PARP Inhibitor Rucaparib Research Articles

P048 - The TRITON clinical trial programme: evaluation of the PARP inhibitor rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

P048 - The TRITON clinical trial programme: evaluation of the PARP inhibitor rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

The TRITON clinical trial programme: evaluation of the PARP inhibitor rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

The TRITON clinical trial programme: evaluation of the PARP inhibitor rucaparib in patients with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

PARP inhibitors for targeted treatment in ovarian cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors emerged as the first targeted treatment for ovarian cancer, and are selectively active for women with mutations in BRCA1 and BRCA2 (mBRCA). On the basis of data showing activity (measured by the proportion of patients who achieved an objective response), 1 Kim G Ison G McKee AE et al. FDA approval summary: olaparib monotherapy in patients with deleterious germline BRCA-mutated advanced ovarian cancer treated with three or more lines of chemotherapy. Clin Cancer Res. 2015; 21: 4257-4261 Crossref PubMed Scopus (379) Google Scholar the PARP inhibitor olaparib received US Food and Drug Administration (FDA) approval in 2014 specifically for women with germline mBRCA-associated (gBRCA) recurrent ovarian cancer. 2 US Food and Drug AdministrationFDA approves Lynparza to treat advanced ovarian cancer. http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm427554.htm Google Scholar However, subsequent findings from clinical trials of PARP inhibitors have suggested that the importance of mBRCA as a predictive biomarker has diminished. For instance, among women without a germline mBRCA, predictors of PARP activity include a somatic (tumour) mutation in BRCA (sBRCA) and evidence of homologous recombination deficiency. 3 Swisher EM Lin KK Oza AM et al. Rucaparib in relapsed, platinum-sensitive high-grade ovarian carcinoma (ARIEL2 part 1): an international, multicentre, open-label, phase 2 trial. Lancet Oncol. 2017; 18: 75-87 Summary Full Text Full Text PDF PubMed Scopus (785) Google Scholar , 4 Mirza MR Monk BJ Herrstedt J et al. Niraparib maintenance therapy in platinum-sensitive, recurrent ovarian cancer. N Engl J Med. 2016; 375: 2154-2164 Crossref PubMed Scopus (1490) Google Scholar Indeed, FDA approval of the PARP inhibitor rucaparib in 2016 broadened the eligibility for PARP inhibitor treatment beyond gBRCA and included patients with sBRCA-associated ovarian cancers. 5 US Food and Drug AdministrationFDA grants accelerated approval to new treatment for advanced ovarian cancer. https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm533873.htmDate: Dec 19, 2016 Google Scholar Rucaparib maintenance treatment for recurrent ovarian carcinoma after response to platinum therapy (ARIEL3): a randomised, double-blind, placebo-controlled, phase 3 trialAcross all primary analysis groups, rucaparib significantly improved progression-free survival in patients with platinum-sensitive ovarian cancer who had achieved a response to platinum-based chemotherapy. ARIEL3 provides further evidence that use of a poly(ADP-ribose) polymerase inhibitor in the maintenance treatment setting versus placebo could be considered a new standard of care for women with platinum-sensitive ovarian cancer following a complete or partial response to second-line or later platinum-based chemotherapy. Full-Text PDF

836TiP - The TRITON clinical trial programme: Evaluation of the PARP inhibitor rucaparib in patients (Pts) with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

836TiP - The TRITON clinical trial programme: Evaluation of the PARP inhibitor rucaparib in patients (Pts) with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD)

988TiP - ARIEL4: An international, randomised phase 3 study of the PARP inhibitor rucaparib vs chemotherapy for the treatment of BRCA-mutated, relapsed, high-grade ovarian cancer

988TiP - ARIEL4: An international, randomised phase 3 study of the PARP inhibitor rucaparib vs chemotherapy for the treatment of BRCA-mutated, relapsed, high-grade ovarian cancer

Abstract 2475: In vitro and in vivo assessment of the mechanism of action of the PARP inhibitor rucaparib

Abstract Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) that is being developed for patients with BRCA1 and BRCA2 mutated and homologous recombination deficient ovarian cancer (Swisher et al., 2016; Lancet Oncol. Epub). The mechanism of action (MOA) of rucaparib was characterized in the studies reported here. Methods: Enzymatic and cellular reporter assay profiling were performed by BPS Biosciences. Rucaparib potency was assessed using 6-day cell viability assays (CellTiter-Glo). In vivo PK/PD and efficacy studies in the MDA-MB-436 (BRCA1 mutant) and the HBCx-17 (BRCA2 mutant) TNBC models were performed at Crown Biosciences and XenTech, respectively. Results: In biochemical assays rucaparib inhibited PARP-1, PARP-2 and PARP-3, with IC50 values of 0.8, 0.5, and 28 nM, respectively. Rucaparib weakly inhibited PARP5a (tankyrase 1[TNKS1]) and PARP5b (tankyrase 2[TNKS2]) with an IC50 of 796 and 486 nM; however rucaparib did not inhibit TNKS1/2 in a cellular reporter assay evaluating Wnt pathway signaling (IC50 >10 µM). Rucaparib cytotoxicity was evaluated in the BRCA1 mutant (UWB1.289) and wild-type (UWB1.289+BRCA1) isogenic pair, and a panel (n=26) of breast and ovarian cancer cell lines. The UWB1.289 cell line was more sensitive to rucaparib (IC50 = 375 nM) than the UWB1.289+BRCA1 cell line (IC50 = 5430 nM). Rucaparib also decreased the cell viability of several BRCA1 and BRCA2 mutant ovarian and breast cancer cell lines. Mechanistic analyses in the UWB1.289 cell line demonstrated a dose dependent decrease in poly (ADP) ribosylation (PAR; IC50 = 2.8 nM), and an increase in DNA damage and apoptosis. Taken together, these results are consistent with the concept of synthetic lethality, where simultaneous inhibition of multiple DNA repair pathways results in irreparable DNA damage and cell death. In vivo PK/PD and efficacy studies assessed the MOA and activity of rucaparib in the MDA-MD-436 (BRCA1 mutant) xenograft model. In a PK/PD study, dose dependent inhibition of PAR was observed in the tumors of mice treated with 15, 50 and 150 mg/kg BID rucaparib, resulting in statistically significant (p < 0.05) inhibition of 45%, 86% and 96%, respectively. An inverse and dose dependent correlation between PAR and rucaparib levels in the plasma and tumor was observed, and the levels of rucaparib in the tumor were consistently higher (> 2-fold) than the levels of rucaparib measured in plasma. Dose dependent and statistically significant tumor growth inhibition was observed in both subcutaneous and orthotopic xenograft studies evaluating the anti-tumor activity of rucaparib in the MDA-MB-436 model. Rucaparib also demonstrated potent activity in the BRCA2 mutant HBCx-17 patient-derived xenograft model. Conclusion: These results demonstrate that rucaparib is a potent and selective inhibitor of PARP1, PARP-2, and PARP-3 with in vitro and in vivo activity in BRCA1/2 deficient tumors. Citation Format: Liliane Robillard, Minh Nguyen, Thomas C. Harding, Andrew D. Simmons. In vitro and in vivo assessment of the mechanism of action of the PARP inhibitor rucaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2475. doi:10.1158/1538-7445.AM2017-2475

Abstract 2476: Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models

Abstract Rucaparib (CO-338) is an oral small molecule inhibitor of the poly(ADP-ribose) polymerase (PARP) enzymes PARP-1, PARP-2 and PARP-3 that is being developed for cancer patients with evidence of homologous recombination (HR) deficiency, including BRCA1 and BRCA2 mutations. PARP inhibitors have demonstrated clinical activity in BRCA1, BRCA2, and ATM mutant prostate cancer patients (Mateo et al. N Engl J Med. 2015;373:1697-708); however, limited preclinical validation of PARP inhibitors in prostate cancer has been reported to date, since no BRCA mutant nor HR deficient prostate cell lines have been identified. In this study, we modelled the functional inactivation of DNA repair genes using siRNA and CRISPR/Cas9, and examined the impact on PARP inhibition, in the DU145 and PC-3 prostate cancer cell lines. These cell lines were chosen based on transfection efficiency, lack of obvious genetic defects in DNA repair pathways, and baseline insensitivity to rucaparib (IC50 > 10 µM). A panel of 26 DNA repair genes were individually knocked down by siRNA, and changes to rucaparib potency were examined in a 7-day cell viability assay. The knockdown of BRCA1 or BRCA2 increased rucaparib cytotoxicity in both prostate cell lines. For example, the IC50 of rucaparib in DU145 and PC-3 cells with reduced BRCA2 levels was 275 and 297 nM, representing a 36- and 37-fold increase in rucaparib sensitivity, respectively. In addition to BRCA1 and BRCA2, the knockdown of several other genes including BARD1, CDK12, FANCA, PALB2, RAD51, RAD51C, RAD51D, and RAD54L increased rucaparib efficacy ≥ 2-fold in at least 1 cell line examined. To further investigate HR deficiency in prostate cancer, BRCA2 was knocked out in the human 22Rv1 prostate cancer cell line using CRISPR/Cas9. Clone 1-50 displayed biallelic BRCA2 inactivation due to the introduction of a deleterious premature stop codon, and immunoblot analysis verified lack of the full-length BRCA2 protein. The IC50 of rucaparib in the parental and clone 1-50 cells was 9,504 and 287 nM, respectively, demonstrating a 33-fold increase in rucaparib cytotoxicity in this BRCA2 deficient model. Androgen receptor (AR) signaling and proliferation were also examined in clone 1-50. In contrast to published reports (Schiewer et al. Cancer Discov. 2012;2:1134-49), no impact of PARP inhibition on dihydrotestosterone (DHT) induced gene expression was observed in the parental 22Rv1 and BRCA2 deficient clone 1-50 cell lines. In vivo model development and efficacy studies with the 22Rv1 and clone 1-50 cell lines are ongoing. Taken together, these findings support the hypothesis that deficiencies in BRCA1 and BRCA2, as well as additional HR genes, may sensitize prostate cancer cells to rucaparib. A phase 2 study investigating rucaparib in patients with metastatic castration-resistant prostate cancer and HR gene deficiency (TRITON2) is ongoing (NCT02952534). Citation Format: Minh Nguyen, Andrew D. Simmons, Thomas C. Harding. Preclinical assessment of the PARP inhibitor rucaparib in homologous recombination deficient prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2017-2476

Abstract 3650: Preclinical evaluation of the PARP inhibitor rucaparib in combination with PD-1 and PD-L1 inhibition in a syngeneic BRCA1 mutant ovarian cancer model

Abstract Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly(ADP-ribose) polymerase (PARP)-1, PARP-2 and PARP-3 that has shown clinical activity in patients with BRCA1 and BRCA2 mutated advanced ovarian cancer. Monoclonal antibodies against programmed death receptor-1 (PD-1) and programmed death-ligand (PD-L1) have also shown efficacy in advanced ovarian cancer patients. It has been reported that BRCA1 and BRCA2 mutated tumors have a higher mutational load and increased CD8+ T cell infiltration, suggesting that the combination of rucaparib and immune checkpoint inhibition may be complementary. However, PARP inhibition has also been reported to have an immunosuppressive effect in preclinical studies. Methods: Subcutaneous syngeneic models using the BRCA1 wild-type C2Km (P53-/-, myc, Kras-G12D, Akt-myr) and BRCA1 mutant BrKras (BRCA1-/-; P53-/-; myc; Kras-G12D; Akt-myr) murine ovarian cell lines were developed in the murine FVB/N background. Antibodies targeting PD-1 (RMP1-14) and PD-L1 (10F.9G2) were dosed by intraperitoneal injection at 5-10 mg/kg twice weekly, while rucaparib was administered by oral gavage at 150 mg/kg twice daily. Treatment was initiated at a tumor volume of ~150mm3 (n=15/group). Animals were dosed for 21 days, and tumors were allowed to regrow to day 76. Results: In vitro cytotoxicity assays demonstrated that rucaparib was 155-fold more potent in the BRCA1 deficient BrKras cell line (IC50 = 84 nM) than the isogenic BRCA1 wild-type C2Km cell line (IC50 = 13 μM). An in vivo study using the syngeneic BrKras model was performed in mice treated with: vehicle, rucaparib, PD-1, PD-L1, rucaparib+PD-1, and rucaparib+PD-L1. All monotherapy and combination groups resulted in significant tumor growth inhibition and were followed for survival analysis. The median survival time (MST) and % cures (defined as undetectable growth at Day 76 post-tumor implantation) for vehicle, PD-L1, PD-1 and rucaparib monotherapy treated animals was 34 days (0%), 41 days (13%), 76 days (40%) and >76 days (56%), respectively. The rucaparib+PD-1 and rucaparib+PD-L1 combination groups demonstrated greater efficacy than the monotherapies, with a MST of >76 days (100%) and >76 days (88%), respectively. Dose response and immune profiling studies are ongoing. In vivo studies were also performed in the BRCA1/2 wild-type models EMT-6, Pan02, and MC38. As expected, as a single agent, rucaparib showed limited activity in these homologous recombination competent models, whereas a range of tumor growth inhibition was observed with monotherapy PD-L1 treatment. No impact on anti-tumor activity was observed in animals treated with rucaparib+PD-L1 as compared to PD-L1 monotherapy in the BRCA1/2 wild-type syngeneic models examined. Conclusions: The combination of rucaparib with PD-1 and PD-L1 inhibition improved survival in a BRCA1 mutant syngeneic model. Citation Format: Liliane Robillard, Minh Nguyen, Andrea Loehr, Sandra Orsulic, Rebecca S. Kristeleit, Kevin Lin, Mitch Raponi, Thomas C. Harding, Andrew D. Simmons. Preclinical evaluation of the PARP inhibitor rucaparib in combination with PD-1 and PD-L1 inhibition in a syngeneic BRCA1 mutant ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3650. doi:10.1158/1538-7445.AM2017-3650

Abstract 4676: DNA repair protein expression and response of homologous recombination deficient ovarian cancer to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib in the ARIEL2 Part 1 study

Abstract Background: PARP inhibitors (PARPis) are active in cancers with homologous recombination (HR) defects. Preclinical studies have shown that secondary mutations or alterations in gene expression (e.g., downregulation of 53BP1, Ku70, Ku80 or DNA-PKcs) restore HR and confer PARPi resistance. In addition, low PARP1 expression can diminish PARP trapping and cause PARPi resistance. ARIEL2 Part 1 is a phase 2 study of the PARPi rucaparib in platinum sensitive, relapsed high grade serous or endometrioid ovarian cancer (OC). Pretreatment OC biopsies were previously assayed for HR gene mutations and loss of heterozygosity (LOH), a genomic scar that reflects HR deficiency. Two tumor groups (BRCA wildtype [wt]/LOH high and BRCA1 or BRCA2 mutant) have objective response rates of 29.3% and 80%, respectively, as well as progression free survival (PFS) of 5.7 and 12.8 months, respectively, on rucaparib (E. Swisher et al., Lancet Oncol., in press). Common AEs included nausea (80%), asthenia/fatigue (78%), constipation (46%), and vomiting (44%). The present studies tested the hypotheses that i) lower PARP1 expression and/or ii) lower expression of NHEJ components 53BP1, DNA-PKcs, Ku80, Ku70, or LIG4, may correlate with diminished response rate as well as PFS in pts treated with rucaparib on ARIEL2. Methods: Immunohistochemical assays were developed for 53BP1, DNA-PKcs, Ku80, Ku70, LIG4, and PARP1 and validated in formalin fixed, paraffin embedded cell lines differing in analyte expression. Available pretreatment OC biopsies from ARIEL2 Part 1 were stained and scored for % of tumor nuclei that were negative (0), weak (1+), moderate (2+) or strong (3+). Modified H-scores were correlated with clinical characteristics and outcome measures. Results: Pretreatment biopsies from 62-68 pts were successfully stained for each repair protein. Across the samples, PARP1 H-scores varied from 0 to 300 (median 160). Focusing on the BRCA wt/LOH high group (n = 38), there was no significant difference in PFS of pts with low (<100), intermediate (100-200) or high (>200) PARP1 H-score (p = 0.57). Expression of DNA-PKcs, Ku70, Ku80, and LIG4 was generally lower (median H-scores 20-60) and did not individually correlate with response or PFS. In contrast, there was a trend toward improved PFS in BRCA wt/LOH high OC pts expressing intermediate or high 53BP1 (H-score ≥ 100, n = 10) compared to pts with low 53BP1 (H-score <100, n = 25), with median PFS 20.7 vs. 5.5 months (p = 0.073), respectively. Staining of additional pretreatment biopsies is planned. Conclusions: In the BRCA wt/LOH high group, pretreatment PARP1 expression does not correlate with rucaparib response. In contrast, BRCA wt/LOH high OC pts with low 53BP1 have a trend toward shorter PFS with rucaparib, suggesting that 53BP1 downregulation might correlate with clinical PARPi resistance in BRCA wt/LOH high OC. Citation Format: Andrea E. Wahner Hendrickson, Kevin K. Lin, Daniel W. Visscher, Rachel M. Hurley, Mitch Raponi, Thomas C. Harding, Linda M. Murphy, Jill M. Wagner, Heidi Giordano, Iain McNeish, Elizabeth M. Swisher, Scott Kaufmann. DNA repair protein expression and response of homologous recombination deficient ovarian cancer to the poly(ADP-ribose) polymerase (PARP) inhibitor rucaparib in the ARIEL2 Part 1 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4676. doi:10.1158/1538-7445.AM2017-4676

Rucaparib: the past, present, and future of a newly approved PARP inhibitor for ovarian cancer.

Rucaparib camsylate (CO-338, AG-014699, PF-01367338) is a potent PARP-1, PARP-2, and PARP-3 inhibitor. Phase I and II studies demonstrated clinical efficacy in both BRCA-mutated (inclusive of germline and somatic) ovarian tumors and ovarian tumors with homologous recombination deficiency (HRD) loss of heterozygosity (LOH). Rucaparib has received the US Food and Drug Administration (FDA) approval for patients with deleterious BRCA mutation (germline and/or somatic)-associated advanced ovarian cancer who have been treated with two or more chemotherapies. There is evidence to suggest that rucaparib has clinical efficacy against ovarian tumors with high HRD-LOH. Rucaparib’s companion diagnostic FoundationFocus™ CDxBRCA test is the first FDA-approved next-generation sequencing-based companion diagnostic test designed to identify patients likely to respond to rucaparib. This article reviews the mechanisms of action, safety, approval, and indications for use of the PARP inhibitor rucaparib as well as future trials and use of rucaparib’s companion diagnostic test.

BRCA1 and RAD51C promoter hypermethylation confer sensitivity to the PARP inhibitor rucaparib in patients with relapsed, platinum-sensitive ovarian carcinoma in ARIEL2 Part 1

BRCA1 and RAD51C promoter hypermethylation confer sensitivity to the PARP inhibitor rucaparib in patients with relapsed, platinum-sensitive ovarian carcinoma in ARIEL2 Part 1

Abstract AP24: CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA

Abstract BACKGROUND: Ovarian high grade serous carcinoma (HGSC) is the commonest subtype of human ovarian cancer, and prognosis remains poor. Transplantable murine models of HGSC that recreate key mutations seen in the human disease are greatly needed. These models would assist investigation of the relationships between tumor genotype, chemotherapy response and immune microenvironment. ID8 is the most widely-used mouse model of ovarian cancer, but it has not been characterized in light of current understanding of HGSC biology. METHODS: We performed whole exome sequencing of ID8, covering 194,000 exons at a mean depth of 400x with 90% exons sequenced >50x. Using CRISPR/Cas9 gene editing, we have generated novel ID8 derivatives with single (Trp53-/-) and double (Trp53-/-;Brca2-/-, Trp53-/-;Brca1-/- and Trp53-/-;Pten-/-) deletions. We have characterized intra-peritoneal growth of these novel mutants, and investigated platinum and PARP inhibitor sensitivity and immune cell infiltration into the tumor microenvironment. RESULTS: Whole exome sequencing showed no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, Rb1). In addition, mutations typical of clear cell (Arid1A, Pik3ca), low grade serous (Braf), endometrioid (Ctnnb1) and mucinous (Kras) carcinomas were notably absent. ID8 also demonstrated intact p53 function and homologous recombination in functional assays. We show that loss of p53 alone significantly increases tumor growth rate within the peritoneal cavity of C57Bl/6 mice, and has marked effects upon immune microenvironment. In particular, loss of p53 increases CCL2 expression and induces immunosuppressive myeloid cell infiltration into tumor and ascites. Trp53-/-;Brca2-/- and Trp53-/-;Brca1-/- cells show significantly increased sensitivity to the PARP inhibitor rucaparib in vitro compared to parental and Trp53-/- cells. In vivo, Trp53- /-;Brca2-/- tumors are more sensitive to platinum chemotherapy than Trp53-/-. They also exhibit slower intraperitoneal growth, with appearance of intra-tumoral CD3+ cell-rich tertiary lymphoid structures, a phenotype observed in human tumors with high mutational burden. Mice bearing Trp53-/-;Pten-/- tumors have significantly reduced survival compared to Trp53-/-. Full in vivo assessment of the Trp53-/-;Brca1-/- and new tripleTrp53-/-;Brca2-/- ;Pten-/- lines is on-going. CONCLUSIONS: These models represent a new and simple tool to investigate the biology of HGSC. All cells will be made available to other researchers upon request. Citation Format: Josephine Walton, Julianna Blagih, Malcolm Farquharson, Darren Ennis, Elaine Leung, Suzanne Dowson, Laura A. Tookman, Clare Orange, Dimitris Athineos, Susan Mason, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R. Balkwill, Karen Vousden, Michelle Lockley, Iain A. McNeish. CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP24.

Abstract AP27: FEASIBILITY OF MONITORING RESPONSE TO THE PARP INHIBITOR RUCAPARIB WITH TARGETED DEEP SEQUENCING OF CIRCULATING TUMOR DNA (CTDNA) IN WOMEN WITH HIGH GRADE OVARIAN CARCINOMA ON THE ARIEL2 TRIAL

Abstract BACKGROUND: TP53 mutations are present in >97% cases of high-grade serous ovarian cancer (HGSOC). Detection of TP53 mutations in ctDNA extracted from plasma has the potential to monitor disease course and treatment response. We have developed targeted amplicon deep sequencing (TADS) to detect low frequency mutations throughout the TP53 gene in ctDNA. Rucaparib is a PARP inhibitor in development for treatment of tumors with HR pathway deficiency. We used TADS to assess TP53 mutant allele fraction (MAF) in ctDNA from patients in ARIEL2, a phase 2 study of rucaparib for treatment of relapsed high-grade ovarian cancer (NCT01891344). MATERIAL AND METHODS: Plasma samples (n=65) from 18 patients were collected during screening, on day 1 of each cycle, and at the end of rucaparib treatment. DNA extracted from plasma underwent TADS of TP53 (median depth 6916×). FFPE tumor specimens were profiled using an NGS-based assay with a targeted gene panel including TP53. Investigator-assessed clinical response rates were evaluated by RECIST v1.1 and GCIG CA-125 criteria. RESULTS: Concordant TP53 mutations were detected in tumor and ctDNA from plasma for all 18 patients. Median TP53 MAF at screening and cycle 1 day 1 was 5.1% (interquartile range: 1.1–17.5, n=16) and 3.8% (IQR: 0.68–10.3, n=16), respectively. Fourteen patients were evaluable for response measured by quantification of TP53 MAF between cycle 1 and 2 (missing sample: n=2; TP53 MAF <0.5%; n=2). 7/9 patients with >50% reduction of TP53 MAF in ctDNA at cycle 2 achieved a RECIST confirmed PR (see Table); this included 5/6 patients with either a germline or somatic mutation in BRCA1/BRCA2. No patients with <50% reduction at cycle 2 (n=5) achieved a RECIST response. CONCLUSIONS: Noninvasive detection of TP53 mutations by TADS is feasible, using plasma samples collected from women with relapsed platinum-sensitive high-grade ovarian cancer participating in an international multicenter trial. Circulating tumor DNA is a promising biomarker for monitoring response to the PARP inhibitor rucaparib. We are now testing the pre-specified hypothesis that a >50% reduction in TP53 MAF between baseline and cycle 2 is predictive of response to rucaparib using 560 plasma samples from 139 ARIEL2 subjects. Updated results will be presented at the meeting. Citation Format: Anna Piskorz, Kevin K. Lin, James Morris, Elaina Mann, Amit Oza, Robert L. Coleman, David M. O'Malley, Michael Friedlander, Janiel M. Cragun, Ling Ma, Heidi Giordano, Nitzan Rosenfeld, Mitch Raponi, Iain A. McNeish, Elizabeth Swisher, James D. Brenton. FEASIBILITY OF MONITORING RESPONSE TO THE PARP INHIBITOR RUCAPARIB WITH TARGETED DEEP SEQUENCING OF CIRCULATING TUMOR DNA (CTDNA) IN WOMEN WITH HIGH GRADE OVARIAN CARCINOMA ON THE ARIEL2 TRIAL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP27.

Abstract AP28: BRCA1 and RAD51C Promoter Hypermethylation Confer Sensitivity to PARP Inhibitors in Patients with Platinum Sensitive Ovarian Carcinoma

Abstract Germline and somatic mutations in BRCA1 and BRCA2 (BRCA) confer PARP inhibitor sensitivity. Promoter hypermethylation is an alternate mechanism of gene down-regulation, and BRCA1 promoter methylation is relatively common in sporadic ovarian cancer. The clinical significance of BRCA1 methylation is less clear than for mutations, as the Cancer Genome Atlas (TCGA) and others have failed to show improved survival in ovarian carcinomas with BRCA1 methylation. No one has previously tested whether BRCA1 methylation confers in vivo sensitivity to PARP inhibitors in patients with ovarian cancer. ARIEL2 is a phase 2 study of the PARP inhibitor rucaparib in patients with recurrent platinum sensitive high-grade ovarian, peritoneal or fallopian tube carcinoma. At enrollment, ARIEL2 required pre-treatment tumor biopsies with the goal of developing tissue predictors of PARP inhibitor sensitivity other than BRCA mutations. The number of women with known germline mutations was capped at 15 patients in order to predominantly enroll BRCA wildtype cases. As presented at ASCO 2016, in cases with no BRCA mutations, a high fraction of genomic loss of heterozygosity (LOH) significantly predicted a better progression-free survival (the primary endpoint), longer duration of response, and a higher fraction of responders compared to cases with low LOH. We assessed BRCA1 and RAD51C promoter hypermethylation using methylation-sensitive polymerase chain reaction in paired archival and pre-treatment biopsies from patients on ARIEL2. Of 165 cases for which methylation analyses were completed, 21 (12.7%) were methylated at the BRCA1 promoter and four (2.4%) at the RAD51C promoter. Methylation of BRCA1 and RAD51C was mutually exclusive with mutation in BRCA or other homologous recombination genes. All four cases with RAD51C methylation and 15/19 (78.9%) with BRCA1 methylation were associated with high LOH. In 90 paired samples archival and pre-treatment tissues, RAD51C methylation was 100% concordant and BRCA1 methylation was highly concordant (p<0·001). For 13 cases with BRCA1 methylation in the archival specimen, 4 (30·8%) were unmethylated in the paired pretreatment tumor, but for 77 unmethylated archival specimens, gain of methylation in the pretreatment biopsy was observed just once. Confirmed RECIST responses were seen in 52.4% (11/21) BRCA1 methylated and 75.0% (3/4) RAD51C methylated cases. In conclusion, BRCA1 and RAD51C methylation in ovarian carcinomas correlates with a high response rate to PARP inhibitors. If methylation was to be used as a predictor of PARP inhibitor sensitivity, it would need to be assessed in a pre-treatment (not archival) specimen. The loss of BRCA1 methylation in recurrent ovarian carcinoma, which was common even in these platinum sensitive cases, could explain why BRCA1 methylation is associated with similar survival to methylated cases, despite initial improved therapeutic sensitivity. Citation Format: Elizabeth Swisher, Maria Harrell, Kevin K. Lin, Clare Scott, Sandra Goble, Amit Oza, Robert L. Coleman, Gottfried Konecny, Anna V. Tinker, David M. O'Malley, Rebecca Kristeleit, Ling Ma, James Brenton, Katherine Bell-McGuinn, Ana Oaknin, Alexandra Leary, Elaina Mann, Heidi Giordano, Mitch Rapon, Iain McNeish, Scott H. Kaufmann. BRCA1 and RAD51C Promoter Hypermethylation Confer Sensitivity to PARP Inhibitors in Patients with Platinum Sensitive Ovarian Carcinoma [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP28.

Trial of rucaparib in prostate indications 3 (TRITON3): An international, multicenter, randomized, open-label phase 3 study of rucaparib vs physician’s choice of therapy for patients (Pts) with metastatic castration-resistant prostate cancer (mCRPC) associated with homologous recombination deficiency (HRD).

TPS5087 Background: Pts with mCRPC often initially receive androgen receptor-targeted therapy (eg, abiraterone or enzalutamide), but they almost always progress. Poly(ADP-ribose) polymerase (PARP) inhibitors (eg, olaparib) have demonstrated clinical activity in pts with mCRPC with a deleterious mutation in a homologous recombination (HR) DNA repair gene; 14 of 16 evaluable pts with mCRPC and a tumor alteration in an HR gene, including BRCA1, BRCA2, and ATM, responded to olaparib (Mateo et al. N Engl J Med. 2015;373:1697-708). The PARP inhibitor rucaparib is approved in the US for treatment of pts with ovarian carcinoma that harbors a deleterious BRCA1 or BRCA2 mutation (germline and/or somatic) who have received ≥2 chemotherapies. These data support investigating rucaparib as a treatment option in pts with mCRPC with HRD. Methods: TRITON3 (NCT02975934)is a phase 3 study evaluating rucaparib (600 mg BID) vs physician’s choice (abiraterone, enzalutamide, or docetaxel) in pts with mCRPC who have a deleterious germline or somatic mutation in BRCA1, BRCA2, or ATM as determined by local and/or central testing. All pts must have progressed on abiraterone or enzalutamide in the mCRPC setting; pts who received prior chemotherapy for mCRPC or PARP inhibitor treatment are excluded. Pts will be randomized 2:1 to rucaparib or physician’s choice of comparator therapy; pts randomized to physician’s choice may cross over to rucaparib on radiographic progression confirmed by independent radiology review (IRR). The primary endpoint is IRR-confirmed radiographic progression-free survival (modified RECIST version 1.1/PCWG3 criteria). Secondary objectives include overall survival, objective response rate, duration of response, clinical benefit rate, pt-reported outcomes, and safety. Pretreatment blood samples will be collected from all pts to enable development of a plasma-based companion diagnostic to identify pts who may benefit from rucaparib treatment. Pts (≈400) will be enrolled at > 100 sites worldwide. Clinical trial information: NCT02975934.

Ovarian Cancers Harbor Defects in Nonhomologous End Joining Resulting in Resistance to Rucaparib.

Purpose: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focused on homologous recombination (HR), DNA double-strand breaks are repaired primarily by nonhomologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance.Experimental Design: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay.Results: NHEJ was defective (D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P = 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, induced resistance to rucaparib (P = 0.014) and HR function recovery in a BRCA1-defective cell line.Conclusions: This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin Cancer Res; 23(8); 2050-60. ©2016 AACR.

Abstract A23: BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance

Abstract Introduction: The BRCA1 protein harbors two C-terminal tandem BRCT domains. The first is encoded by BRCA1 gene sequences located toward the end of exon 16, and the second BRCT domain initiated at exon 21. BRCA1 BRCT domains bind to proteins containing a phosphorylated serine-proline-x-phenylalanine (pSPxF) motif, including Abraxas, BACH1 and CtIP. Protein–protein interactions mediated by BRCT domains regulate DNA damage repair and cell cycle checkpoint signaling. Many cancer-associated mutations located in the BRCT domains of BRCA1 result in protein structural defects, and consequently misfolding and proteasomal degradation. Experimental Procedures: In this study, we examined PARP inhibitor (PARPi) resistance mechanisms utilizing MDA-MBA-436, HCC-1395 and SNU-251 human breast cancer and ovarian cancer cell lines that harbor frameshift mutations in BRCA1 exon 20, exon 20 and exon 23, respectively; predicted to generate BRCT domain disruptions. Cells were cultured in the presence of increasing concentrations of the PARPi rucaparib until resistant clones emerged. Quantitative RT-PCR and Western blotting were used to measure BRCA1 mRNA and protein levels. Results: Despite BRCA1 mRNA being abundant in all parental cell lines, BRCA1 protein was low or undetectable. However, PARPi resistant cell lines all demonstrated elevated BRCA1 protein levels that could be detected with N- but not C-terminal specific antibodies. Notably, the gel migration and molecular weight of BRCA1 proteins were markedly lower than the mutation-induced stop codon expected size. To identify potential protein products, we carried out immunoprecipitation and mass spectrometry to analyze BRCA1 peptide sequences. BRCA1 peptides encoded by exons 2-16 were readily detected; however, no peptides encoded by exon 17-24 were present in any cell line. qRT-PCR analyses suggested that BRCA1 mRNA was subject to alternative splicing and removal of exons 16-24. To understand why PARPi selection pressure generates BRCT-less BRCA1 proteins, we ectopically overexpressed BRCA1 cDNA that harbored stop codons located in BRCT domains or prior to the first BRCT domain. BRCT mutation containing constructs had undetectable protein levels, presumably due to protein misfolding, and cells were highly PARPi sensitive. In contrast, cells expressing BRCA1 constructs with stop codons prior to the BRCT domains had robust protein expression and demonstrated residual RAD51 foci and PARPi resistance. Conclusions: Our findings indicate that alternative splicing can remove deleterious mutations that disrupt BRCT peptide folding, generating more truncated but functional proteins capable of restoring residual DNA repair and PARPi resistance. Citation Format: Yifan Wang, Andrea J. Bernhardy, Neil Johnson. BRCA1 mutations in the BRCT domain can be removed through alternative splicing and induce PARP inhibitor resistance [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A23.

Rucaparib Approved for Ovarian Cancer.

The FDA approved the PARP inhibitor rucaparib to treat women with advanced ovarian cancer who have already been treated with at least two chemotherapies and have a BRCA1 or BRCA2 gene mutation identified by an approved companion diagnostic test. The agency also gave a nod to the FoundationFocus CDxBRCA test to detect BRCA alterations.

Genomic LOH May Predict Rucaparib Response in Ovarian Cancer

Abstract High LOH is associated with response to the PARP inhibitor rucaparib in BRCA wild-type ovarian cancer.

373 - Feasibility of monitoring response to the PARP inhibitor rucaparib with targeted deep sequencing of circulating tumor DNA (ctDNA) in women with high grade ovarian carcinoma on the ARIEL2 trial

373 - Feasibility of monitoring response to the PARP inhibitor rucaparib with targeted deep sequencing of circulating tumor DNA (ctDNA) in women with high grade ovarian carcinoma on the ARIEL2 trial