntrl number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 3. DATES COVERED (Frm - T) Octber REPORT TYPE Annual 4. TITLE AND SUBTITLE A Gene Expressin Prfile f BRCAness That Predicts fr Respnsiveness t Platinum and PARP Inhibitrs 15 July July a. CONTRACT NUMBER W81XWH b. GRANT NUMBER OC c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Panagitis Knstantinpuls 5d. PROJECT NUMBER 5e. TASK NUMBER panagitis_knstantinpuls@dfci.harvard.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER Dana Farber Cancer Institute 44 Binney St, Bstn, MA, SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Cmmand Frt Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Apprved fr Public Release; Distributin Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT In this annual prgress reprt f the wrk perfrmed frm 7/15/2014 t 7/14/2015, we: i) present data n a unique subset f varian cancers which are nt BRCAlike, i.e. nt HR deficient and are resistant t PARPis but are sensitive t platinum. These tumrs exhibit alteratins in anther DNA repair pathway called nucletide excisin repair (NER) pathway. ii) present data that HSP90-inhibitrs which were identified via ur BRCAness prfile may sensitize PARPi-resistant xengraft mdels t PARPis. iii) update ur prgress n validating ur prfile in a chrt f mlecularly defined BRCA-mutated and HR-prficient tumrs. 15. SUBJECT TERMS varian cancer, hmlgus recmbinatin, brcaness, PARP inhibitrs, platinum analgues, gene expressin prfiling, HSP90 inhibitrs 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT Unclassified b. ABSTRACT Unclassified c. THIS PAGE Unclassified 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC Unclassified 14 19b. TELEPHONE NUMBER (include area cde) 2 Standard Frm 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

3 Table f Cntents Page 1. Intrductin Keywrds Accmplishments Impact Changes/Prblems Prducts Participants & Other Cllabrating Organizatins Special Reprting Requirements

4 1. INTRODUCTION Patients with BRCA1/2-assciated EOCs exhibit imprved verall survival and high sensitivity t duble strand DNA break inducing agents due t an underlying defect in DNA repair via HR. Hwever, it is increasingly recgnized that a subset f patients with spradic EOCs als exhibit defective HR caused by mechanisms that are unrelated t germline BRCA1 r BRCA2 mutatins. These tumrs may behave similarly t BRCA1/2-mutated EOCs and are cmmnly referred t as having a "BRCAness" phentype (i.e. BRCAlike). Identifying tumrs with a BRCAness phentype is f increased clinical imprtance nt nly due t the advent f PARPinhibitrs but als because patients with this phentype may need t be managed differently than the remaining patients. We have develped a 60-gene expressin prfile that may identify tumrs with a BRCAness phentype and may als be used t identify cmpunds that can enhance respnsiveness t platinum and PARP-inhibitrs. In this annual prgress reprt f the wrk perfrmed frm 7/15/2014 t 7/14/2015, we: i) present data n a unique subset f varian cancers which are nt BRCAlike, i.e. nt HR deficient and thus are resistant t PARPis but are sensitive t platinum. ii) present data that HSP90-inhibitrs which were identified via ur BRCAness prfile may sensitize PARPi resistant xengraft mdels t PARPis. iii) update ur prgress n validating ur prfile in a chrt f mlecularly defined BRCAmutated and HR-prficient tumrs. 2. KEYWORDS Ovarian cancer, Hmlgus recmbinatin, BRCAness, Gene expressin prfiling, PARP inhibitrs, Platinum analgues, HSP90 inhibitrs, Platinum Resistance, Nucletide Excisin Repair 4

5 3. ACCOMPLISHMENTS What were the majr gals f the prject? The majr gals f this prject during this perid were t cntinue ur wrk alng Tasks 1, 3 and 4. The first gal was t identify subsets f varian cancers which are BRCAlike r nnbrcalike and t further characterize their sensitivity t platinum and PARPi (Task 1). Anther gal was t prvide further data shwing that HSP90-inhibitrs sensitize varian cancers t PARPis (Task 3). Finally, in ur Task 4, ur gal was t cntinue wrking twards validating ur prfile in a chrt f mlecularly defined BRCA-mutated and HR-prficient tumrs. What was accmplished under these gals? We have fully accmplished the gals f this prject during this perid. Task 1. Determine whether the BRCAness gene expressin prfile is capable f prspectively identifying spradic patients whse tumrs exhibit defects in hmlgus recmbinatin and increased sensitivity t platinum and PARP inhibitrs in vitr. In additin and in cntinuatin f the wrk we have perfrmed the previus years, we were able t define a unique subset f varian cancers which are nt BRCAlike, i.e. nt HR deficient and thus are resistant t PARPis but are sensitive t platinum. Specifically, we curated the EOC TCGA dataset t assess ptential inactivating events f the nucletide excisin repair (NER) pathway including mutatins, hmzygus deletins and prmter hypermethylatin f NER genes. We fund that a ttal f 24 (8%) f 316 EOCs harbred either NER mutatins r hmzygus deletins f NER genes (Fig. 1A). Specifically, we identified 19 cases with nnsynnymus r splice site NER gene mutatins (all smatic) and 6 cases with hmzygus deletins f NER genes amng the 316 sequenced EOCs f the TCGA dataset. Nne f the NER genes were fund t harbr prmter hypermethylatin. All NER mutatins were mutually exclusive, i.e. n individual tumr harbred mutatins in mre than ne NER gene. Furthermre, NER mutatins were mutually exclusive with hmzygus deletins f the NER genes with the exceptin f ne case that harbred bth an ERCC5 mutatin and hmzygus deletin f ERCC2. Of the 19 cases with NER mutatins, 7 (36.8%) were accmpanied by heterzygus lss f the respective NER gene, indicating that in these cases bth wild-type alleles had been lst. Imprtantly, patients with tumrs with NER alteratins exhibited higher median OS (63.5 vs 5

6 41.5 mnths respectively, lg rank p = 0.048) and PFS (30.4 vs 14.7 mnths respectively, lg rank p = 0.069) cmpared t patients with tumrs withut NER alteratins and BRCA1/2 mutatins (Fig. 1B and 1C). Furthermre, patients with tumrs with NER alteratins exhibited similar utcme (OS and PFS) with tumrs harbring BRCA1 r BRCA2 mutatins (Fig. 1B and 1C). As a prf f principle that NER alteratins are functinally assciated with platinum sensitivity, we evaluated ne NER mutatin (ERCC6-Q524*) that was present in a patient with stage IV subptimally-debulked high grade serus tumr that had cmplete respnse t first line platinum chemtherapy and remained in cmplete remissin fr 31.5 mnths after diagnsis. Figure 1. In rder t determine the functinal significance f ERCC6-Q524* n platinum sensitivity, we evaluated whether this variant culd rescue platinum sensitivity in an ERCC6-deficient cell line. An ERCC6-deficient immrtalized fibrblast cell line (GM16095) was cmplemented with either 6

7 wild-type ERCC6 r the mutant ERCC6-Q524*. Expressin f wild-type ERCC6 rescued cisplatin sensitivity f ERCC6-deficient cells while cmplementatin with mutant ERCC6-Q524* did nt impact cisplatin sensitivity (Fig. 2A). In rder t cnfirm that ERCC6 lss alne is slely sufficient t induce cisplatin sensitivity, we assessed cisplatin cyttxicity fllwing sirna knckdwn f ERCC6. ERCC6 depletin significantly increased platinum sensitivity, cmparable t BRCA2 lss, a majr mediatr f DNA crsslink repair. Given that ERCC6-Q524* was a smatic mutatin and nt assciated with heterzygus lss, we evaluated whether this mutatin may exert a dminant negative effect. We pstulated that ERCC6-Q524* may interfere with the functin f the wild-type allele and hence increase sensitivity t cisplatin. Indeed, intrductin f the ERCC6-Q524* variant in ERCC6 wild-type 293T cells dramatically increased cisplatin sensitivity cmpared t cells transfected either with wild-type ERCC6 r cntrl empty vectr suggesting that this mutatin sensitizes cells t cisplatin by a dminant negative mechanism (Fig. 2B). Figure 2. We evaluated the assciatin f this NER mutatin with sensitivity t the PARPi rucaparib. Unlike in the case f cisplatin, expressin f wild-type r mutant ERCC6 did nt affect PARPi 7

8 sensitivity f ERCC6-deficient fibrblasts. Furthermre, since defective HR is a critical mediatr f platinum and PARPi sensitivity in EOC, we evaluated whether deficiency in ERCC6 affected HR in vitr. Inhibitin f ERRC6 did nt affect HR efficiency in vitr, as measured by directrepeat GFP recmbinatin (DR-GFP) assay and by IR-induced RAD51 fci frmatin, a surrgate fr HR efficiency. Tgether, these results indicate that functinal lss f ERCC6 des nt impair HR efficiency nr alters sensitivity t PARPi r ther duble strand break-inducing agents such as dxrubicin. In cnclusin, we reprt fr the first time that NER pathway alteratins (mutatins and hmzygus deletins) ccur in EOC and that these alteratins are assciated with a phentype f clinical platinum sensitivity that is similar t that f BRCA1/2-mutated tumrs characterized by imprved verall and prgressin free survival. We shwed that the NER mutatin identified in a very platinum sensitive tumr (ERCC6-Q524*) was functinally assciated with platinum sensitivity in vitr. Imprtantly, this mutatin did nt affect HR and did nt cnfer sensitivity t PARPi thus prviding a nvel mechanism f discrdance between platinum and PARPi sensitivity in EOC. Our findings suggest that NER alteratins may have a previusly unrecgnized rle as bimarkers fr selectin f patients fr participatin in PARPi trials as well as fr deciding therapy after develpment f PARPi resistance. This wrk was published at Cancer Research (Ceccaldi R, O'Cnnr KW, Muw KW, Li AY, Matulnis UA, D'Andrea AD, Knstantinpuls PA. Cancer Res Feb 15;75(4):628-34). Task 2. Determine whether the BRCAness gene expressin prfile is assciated with clinical respnse t platinum and survival in patients with spradic varian cancer. This task has already been cmpleted. Task 3. Evaluate whether the cmpunds identified by the Cnnectivity Map can reverse PARP resistance in vitr, and t investigate the mechanism fr this effect. In additin t the in vitr data, we have nw perfrmed experiments t assess whether HSP90 inhibitrs identified via the Cnnectivity Map can revert PARPi resistance in viv. Specifically, we used a PARPi resistant xengraft mdel and we perfrmed efficacy studies. Strikingly, this mdel was PARPi resistant and was als resistant t cmbinatins f laparib and the PI3K inhibitrs BKM120 r BYL719. 8

9 Olaparib was dsed at 100mg/kg p daily x 4 weeks, AT13387 (HSP90i) was administered at 45mg/kg p fr 2 days (D1,D2) n / 5 days ff x 4 weeks (i.e. Days 1, 2, 8, 9, 15, 16, 22, 23). As shwn in Figure 3, the cmbinatin f AT13387 and laparib induced inhibitin f tumr grwth as ppsed t vehicle cntrl, laparib alne and AT13387 alne. Figure 3. Task 4. Determine the reprducibility f the BRCAness prfile when using the DASL mrna assay in a chrt f FFPE varian cancer specimens with knwn clinical utcme and platinum respnsiveness. A very imprtant step twards assessing the reprducibility f ur BRCAness prfile is t identify the right chrt f mlecularly defined patients that are BRCAlike and nnbrcalike. T achieve this, we defined tw grups f patients, a BRCA1/2-mutated grup which is expected t be BRCAlike and a HR-prficient grup which is expected t be nnbrcalike. The BRCA1/2-mutated grup was cmprised f 37 HGSOCs (29 with BRCA1 and 8 with BRCA2 mutatins) with BRCA1/2 germline mutatins identified by genetic testing (Figure 2). The HRprficient grup (i.e. grup f tumrs withut HR alteratins) cmprised 16 varian cancers which were identified in a tw-step prcess (Figure 2). First, we perfrmed Next Generatin Sequencing (NGS) t exclude tumrs with mutatins in HR genes; this analysis identified 17 such tumrs (Figure 4). These 17 tumrs were subsequently evaluated fr BRCA1 expressin by immunhistchemistry t exclude the pssibility f BRCA1 prmter hypermethylatin that wuld lead t absent BRCA1 expressin. As a result f this testing, 1 tumr was fund t have staining in less than 5% f tumr cells with the presence f a strng internal cntrl (Figure 3), which was then excluded frm the HR-prficient(HR intact) grup. Interestingly, review f the 9

10 NGS data fr this case demnstrated that this tumr had a single cpy deletin f the BRCA1 gene, suggesting that BRCA1 lss was likely due t single cpy deletin f BRCA1 and epigenetic silencing f the cmplementary allele. Ultimately, the HR prficient grup cnsisted f 16 tumrs withut mutatins in HR pathway genes and withut BRCA1 lss by IHC. Figure 4. Since we have nw identified the right mlecularly-defined chrt f patients, we plan t determine the reprducibility f ur BRCAness prfile. What pprtunities fr training and prfessinal develpment has the prject prvided? Hw were the results disseminated t cmmunities f interest? What d yu plan t d during the next reprting perid t accmplish the gals? In the next funding perid, we plan t cmplete Tasks 1, 3 and 4. 10

11 4. IMPACT What was the impact n the develpment f the principal discipline(s) f the prject? a) We have discvered a nvel mechanism f discrdance between platinum and PARPi sensitivity in varian cancer that invlved NER alteratin. Our findings suggest that NER alteratins may have a previusly unrecgnized rle as bimarkers fr selectin f patients fr participatin in PARPi trials as well as fr deciding therapy after develpment f PARPi resistance. b) The prmise f PARP inhibitrs in the management f varian cancer is tempered by the fact that HR-prficient cancers d nt respnd well t these agents, suggesting that apprximately 50% f varian cancer patients (i.e. thse withut HR alteratins) d nt benefit frm this nvel class f drugs. Cmbinatin f PARPis with agents that inhibit HR may represent an effective strategy t sensitize HR prficient tumrs t PARPis and thus ptentially expand use f these agents beynd patients with HR deficient EOCs. Our in viv findings that HSP90is revert PARPi resistance in xengrafts prvide the preclinical ratinale fr using a cmbinatin f 17-AAG and laparib and/ r carbplatin in varian cancers that are HR prficient either at baseline r at the time f develpment f platinum r PARPi resistance. This can have a significant impact n patients wh develp resistance t PARP-inhibitrs r platinum analgues as the cmbinatin f 17-AAG/PARP-inhibitrs r 17-AAG/carbplatin may effectively vercme this prblem. What was the impact n ther disciplines? What was the impact n technlgy transfer? What was the impact n sciety beynd science and technlgy? 11

12 5. CHANGES/PROBLEMS Changes in apprach and reasns fr change Actual r anticipated prblems r delays and actins r plans t reslve them Changes that had a significant impact n expenditures Significant changes in use r care f human subjects, vertebrate animals, bihazards, and/r select agents Significant changes in use r care f human subjects Significant changes in use r care f vertebrate animals. Significant changes in use f bihazards and/r select agents 6. PRODUCTS Publicatins, cnference papers, and presentatins Jurnal publicatins. 1. Ceccaldi R, O'Cnnr KW, Muw KW, Li AY, Matulnis UA, D'Andrea AD, Knstantinpuls PA. A Unique Subset f Epithelial Ovarian Cancers with Platinum Sensitivity and PARP Inhibitr Resistance. Cancer Res Feb 15;75(4): Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcrin MI, O'Cnnr KW, Knstantinpuls PA, Elledge SJ, Bultn SJ, Yusufzai T, D'Andrea AD. Hmlgus- 12

13 recmbinatin-deficient tumurs are dependent n Plθ-mediated repair. Nature Feb 12;518(7538): di: /nature Guillemette S, Serra RW, Peng M, Hayes JA, Knstantinpuls PA, Green MR, Cantr SB. Resistance t therapy in BRCA2 mutant cells due t lss f the nuclesme remdeling factr CHD4. Genes Dev Mar 1;29(5): Hwitt BE, Shukla SA, Shll LM, Ritterhuse LL, Watkins JC, Rdig S, Stver E, Strickland KC, D'Andrea AD, Wu CJ, Matulnis UA, Knstantinpuls PA. Assciatin f Plymerase e- Mutated and Micrsatellite-Instable Endmetrial Cancers With Neantigen Lad, Number f Tumr-Infiltrating Lymphcytes, and Expressin f PD-1 and PD-L1. JAMA Oncl Jul 9. di: /jamancl Knstantinpuls PA, Ceccaldi R, Shapir GI, D'Andrea AD. Hmlgus Recmbinatin Deficiency: Expliting the Fundamental Vulnerability f Ovarian Cancer. Cancer Discv Oct 13 Bks r ther nn-peridical, ne-time publicatins. Other publicatins, cnference papers, and presentatins. 1. Kyle Strickland, Brke E. Hwitt, Sctt J. Rdig, Lauren Ritterhuse, Alan D. D'Andrea, Ursula Matulnis, Panagitis Knstantinpuls. Tumr infiltrating and peritumral T cells and expressin f PD-L1 in BRCA1/2-mutated high grade serus varian cancers. ASCO 2015 Meeting, Oral Presentatin Website(s) r ther Internet site(s) Technlgies r techniques Inventins, patent applicatins, and/r licenses 13

14 Other Prducts Nthing t reprt 7. PARTICIPANTS & OTHER COLLABORATING ORGANIZATIONS What individuals have wrked n the prject? Panagitis Knstantinpuls: N change As a reminder, given that Dr Knstantinpuls mved t Dana Farber Cancer Institute, this award was transferred t Dana Farber Cancer Institute effective 9/1/2014. Has there been a change in the active ther supprt f the PD/PI(s) r senir/key persnnel since the last reprting perid? What ther rganizatins were invlved as partners? 8. SPECIAL REPORTING REQUIREMENTS 14