Weroha, Saravut J.

5077 Background: A standard of care (SOC) for the adjuvant treatment of ovarian and primary peritoneal cancer is a platinum-based intravenous (IV) chemotherapy doublet. Intraperitoneal (IP) chemotherapy is also a SOC, but the high incidence of grade 3/4 adverse events has limited its acceptance among clinicians. To identify genetic markers for completion of IP chemotherapy, we analyzed SNPs in four genes known to play a role in metabolism of platinum or taxane drugs. Methods: Patients diagnosed with primary or recurrent stage III or IV epithelial ovarian or primary peritoneal cancer who had primary or secondary debulking surgery at Mayo Clinic (Rochester, MN) between January 2007 and February 2009 followed by IP chemotherapy were included in this study. A cycle was defined as IV paclitaxel (135mg/m2) on day one, IP cisplatin (100mg/ m2) on day two, and IP paclitaxel (60mg/ m2) on day eight. With prior consent from the patient, peripheral blood was obtained before treatment for extraction of germline DNA. Using a custom Illumina BeadXpress 96-plex panel, SNPs in GSTM1 (N=7), ABCB1 (N=57), CYP3A4 (N=7), and CYP2C8 (N=25) were genotyped. The association between SNPs in these subsets of genes and completion of IP chemotherapy was analyzed using linear regression. Results: Thirty-seven patients were included in this study and 16 (43.2%) completed IP chemotherapy. Twenty-two out of the fifty-seven ABCB1 SNP’s were associated with the number of cycles (p<0.15). There were no significant associations for GSTM1, CYP3A4 and CYP2C8 SNP’s. The minor A allele at missense SNP rs2229109 in the ABCB1 gene was present in five (31.2%) who completed treatment and only one (4.8%) who did not (p<0.007). However, the later was due to catheter complication. There was no association between rs2229109 and overall or progression-free survival. Conclusions: Our findings suggest that SNP rs2229109 in ABCB1 gene is associated with completion of IP chemotherapy, and potentially, less toxicity from chemotherapy. Thus, these patients may be optimal candidates for IP chemotherapy. Further studies in a larger population are needed.

Abstract Poly (ADP) ribose polymerase inhibitors (PARPi) have shown promising activity against recurrent ovarian cancers, particularly in women with germ line mutations of BRCA1/2. The efficacy of PARP inhibitors can, however, be decreased by acquired drug resistance. Autophagy or “self-eating” is one mechanism implicated in cancer progression and therapy resistance. Autophagy is a catabolic mechanism that can degrade organelles and long-lived proteins to provide energy for cancer cells under nutrient poor conditions or in the presence of stress. The energy provided from autophagy is used to fuel the DNA repair process and metabolic needs in cancer cells. Autophagy can protect cancer cells from chemotherapy or can enhance the response to certain drugs. In this study, we have asked whether autophagy contributes to the resistance of ovarian cancer cells to PARPi. Our group has found that four PARP inhibitors (olaparib, niraparib, rucaparib and talazorapib) induce autophagy in 8 ovarian cancer cell lines, detected by LC3A to LC3B conversion on western blot analysis and punctate GFP-LC3 fluorescence. Transmission electron microscopy confirmed the increased autophagosome formation in olaparib treated cells. To test whether autophagy contributes to the resistance of OC cells to olaparib, we determined the impact of autophagy inhibition on the sensitivity of OC cell lines to olaparib by either using chloroquine, hydroxychloroquine or LYS05 to block hydrolysis of proteins and lipids in autophagosomes or using siRNA against ATG5 or ATG7 to prevent formation of autophagosome. Enhancement of PARPi activity was observed in 6 out 8 ovarian cancer cell lines. Moreover, a combination of olaparib and chloroquine proved more effective than either single agent in a xenograft model of the OVCAR8 ovarian cancer cell line and in a patient derived xenograft model. Mechanistically, olaparib decreased PARP activity leading to DNA damage and accumulation of γ-H2AX. Olaparib treatment increased reactive oxygen species (ROS) and phosphorylation of ATM, while decreasing the phosphorylation of AKT and mTOR. We also observed an increase in PTEN, a well-recognized negative regulator of AKT. Inhibition of PTEN decreases LC3 conversion, suggesting PTEN as an important regulator of olaparib-induced autophagy in ovarian cancer cells. Taken together, our data demonstrate for the first time that olaparib induces autophagy in ovarian cancer cells and provides an adaptive mechanism of resistance to PARPi. Olaparib-induced autophagy may depend upon activation of ATM and PTEN by ROS, downregulating pAKT/pmTOR signaling. Moreover we have identify unique combinations of therapy with PARPi and CQ or other inhibitors of autophagy that could improve outcomes for ovarian cancer patients. Citation Format: Janice M. Santiago-O'Farrill, Saravut J. Weroha, Xiaonan Hou, Lan Pang, Philip Rask, Zhen Lu, Robert C. Bast. PARP inhibitor-induced autophagy provides an adaptive mechanism of drug resistance in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4768.

Ovarian cancer is the most lethal gynecologic malignancy. About 75% of ovarian cancer patients relapse and/or develop chemo‐resistant disease after initial response to standard‐of‐care treatment with platinum‐based therapies. HER2 amplifications and overexpression in ovarian cancer are reported to vary, and responses to HER2 inhibitors have been poor. Next generation sequencing technologies in conjunction with testing using patient‐derived xenografts (PDX) allow validation of personalized treatments. Using a whole‐genome mate‐pair next generation sequencing (MPseq) protocol, we identified several high grade serous ovarian cancers (HGS‐OC) with DNA alterations in genes encoding members of the ERBB2 pathway. The efficiency of anti‐HER2 therapy was tested in three different PDX lines with the identified alterations and high levels of HER2 protein expression. Treatment responses to pertuzumab or pertuzumab/trastuzumab were compared in each PDX line WITH standard carboplatin and paclitaxel combination treatment. In all three PDX models, HER2‐targeted therapy resulted in significant inhibition of tumor growth compared with untreated controls. However, the responses in each case were inferior to those to chemotherapy, even for chemo‐resistant lines. When chemotherapy and HER2‐targeted therapy were administered together, a significant regression of tumor was observed after 6 weeks of treatment compared with chemotherapy alone. Post‐treatment analysis of these tissues revealed that inhibition of the ERBB2 pathway occurred at the level of phosphorylation and expression of downstream targets. In conclusion, while targeting of presumably activated ERBB2 pathway alone in HGS‐OC results in a modest treatment benefit, a combination therapy including both chemotherapy drugs and HER2 inhibitors provides a far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to HER2‐targeted therapy.

Abstract Ovarian cancer (OC) is most lethal gynecologic malignancy. About 75% of OC patients relapse and/or develop chemo-resistant disease after initial response to standard-of-care treatment with platinum-based therapies. HER2 amplifications and overexpression in OC are reported to vary and responses to HER2 inhibitors have been feeble. Next generation sequencing technologies in conjunction with testing using patient-derived xenografts (PDX) allow validation of personalized treatments. Using whole-genome mate-pair next generation sequencing protocol we profiled a set of serous OC to identify copy number changes and gene fusions. Specific focus was on DNA alterations which were therapeutically targetable. The efficiency of anti-Her2 therapy was tested in three different PDX lines with alterations at genes of ERBB pathway and various levels of HER2 protein expression. Tumor in one line harbored a duplication of ERBB2 and high expression of HER2 protein. Second PDX model had a fusion at NRG1 gene, a ligand for HER4 and HER4 receptors, that was highly expressed on protein level. Third line harbored an amplification of NRG3 gene, a known ligand for HER4 receptor. Treatment responses to pertzumab/trastuzumab were compared in each PDX line to standard carboplatin and paclitaxel combination treatment. In all three PDX models HER2-targeted therapy resulted in significant inhibition of tumor growth compared to untreated control. However, the responses in each were inferior to chemotherapy, even in chemo-resistant case. There was an extra benefit when chemotherapy and HER-2 targeted therapy were administered together, as a significant regression of tumor was observed after 6 weeks of treatment using this regiment compared to chemotherapy alone. Post-treatment analysis of the tissues, nevertheless, revealed inhibition of ERBB2 pathway on the level of phosphorylation and expression of downstream targets. In conclusion, targeting of presumably activated ERBB2 pathway alone in OC results in a modest treatment benefit. A combination therapy including both chemo drugs and Her2 inhibitors provides far better response. Further studies are needed to address development of recurrence and sensitivity of recurrent disease to Her2 targeted therapy. Citation Format: Irina V. Kovtun, Zhang Piyan, Faye R. Harris, Xiaonan Hou, Saravut J. Weroha, George Vasmatzis. Targeting ERBB2 pathway in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A19.

Abstract Ovarian cancer (OC) is the most lethal gynecologic malignancy and the fifth leading cause of cancer death in the United States. Metformin, an antidiabetic drug, has been shown to decrease the risk of mortality in cancer patients, particularly those with OC. Though the drug acts through inhibition of the PI3K/mTOR/AKT pathway, which is commonly altered in OC, knockout of key proteins in the pathway does not attenuate metformin’s antidiabetic effects. Further, intravenous metformin administration is less effective than oral, implying that some crucial interaction takes place in the gut. The gut is home to some 1013 bacterial cells, collectively known as the microbiota. These bacteria play important roles in fermentation of fiber, producing short-chain fatty acids (SCFA) that have positive health benefits, including prevention of metabolic syndrome, bowel disorders, and certain cancers. Mechanistically, SCFAs inhibit the PI3K/mTOR/AKT pathway, similar to metformin. Conditions such as obesity, metabolic syndrome, and diabetes significantly alter gut microbiota composition, possibly leading to a reduction in beneficial SCFAs. However, in type II diabetes, treatment with metformin leads to an increase in SCFA-producing bacteria, suggesting that metformin may positively influence the microbiota to improve patient metabolic profiles. In OC, metformin may play a similar role in promoting the presence of SCFA-producing bacteria. We hypothesized that metformin increases the abundance of SCFA-producing bacteria in the gut, with which it acts synergistically to decrease growth of OC cells in combination with OC chemotherapy. To investigate our hypothesis, we have employed the OC patient-derived xenograft (PDX) mouse model established at Mayo Clinic. Immunocompromised SCID mice received OC cells from Mayo Clinic patients and the tumors were allowed to grow to an appropriate size before treatment was started. Cage mates were split into one of four treatment arms: a control (no treatment) group, metformin only, carboplatin/paclitaxel (CP) chemotherapy only, or CP with metformin. Mice received 2mg/ml metformin in their water continuously and/or 51mg/kg carboplatin and 15mg/kg paclitaxel intravenously weekly. Stool was collected weekly prior to tumor injection, during tumor growth, and through the four weeks of treatment. DNA was extracted from stool and bacterial 16S rRNA amplicon sequencing was used to determine microbiota profiles. Mice with chemosensitive tumors treated with only CP chemotherapy had significant reductions in tumor growth along with significantly less microbial gut diversity than pretumor and pretreatment mice. Chemosensitive mice treated with metformin alone did not lose significant microbial diversity but did not have significant reductions in tumor growth as compared to the control mice. CP in combination with metformin both reduced tumor growth and protected against loss of microbiota diversity. Further, mice treated with only metformin had significant increases in abundance in the Firmicutes genera Ruminococcus and Oscillospira, SCFA producers that are associated with leanness and health. Mice treated with both metformin and CP had significant increases in another SCFA-producing Firmicute, Moryella. Finally, mice with chemoresistant tumors showed improved tumor growth inhibition with combination CP/metformin therapy, suggesting that metformin-associated changes in the gut microbiota may increase sensitivity to CP. These data support the hypothesis that metformin protects against detrimental loss of gut microbiota diversity induced by chemotherapy treatment, enriching beneficial SCFA producers. This work implies a role for the gut microbiota in OC treatment and provides support for metformin’s potential use as a beneficial addition to chemotherapy treatment. Citation Format: Dana M. Walsh, Ismail Mert, Chen Jun, Xiaonan Hou, Saravut John Weroha, Marina Walther-Antonio. Metformin alters the gut microbiota of ovarian cancer patients treated with carboplatin/paclitaxel chemotherapy and enhances sensitivity in resistant tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A70.

Transforming growth factor beta (TGF-β) signaling has pleiotropic functions regulating cancer initiation, development, and metastasis, and also plays important roles in the interaction between stromal and cancer cells, making the pathway a potential therapeutic target. LY2157299 monohydrate (LY), an inhibitor of TGF-β receptor I (TGFBRI), was examined for its ability to inhibit ovarian cancer (OC) growth both in high-grade serous ovarian cancer (HGSOC) cell lines and xenograft models. Immunohistochemistry, qRT-PCR, and Western blot were performed to study the effect of LY treatment on expression of cancer- and fibroblast-derived genes. Results showed that exposure to TGF-β1 induced phosphorylation of SMAD2 and SMAD3 in all tested OC cell lines, but this induction was suppressed by pretreatment with LY. LY alone inhibited the proliferation, migration, and invasion of HGSOC cells in vitro. TGF-β1-induced fibroblast activation was blocked by LY. LY also delayed tumor growth and suppressed ascites formation in vivo. In addition, independent of tumor inhibition, LY reduces ascites formation in vivo. Using OVCAR8 xenograft specimens we confirmed the inhibitory effect of LY on TGF-β signaling and tumor stromal expression of collagen type XI chain 1 (COL11A1) and versican (VCAN). These observations suggest a role for anti-TGF-β signaling-directed therapy in ovarian cancer.

Abstract Malignant ascites is one of the most common causes of morbidity in end stage ovarian cancer patients with negative impact on quality of life. Novel non-invasive palliative therapeutic options are lacking for such patients. The zinc metalloprotease, pregnancy-associated plasma protein-A (PAPP-A), plays a key role in the insulin-like growth factor (IGF) pathway, promoting ovarian cancer cellular transformation, growth and invasiveness. Furthermore, patient primary malignant ascites is known to contain high levels of PAPP-A by ELISA. Preliminary data shows that inhibition of PAPP-A through a neutralizing monoclonal PAPP-A antibody (mAb-PA) inhibits the accumulation and promote the regression of ascites in an ovarian cancer patient-derived xenograft (PDX) models. In the current study, we investigated whether mAb-PA can promote ascites regression in an additional ovarian PDX model with measurable ascites. Patient derived ascites xenograft (PDAX) models were defined as SCID mice that developed ascites after intraperitoneal heterotransplantation of patient solid tumor collected at the time of primary cytoreduction. Ascites from these models (n=51) was screened for human PAPP-A protein by ELISA and models were divided into two groups by the relative concentration of PAPPA-A: high (n=18) and low (n=33). PAPP-A High PDAX model PH438 was re-established intraperitoneal in 20 SCID mice by ascites injection (0.1 ml per mouse). When ascites area reached a threshold of &gt;0.60 cm2 by ultrasound, mice were treated with 60mg/Kg of mAb-PA (n=10) or IgG2a control (n=10) on day one and three. On day four, a second ultrasound measurement was obtained and the mice were euthanized. Ascites burden was measured at necropsy. Personnel involved with the acquisition of ultrasounds measurements, subsequent ascites harvests, and post hoc analyses were blinded to the treatments. The ratio of mean ascites (mAb-PA/IgG2a in grams) collected at necropsy was 2.04/2.51, indicating that the mAb-PA arm had less ascites compared to control. These data were consistent with pre- and post-treatment ultrasound measurements of ascites burden; a statistically significant (p=0.0283 by paired t test) reduction of ascites burden of 52.3% was observed, compared to the starting baseline, indicating that mAb-PA treatment causes ascites regression. Ascites weight at necropsy was compared to the area of greatest fluid echogenicity by ultrasound and the Pearson correlation R value achieved was of 0.859 (p&lt;0.0001). These PDAX data implicate PAPP-A attenuation as a potential strategy to treat malignant ascites in OC. Additional PDAX models are under examination to confirm these findings and support the translational development of PAPP-A as a new therapeutic target for women with refractory ascites. Citation Format: Valentina Zanfagnin, Laurie K. Bale, Marc A. Becker, Xiaonan Hou, Diogo Torres, Cheryl A. Conover, Saravut J. Weroha. Regression of malignant ascites via PAPP-A inhibition in ovarian cancer patient-derived xenograft 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 4817. doi:10.1158/1538-7445.AM2017-4817

Abstract Purpose: The treatment of ovarian cancer poses a challenge since 70% of patients will relapse with incurable platinum resistant disease. Although ovarian cancers lack frequently-occurring driver mutations or amplifications, the study presented herein examines the concept of individualized therapy based on mate pair sequencing and protein expression in ovarian cancer patient-derived xenografts. Experimental Procedure: Macrodissection of patient tumor was followed by genomic DNA isolation and next-generation sequencing using an Illumina Mate Pair protocol to identify structural genomic changes and copy number variations. Secondary analyses were carried out to select potential therapeutic targets among those. The tumors were also propagated intraperitoneally in immunocompromised mice and treated with standard chemotherapy or/and targeted therapy chosen based on genomic analyses. Results: Mate pair analysis revealed that, in general, copy number changes (amplifications, gains and losses) were common in ovarian tumors and included genes potentially targetable. Tumors from early generation patient- derived xenografts showed a landscape of genomic aberrations identical to that of original patient tumor. One PDX model in which high expression of HER2 and copy number gains at RICTOR and AKT genetic loci were revealed, was treated with either chemotherapy and Pertuzumab/trastuzumab (HER2 inhibitors), MK-8669 (mTOR inhibitor) or MK-2206 (AKT inhibitor) or chemotherapy alone. Tumor size was measured by serial abdominal ultrasound and after 28 days, the final tumor size ratio compared to baseline was 0.23, 0.36, 0.55 and 0.56, respectively, indicating that the best response was seen in the combined chemotherapy and Pertuzumab/trastuzumab cohort and that was better than in the chemotherapy only cohort. Conclusions: Patient-derived xenografts models in conjunction with genomic and protein analyses of patient tumors are valuable for testing of combination therapies including specific targeting drugs. This approach also provides actionable information for more tailored treatment of ovarian cancer patients. Citation Format: Konstantinos Leventakos, Faye R. Harris, Lin Yang, Xiaonan Hou, Saravut Weroha, Irina V. Kovtun. Testing personalized therapies for ovarian cancer using mate pair genomic analysis and patient-derived xenografts [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 4814. doi:10.1158/1538-7445.AM2017-4814

Abstract Over the recent years significant knowledge has been accumulated on which drugs can be therapeutically beneficial for a given patient based on mutational profiling of their tumors using selected gene panels. Much less, however, is known under which conditions targeting of genes which are amplified could be beneficial. Apart from successfully targeting HER2-positive cancers, many of which frequently carry amplified ERBB2 gene, no other targeted therapy based on DNA amplifications or gains of the genes is offered to the patients due to lack of clinical response data. Using mate pair sequencing we have profiled 60 high grade serous ovarian tumors to compare genomic rearrangements and copy number changes. The analyses revealed that these tumors carry highly rearranged genome. Copy number changes amplifications, gains and losses were very common and included genes potentially therapeutically targetable. In contrast, only a few of targetable fusion genes were identified in a set of 60 cases. The tumors were propagated in immunocompromised mice (mouse avatars), sequenced and showed identical to original patient tumor landscape of rearrangements. Using these mouse avatar models we tested whether a therapy inhibiting selected based on genomic amplification targets provided a benefit compared to standard chemotherapy. Mice which harbored tumors showing amplifications of CDKs (CDK2, 4 and 9) and ERBB3 were treated with flavopiridol and pertuzumab respectively, and response was compared to that of standard for ovarian cancer chemotherapy (carboplatin and paclitaxel combination). We observed that application of either agent, significantly inhibited tumor growth in treated mice as compared to untreated. However, neither drug alone caused tumor regression. The treatment regimen then was modified to administer chemotherapy and targeted therapy together. A mouse avatars in which genomic analyses revealed amplification at loci involving AKT3 and RICTOR genes was treated with chemotherapy alone and in combination with MK2206 (AKT inhibitor) or MK8669 (mTOR inhibitor). The high level of both target proteins was confirmed by immunoblotting. Significant regression of tumor was observed after 6 weeks of treatment. Addition of either targeted drug provided an extra benefit in killing tumor cells compared to chemotherapy alone. The mice are followed to study tumor recurrence. These data indicated that genomic information obtained using next generation sequencing can aid therapy decisions by tailoring treatments to individual tumor makeup. Avatar models in conjunction with genomic analyses of the original tumors are invaluable for testing specific targeted therapies selected based on amplification/gains of putative genes-targets and for studying mechanisms of tumor recurrence and therapy resistance. Citation Format: Faye R. Harris, Lin Yang, Xiaonan Hou, Konstantinos Leventakos, Saravut J. Weroha, George Vasmatzis, Irina V. Kovtun. Targeting genes which are amplified in ovarian cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A34.