Erin George

Targeting the ATR/CHK1 Axis with PARP Inhibition Results in Tumor Regression in BRCA-Mutant Ovarian Cancer Models

Purpose: PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA-mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression. Experimental Design: Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776), or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell cycle, genome instability, and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived xenograft (PDX) model. Results: PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum tolerated dose (MTD) in a BRCA2MUT PDX. PARPi treatment increased pATR and pCHK1, indicating activation of the ATR–CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared with single-agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA2MUT PDX with the PARPi–ATRi combination inducing tumor regression and in most cases, complete remission. Conclusions: PARPi causes increased reliance on ATR/CHK1 for genome stability, and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models. Clin Cancer Res; 23(12); 3097–108. ©2016 AACR.

RING domain–deficient BRCA1 promotes PARP inhibitor and platinum resistance

Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain-deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.

A patient-derived-xenograft platform to study BRCA-deficient ovarian cancers

Approximately 50% of high-grade serous ovarian cancers (HGSOCs) have defects in genes involved in homologous recombination (HR) (i.e., BRCA1/2). Preclinical models to optimize therapeutic strategies for HR-deficient (HRD) HGSOC are lacking. We developed a preclinical platform for HRD HGSOCs that includes primary tumor cultures, patient-derived xenografts (PDXs), and molecular imaging. Models were characterized by immunohistochemistry, targeted sequencing, and reverse-phase protein array analysis. We also tested PDX tumor response to PARP, CHK1, and ATR inhibitors. Fourteen orthotopic HGSOC PDX models with BRCA mutations (BRCAMUT) were established with a 93% success rate. The orthotopic PDX model emulates the natural progression of HGSOC, including development of a primary ovarian tumor and metastasis to abdominal viscera. PDX response to standard chemotherapy correlated to that demonstrated in the patient. Pathogenic mutations and HGSOC markers were preserved after multiple mouse passages, indicating retention of underlying molecular mechanisms of carcinogenesis. A BRCA2MUT PDX with high p-CHK1 demonstrated a similar delay of tumor growth in response to PARP, CHK1, and ATR inhibitors. A poly (ADP-ribose) polymerase (PARP) inhibitor radiotracer correlated with PARP1 activity and showed response to PARP inhibition in the BRCA2MUT PDX model. In summary, the orthotopic HGSOC PDX represents a robust and reliable model to optimize therapeutic strategies for BRCAMUT HGSOC.

Personalized vaccination against ovarian cancer: What are the possibilities?

The basis of immunology is the discrimination between self and nonself. All pathogens have molecular signatures that can be recognized by the host’s immune system and activate immune responses. Vaccinations against viruses and bacteria, which have successfully exploited this vulnerability, have become an integral part of preventive healthcare. With regard to cancer therapy, prophylactic vaccines against human papilloma virus have evolved over the last decade for the prevention of cervical cancer and have become part of routine vaccination schedules [1]. Theoretically, vaccines for ovarian cancer patients directed against private tumor neo-antigens derived from nonsynonymous somatic mutations is a promising treatment strategy. Such an approach will activate endogenous immune cells to recognize specific tumor associated antigens (TAAs), thus killing cancer cells with minimal harm to the surrounding healthy tissue. Additionally, by increasing the pool of available tumor-specific T cells, therapeutic cancer vaccines could contribute to combination immunotherapy. There is still an ongoing debate about the immunogenicity of ovarian cancer. Some evidence indicates that a proportion of these tumors express a large number of known TAAs [2], have immunoreactive gene signatures, and are infiltrated by intraepithelial tumor–infiltrating lymphocytes (TILs) [3]. On the other hand, others have demonstrated that ovarian tumors have a very heterogeneous and comparatively low mutational load, thus making immune recognition of neo-antigens dubious [4]. In spite of this comparatively low mutational load, however, one type of ovarian tumor, epithelial ovarian cancer (EOC), is susceptible to immune recognition [2,3]. Unfortunately, the projected potential of cancer vaccines based on these preclinical findings has not translated into the clinical setting thus far. The explanation for this can be the wrong choice of antigens or can be related to the inability of tumor-specific T cells to home in the tumor microenviroment. For example, barriers that prevent T cell homing in ovarian tumors include the peripheral tolerance mediated by regulatory T cells (T Reg) or the vascular endothelial growth factor A (VEGF-A) mediated vascular endothelial barrier [5]. Another major reason for clinical failure includes that most molecularly defined tumor vaccines, up till now, have used a single ‘self’ antigen. Therefore, the poor outcome of this approach in the clinical trials could be credited, at least in part, to immune-selection of antigen loss variants after vaccination. There are two main classes of targetable TAAs in ovarian cancer: the shared common TAAs and the exclusive, individually mutated neo-antigens. The shared antigens can be separated into three main groups: the overexpressed antigens, which are normal surface proteins expressed in elevated levels on cancer cells but in lower levels in normal cells (i.e. mesothelin); tissue-specific TAAs, which are shared between tumors and the normal tissue of their origin; and TAAs whose expression is normally restricted to male germline cells (i.e. cancer testes (CT) antigens such as NY-ESO-1) [6]. In the past decades, the mutation-based cancer vaccines were restricted to using synthetic peptides featuring frequent driver mutations in many human cancers such as mutated p53 or Ras [7]. Also, the available pool of immunogenic mutations increased very slowly and most of the published shared immunogenic mutations were limited to small subsets of cancer patients [8]. Further, there was no feasible concept to use individual mutations. Instead, the therapeutic application of TAAs was focused mainly on shared TAAs. One example of a shared TAA tested in early phase clinical trials is the cancer testes antigen NY-ESO -1 [9]. In this small phase I trial decitabine, which has been shown to enhance NY-ESO-1 expression, was used in combination with a NY-ESO-1 vaccine and liposomal doxorubicin chemotherapy in recurrent ovarian cancer. Stable disease was noted in 50% (5/10 patients) that lasted a median of 6.3 months and a partial response was seen in 1 patient with duration of response of only 5.8 months [9]. Since then others have studied cancer testes antigens with conflicting results in clinical trials [10]. They have also found that some of the CT antigens are expressed at low levels in some normal tissues and that no single CT antigen is universally expressed and that the frequency and expression levels can be heterogeneous across tumors [10]. The high-avidity T cell clones recognizing these shared antigens may be deleted by central tolerance mechanisms, which may be one other explanation of the unsatisfactory outcome of clinical vaccination trials [11]. On the other hand, the current technological breakthroughs and extensive deep sequencing analyses have discovered that solid tumors contain as few as tens to several thousand private non-synonymous somatic mutations, and these mutations are different even among tumors from the same histologic subtype [12]. These neo-antigens result from the large number of somatic mutations that are fully tumor

Abstract A16: Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers

Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. We and others have shown that inhibition of the ATR checkpoint kinase is synthetically lethal with multiple distinct cancer-associated mutations, including p53 loss, oncogenic stress (HRAS-G12V, KRAS-G12D, NRAS-G12D, MYC and CCNE1), deficiency in homologous recombination (BRCA1/2, PALB2, ATM loss), alternative lengthening of telomeres (ALT), chromatin modification (SETD2 loss), and others. The specificity of ATR inhibitors is vital to their successful clinical application, since off-targeting increases toxicity to normal cells and limits value in personalized treatments. We report the generation of a novel class of highly potent and specific ATR inhibitors (ATRN series) that exhibit low nanomolar activity in cultured cells (IC50 = 2-8 nM) and do not detectably inhibit ATM, DNA-PK or mTOR (IC50 > 10 µM). In side-by-side comparisons, both the potency and selectivity of the ATRN series is superior to previously reported ATR inhibitors (VE821, VE822, AZD20, AZD6738, ETP-46464). In addition, the ATRN series has sufficient bioavailability and stability for in vivo application. Our lead compound (ATRN-119) slows progression of human BRCA2-deficient PDAC (CAPAN1) and RAS oncogene-driven p53-null colon tumors in mice with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived xenograft (PDX) ovarian tumors show a significant reduction in tumor progression after 5 weeks treatment of ATRN-119, and display no toxicity or significant weight loss. In addition, cancers that maintain or reacquire HR function, and thus are resistant to treatment with PARPi or cisplatin, remain responsive to ATRN series inhibitors. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian, and PARP resistant cancers, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. In summary, the ATRN series is a highly selective and potent class of ATR inhibitors with therapeutic potential for treating a broad range of cancers. The potentially identified biomarkers, in particular HR-deficiency, will inform patient selection (a critical component for delivering medical benefit) for treatment with our agent and could reduce future clinical risk and side-effects. Thus targeting patients with HR-deficient cancers is a particularly promising strategy in treating a broad range of cancers. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Erin George, Tina Gill, Matthew Scheiwer, Nicolas Gordon, Karen Knudsen, Fiona Simpkins, Oren Gilad and Eric J. Brown. Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers [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 A16.

Abstract AP29: BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE

Although cancers harboring BRCA1 mutations initially respond well to platinum and poly(ADP-ribose)-polymerase inhibitor (PARPi) therapy, resistance invariably arises and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. In this study, we utilized the SUM1315MO2 cancer cell line that harbors a hemizygous BRCA1185delAG mutation to study PARPi and platinum resistance. SUM1315MO2 cells were initially PARPi and cisplatin sensitive but readily acquired resistance. PARPi and cisplatin resistant clones did not harbor secondary reversion mutations. Rather, increased expression of a RING domain-deficient BRCA1 protein (Rdd-BRCA1) was required for resistance. Translation initiation occurred downstream of the frameshift mutation, likely at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BARD1 interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci-formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance in vitro and in vivo. Furthermore, Rdd-BRCA1 protein expression was detectable in recurrent carcinomas from germline BRCA1185delAG mutation carriers. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic, and when expressed at high enough levels are capable of contributing to PARPi and platinum resistance. Citation Format: Yifan Wang, John J. Krais, Andrea J. Bernhardy, Emmanuelle Nicolas, Kathy Q. Cai, Maria I. Harrell, Hyoung H. Kim, Erin George, Elizabeth M. Swisher, Fiona Simpkins and Neil Johnson. BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE [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 AP29.

Abstract B46: Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research.

Introduction: Treatment for cancer is moving from a “one size fits all” to a personalized and targeted approach addressing the unique biology of each patients9 tumor. Preclinical research in ovarian cancer (OVCA) has relied on established cell lines that have recently been shown to have marked differences in molecular profiles when compared to ovarian tumors from the TCGA. Patient derived xenografts (PDXs) are emerging as a reliable preclinical model that can recapitulate the principal characteristics of the patients9 original tumor while remaining biologically stable while passaged in mice. We developed an orthotopic model that involves transplanting tumor directly to the fallopian tube/ovary in order to accurately study ovarian cancer tumorigenicity and metastasis in the native environment. Experimental Procedures: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week female mice. Tumor growth was followed over time with ultrasound. Tumors were evaluated by IHC, genomic and proteomic analysis. Alu II probe staining was performed to evaluate human stroma content. DNA sequencing analysis was performed using a 153 ovarian cancer gene panel, which includes all genes relevant to OVCA, including homologous recombination and all actionable genes. Reverse Phase Protein Array Analysis (RPPA) was used to evaluate signaling pathway activation. Several primary ovarian tumor cultures were also developed from the patients9 tumor for mechanistic studies. Results: To date, we have transplanted tumor from 18 primary, 4 interval, and 6 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 90% success rate in generating tumors in mouse passage 1 (MP1) and 100% in generating MP2 and MP3. The mean time for tumors to reach ~1 cm (first passage in mice) was 8 weeks and ~6 weeks when tumors were then passaged again (MP2/MP3). We have generated 12 BRCA1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrated human cells in the stroma. We have generated several primary tumor cell lines from the original tumors/PDXs. These primary cells express ovarian epithelial markers CK7, and PAX8. Targeted DNA sequencing analysis showed that P53 and BRCA1/2 pathogenic mutations were highly conserved from the patient tumor, to the PDXs (MP1-3) and primary tumor cell lines. We evaluated 280 phospho/total proteins in our tumor samples by RPPA. Unsupervised cluster analysis of 24 patient tumors and correlating PDX (MP1-3) and 6 cell lines showed that several parent tumors clustered together with their MP1-3 PDXs. WO-2-1 BRCA 2 mutant PDXs were sensitive to platinum treatment demonstrating a response similar to that noted in the original patient after platinum chemotherapy. Conclusions: Although technically more challenging the orthotopic transplantation technique is feasible in generating ovarian cancer PDX models that more closely resemble the natural environment for ovarian cancer tumor growth and metastasis. PDXs maintain consistent gene alterations and signaling pathway activation to the original patient tumor. Given PDXs maintain the characteristics of the patients9 original tumor, they are excellent models to study therapeutic response. Citation Format: Erin George, Hyoung Kim, Clemens Krepler, Janos Tanyi, Zhi Wei, Katrin Sproesser, Patricia Brafford, Marilda Beqiri, Adina-Monica Vultur, Rachel Lee, Mark Morgan, Ronny Drapkin, Tan Ince, Meenhard Herlyn, Fiona Simpkins. Use of a novel orthotopic ovarian cancer transplant patient derived xenograft model as a preclinical platform for bench to bedside research. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B46.

Abstract A02: A novel orthotopic ovarian patient derived xenograft model platform to investigate novel therapies for BRCA deficient ovarian cancers

Introduction: To create a personalized, targeted approach to high grade serous ovarian cancers (HGSOC), reliable preclinical models are essential. About ~50% of HGSOC have defects in genes involved in homologous recombination (HR) such as BRCA. PARP inhibitors (PARPi) capitalize on synthetic lethality in HR-deficient tumors, however, clinical efficacy is limited (response rate only ~40%). Patient derived xenografts (PDXs) are emerging as a reliable preclinical model that recapitulates principal characteristics of the patients9 tumor while remaining biologically stable while passaged in mice. We developed a BRCA1/2 orthotopic PDX experimental platform to study alternative strategies for synthetic lethality. We hypothesized that targeting the ATR/CHK1 axis is synthetically lethal in BRCA mutant HGSOC models. Experimental Procedures: Fresh HGSOC tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 wk mice. Tumor growth was followed. Tumors were evaluated by IHC, genomic and proteomic analysis. Alu II probe staining was used to evaluate human stroma content. DNA sequencing was performed using a 153 OVCA gene panel. Reverse Phase Protein Array Analysis (RPPA) was evaluated for signaling pathway activation. Primary ovarian tumor cultures were developed from patients9 tumor for mechanistic studies. To study the ATR/CHK1 axis in HR-deficient HGSOC, PARPi (Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) were evaluated. PEO1 (BRCA2 mutant), PEO4 (BRCA wildtype) and JHOS4 (BRCA1 mutant) HGSOC cells were evaluated for cell proliferation, survival, and genome stability before and after treatment. BRCA2 mutant (8945delAA) PDX (WO-2-1) was expanded in 70 mice. Mice were randomized into 5 gps: untreated, carboplatin, PARPi, CHK1i, and ATRi. Treatment was initiated when tumors were 70-100mm3 and volume was assessed weekly with ultrasound. PARP tumor activity and response to PARPi was assessed with a PET PARP1 radiotracer [18F]FTT (fluorthanatrace). Results: We developed a pipeline to study HR deficient HGSOC. We created an orthotopic PDX platform from 15 BRCA mutant patients in order to accurately study OVCA tumorigenicity and metastasis in the native environment with a 90% take rate in generating tumors in mouse passage 1 (MP1), and 100% take rate for MP2 and MP3. The PDX model (WO-2-1) was similarly platinum sensitive as the patient after platinum treatment. Tumors were evaluated by genomic and proteomic analysis to identify a target population and streamline therapeutic approaches. Pathogenic mutation profiles from the original patient tumor were preserved in PDXs serially passaged (MP1-3). High pCHK1 (s345) was used as a marker for investigation of ATR/CHK1 inhibition in BRCA mutant PDX models. We showed that ATRi and CHK1i are similarly effective to PARPi in a BRCA2 mutant PDX. A novel PET PARP1 radiotracer [18F]FTT was used and demonstrated co-localization of signal in a BRCA2 mutant PDX, which was diminished with olaparib treatment. Conclusions: Although technically more challenging, the orthotopic transplantation technique is feasible in generating HGSOC PDX models with a high success rate that more closely resembles the natural environment for HGSOC progression. Evaluation of genomic and proteomic profiles of a tumor allows one to streamline targeted therapies for testing in PDX preclinical trials that may in the future be translated back to the patient. Citation Format: Erin George, Hyoung Kim, Janos Tanyi, Ryan Ragland, Eric Brown, Rugang Zhang, Patricia Brafford, Katrin Sproesser, Marilda Beqiri, Adina Vultur, Clemens Krepler, Brandon Weis, Kate Nathanson, Yuling Lu, Gordon Mills, Mehran Makvandi, Robert Mach, Mark Morgan, Fiona Simpkins. A novel orthotopic ovarian patient derived xenograft model platform to investigate novel therapies for BRCA deficient ovarian cancers. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A02.

Abstract A08: Targeting the ATR/CHK1 axis in BRCA1/2 mutant ovarian cancer using an orthotopic patient-derived xenograft (PDX) model.

Introduction: Approximately 50% of high grade serous ovarian cancers (HGSOCs) have defects in genes involved in homologous recombination repair (HR). BRCA1/2 mutant HGSOCs have a deficiency in the repair of double strand DNA breaks by HR. Poly(ADP-ribose) polymerase inhibitors (PARPi) impair the repair of single-stranded breaks leading to double strand DNA breaks which cannot be repaired efficiently in BRCA-deficient cancers capitalizing on synthetic lethality. Given, PARPi only have a modest clinical response rate of ~40%, alternative strategies capitalizing on synthetic lethality are needed. The ATR/CHK1 signaling pathway is responsible for arresting cell cycle progression allowing repair of DNA damage. Targeting the ATR/CHK1 axis represents an alternative approach capitalizing on synthetic lethality in BRCA mutant HGSOCs. Experimental Procedures: PARP inhibitor (PARPi, Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) were evaluated in vitro and in vivo. PEO1 (BRCA2 mutant), PEO4 (BRCA1/ 2 wildtype) and JHOS4 (BRCA1 mutant) ovarian cancer cells were used for in vitro studies. Cell proliferation, survival, and genome stability were quantified and drug activity on cell signaling pathways was assessed. Using a BRCA2 deficient PDX model (8945delAA), the tumor was expanded via orthotopic transplantation onto the fallopian tube/ovary into 70 5-8 week female mice. Mice were randomized into 5 groups: untreated, carboplatin, PARPi, CHK1i,and ATRi. Once tumor volumes reached 70-100 mm3, treatment was initiated and tumor volume was assessed by ultrasound. Results: We have preliminary data that ATRi and CHK1i are as effective as PARPi in BRCA1/2 mutant ovarian cancer cells. pH2AX protein, a marker for DNA damage, is increased more so in the BRCA mutant cells compared to wildtype demonstrating selective activity in HR deficient cells capitalizing on synthetic lethality. pCHK1 accumulates with CHK1i (given the decrease in activation of downstream phosphatase PPA2) and is decreased with ATRi confirming drug inhibiting target. In a pilot study using a novel BRCA2 deficient patient derived xenograft (PDX) model, treatment with CHK1i and ATRi have similar anti-tumor effects to that of PARPi (olaparib) at 2 weeks. Reverse Phase Protein Array Analysis (RPPA) and target DNA sequencing results to assess signature of response are pending. Conclusions: Strategies to optimize approaches capitalizing on synthetic lethality are needed. BRCA mutant PDXs serve as novel experimental models for therapeutic studies. ATRi or CHK1i may be equally or more effective than PARPi as a primary treatment option for BRCA1/2-deficient cancers. Citation Format: Hyoung Kim, Erin George, Eric Brown, Rugang Zhang, Clemens Krepler, Janos Tanyi, Rachel Lee, Mark Morgan, Meenhard Herlyn, Fiona Simpkins. Targeting the ATR/CHK1 axis in BRCA1/2 mutant ovarian cancer using an orthotopic patient-derived xenograft (PDX) model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A08.

Abstract 4835: Targeting the ATR/CHK1 axis in combination with PARP inhibition is more effective than PARP inhibition alone in BRCA mutant models

Introduction: Approximately 50% of high grade serous ovarian cancers (HGSOC) have defects in genes involved in homologous recombination repair (HR). BRCA1/2 mutant HGSOCs have a deficiency in the repair of double strand DNA breaks by HR. Poly(ADP-ribose) polymerase inhibitors (PARPi) block the repair of single-stranded breaks leading to double strand DNA breaks which cannot be repaired efficiently in BRCA-deficient cancers capitalizing on synthetic lethality. PARPi have a modest clinical response of only ∼40% in recurrent BRCA mutant HGSOCs. We hypothesize that PARPi alone increases reliance on other DNA repair pathways such as ATR/CHK1 in HR deficient cells, and by targeting ATR/CHK1 in combination with PARPi would be more effective in eradicating tumor growth. Experimental Procedures: Effects of PARP inhibitor (PARPi, Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) on cell cycle, survival, colony formation, genome stability were evaluated in PEO1 (BRCA2 mutant), PEO4 (BRCA wild-type), JHOS4 (BRCA1 mutant), and WO-24 (BRCA wild-type) ovarian cancer cells. A BRCA2 mutant (8945delAA) orthotopic PDX model was used to evaluate PARPi alone or in combination with CHK1/ATRi. Targeted capture massively parallel sequencing, Reverse-Phase Protein Array Analysis (RPPA) and IHC were performed on cells and xenografts to evaluate for biomarkers of response. Results: Monotherapy with PARPi, CHK1i, and ATRi in vitro demonstrated selectivity in mediating cell death and DNA damage in BRCA1/2 mutant cell lines (PEO1, JHOS4) compared to BRCA1/2 wild-type, platinum resistant cell lines (PEO4, WO-24). However, monotherapy only results in ∼40-50% cell death in BRCA1/2 mutant cell lines. PARPi alone resulted in tumor suppression but not tumor eradication in a BRCA2 mutant PDX model. PARPi treatment resulted in an increase in ATR/CHK1 signaling in BRCA1/2 mutant cells. Treatment with ATR/CHK1i in combination with PARPi is synergistic in reducing survival of BRCA1/2 cells. Combination treatment was more effective in targeting cell cycle mediators, and promoting apoptosis. Treatment with either PARPi+ATRi or PARPi+CHK1i combinations was synergistic in causing tumor suppression but PARPi/ATRi combination caused tumor regression in a BRCA2 mutant PDX model. Conclusions: Strategies to optimize approaches capitalizing on synthetic lethality are needed for HR deficient HGSOC. PARPi is effective in BRCA deficient cancers but can potentially be more effective when combined with ATR/CHK1i. Citation Format: Erin George, Hyoung Kim, Janos Tanyi, Ryan Ragland, Rugang Zhang, Patricia Bradford, Clemens Krepler, Katherine Nathanson, Brandon Wenz, Yiling Lu, Gordon Mills, Mark Morgan, Fiona Simpkins. Targeting the ATR/CHK1 axis in combination with PARP inhibition is more effective than PARP inhibition alone in BRCA mutant models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4835.