Preclinical Models Of Ovarian Cancer Research Articles

Using genetically encoded fluorescent biosensors to interrogate ovarian cancer metabolism

BackgroundEpithelial ovarian cancer (OC) is the most lethal gynecological malignancy and patients present with significant metastatic burden, particularly to the adipose-rich microenvironment of the omentum. Recent evidence has highlighted the importance of metabolic adaptations in enabling this metastasis, leading to significant interest in evolving the arsenal of tools used to study OC metabolism. In this study, we demonstrate the capability of genetically encoded fluorescent biosensors to study OC, with a focus on 3D organoid models that better recapitulate in vivo tumor microenvironments.Materials and methodsPlasmids encoding the metabolic biosensors HyPer, iNap, Peredox, and Perceval were transfected into 15 ovarian cancer cell lines to assay oxidative stress, NADPH/NADP+, NADH/NAD+, and ATP/ADP, respectively. Fluorescence readings were used to assay dynamic metabolic responses to omental conditioned media (OCM) and 100 μM carboplatin treatment. SKOV3 cells expressing HyPer were imaged as 2D monolayers, 3D organoids, and as in vivo metastases via an intravital omental window. We further established organoids from ascites collected from Stage III/IV OC patients with carboplatin-resistant or carboplatin-sensitive tumors (n = 8 total). These patient-derived organoids (PDOs) were engineered to express HyPer, and metabolic readings of oxidative stress were performed during treatment with 100 μM carboplatin.ResultsExposure to OCM or carboplatin induced heterogenous metabolic changes in 15 OC cell lines, as measured using metabolic sensors. Oxidative stress of in vivo omental metastases, measured via intravital imaging of metastasizing SKOV3-HyPer cells, was more closely recapitulated by SKOV3-HyPer organoids than by 2D monolayers. Finally, carboplatin treatment of HyPer-expressing PDOs induced higher oxidative stress in organoids derived from carboplatin-resistant patients than from those derived from carboplatin-sensitive patients.ConclusionsOur study showed that biosensors provide a useful method of studying dynamic metabolic changes in preclinical models of OC, including 3D organoids and intravital imaging. As 3D models of OC continue to evolve, the repertoire of biosensors will likely serve as valuable tools to probe the metabolic changes of clinical importance in OC.

Dominant-negative transforming growth factor-β receptor-armoured mesothelin-targeted chimeric antigen receptor T cells slow tumour growth in a mouse model of ovarian cancer

Ovarian cancer is a major cause of death among all gynaecological cancers. Although surgery, chemotherapy and targeted therapy have yielded successful outcomes, the 5-year survival rate remains < 30%. Adoptive immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy, has demonstrated improved survival in acute lymphoblastic leukaemia with manageable toxicity. We explored CAR T-cell therapy in a preclinical mouse model of ovarian cancer. Second-generation CAR T cells were developed targeting mesothelin (MSLN), which is abundantly expressed in ovarian cancer. Cytotoxicity experiments were performed to verify the lethality of CAR T cells on target cells via flow cytometry. The in vivo antitumour activity of MSLN CAR T cells was also verified using a patient-derived xenograft (PDX) mouse model with human tumour-derived cells. We also evaluated the potency of CAR T cells directed to MSLN following co-expression of a dominant-negative transforming growth factor-β receptor type II (dnTGFβRII). Our data demonstrate that anti-MSLN CAR T cells specifically eliminate MSLN-expressing target cells in an MSLN density-dependent manner. This preclinical research promises an effective treatment strategy to improve outcomes for ovarian cancer, with the potential for prolonging survival while minimizing risk of on-target off-tumour toxicity.

Adaptations of key metabolic pathways in primary and metastatic epithelial ovarian cancer (253)

Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005). Objectives: Patients with ovarian cancer (OC) often present with advanced-stage disease at diagnosis; however, the molecular mechanisms which drive the aggressive nature of OC metastases have not been well characterized. Metastasizing cancer cells undergo metabolic reprogramming to utilize available lipids in the metastatic niche of the omentum. This study aimed to understand the compensatory metabolic mechanisms that enable this OC metastasis. Our work focused on understanding the contributions of the pentose phosphate pathway (PPP)—a key mechanism responsible for redox homeostasis—in enabling this evolution. Methods: Using gene expression data of matched human primary and omental tumor samples (n=30), we performed targeted analysis of metabolic gene expression changes, which revealed upregulation of the PPP in omental metastases. We then performed in vitro and in vivo experiments using pre-clinical ovarian cancer models (SKOV3, IGROV, HEYA8). Given the canonical role of the PPP in maintaining redox homeostasis, we assessed oxidative stress in organoid cultures from omental metastases harvested at mouse necropsy and in cultures grown in omental conditioned media. Inhibition of the rate-limiting enzyme of the PPP, glucose-6-phosphate dehydrogenase (G6PD), was performed via shRNA knockdowns and treated with a pharmacological inhibitor. HeyA8 tumor-bearing mice were treated with the G6PD inhibitor, and the metastatic burden was evaluated using IVIS during treatment and in peritoneal organs following the sacrifice. Statistical significance was established using unpaired two-tailed Student’s t-tests. Results: Differential analysis of metabolic gene expression and pathway analysis revealed upregulation of the PPP in omental metastases compared to primary ovarian tumors (p<0.05, EnrichR Score = 800). Primary tumors and omental metastases were harvested from SKOV3, HeyA8, and IGROV1 tumor-bearing mice at necropsy and used to establish organoids. DCFH-DA, a fluorogenic probe for ROS, measured higher levels of oxidative stress in metastatic organoids than primary tumors (normalized F525= 11.3623, p<0.0005). SKOV3 organoids expressing HyPer, a ratiometric biosensor of intracellular H2O2, exhibited higher oxidative stress when grown in omental conditioned media versus basal organoid media (normalized F420/F500=1.27, p<0.05). Pharmacological and shRNA inhibition of G6PD sensitized cells to oxidative stress, reducing cell viability in vitro (p<0.05). Treatment with a G6PD inhibitor in vivo significantly decreased tumor growth and metastases compared to vehicle treatment (p<0.005).

Clinical significance of cadherin-6 expression in primary and recurrent epithelial ovarian cancer and its association with outcomes: A potential therapeutic target for epithelial ovarian cancer (206)

<b>Objectives:</b> Cadherin-6 (CDH6) is a membrane glycoprotein, and aberrant expression of CDH6 has been reported in several human carcinomas, suggesting a possible role in metastasis and invasion. Recently, antibody-drug conjugates (ADCs) targeting CDH6 have drawn attention to new cancer therapies and demonstrated their antitumor activity in preclinical models of epithelial ovarian cancer (EOC). However, limited data are available regarding the frequency of CDH6 expression and its relationship to clinical factors in EOC. Our goal was to understand the clinical significance of CDH6 expression in EOC. <b>Methods:</b> We performed immunohistochemical (IHC) staining on CDH6 expression in a total of 232 EOC surgical samples (including 181 primary tumors and 51 recurrent tumors). CDH6 IHC was performed using the Leica BOND III staining platform with an anti-CDH6 antibody (HPA007456, ATLAS ANTIBODIES). The CDH6 IHC scoring standard refers to the HER2 detection guidelines for gastric cancer (0, 1+, 2+, 3+). We define a score greater than or equal to 1 as positive for the expression of CDH6. <b>Results:</b> We investigated 181 patients with primary EOC for CDH6 expression through IHC. Total 117 (64.6%) patients tested positive. In which, 18 (15.4%), 84 (71.8%) and 15 (12.8%) cases were scored as 1+, 2+ and 3+, respectively. We then investigated the relationship between CDH6 expression and clinical-pathologic features in EOC. CDH6 expression was observed more frequently in high-grade serous carcinomas (89.0%, p < 0.0001), stage III/IV tumors (84.8%, p < 0.0001) and gross residual tumors in primary surgery (84.7%, p < 0.0001). CDH6-positive patients showed shorter progression-free survival (PFS) and overall survival (OS) (p < 0.0001 and p=0.0006) than that of CDH6-negative patients in all EOC patients. In addition, patients with CDH6 3+ had shorter PFS and OS than that of patients who had 0, 1+ and 2+ (p =0.028 and p=0.015) in the high-grade serous subgroup. We also investigated the expression of CDH6 in 51 recurrent EOC; 38/51 (74.5%) recurrent tumors tested positive for CDH6 expression, and nine (23.7%), 17 (44.7%) and 12 (31.6%) received scores of 1+, 2+ and 3+, respectively. CDH6 expression from recurrent tumors was observed in 18/24 (75.0%) high-grade serous, 11/16 (68.8%) clear cell, 4/5 (80.0%) endometrioid and 5/6 (83.3%) other histotypes. <b>Conclusions:</b> CDH6 expression is frequently observed in primary and recurrent EOC of all histotypes, especially in high-grade serous carcinoma. The frequency of CDH6 expression is consistent between primary and recurrent tumors. CDH6 is a promising therapeutic target for the treatment of EOC.

Discovery of a novel ALK/ROS1/FAK inhibitor, APG-2449, in preclinical non-small cell lung cancer and ovarian cancer models

BackgroundTyrosine kinase inhibitors (TKIs) are mainstays of cancer treatment. However, their clinical benefits are often constrained by acquired resistance. To overcome such outcomes, we have rationally engineered APG-2449 as a novel multikinase inhibitor that is highly potent against oncogenic alterations of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 receptor tyrosine kinase (ROS1), and focal adhesion kinase (FAK). Here we present the preclinical evaluation of APG-2449, which exhibits antiproliferative activity in cells carrying ALK fusion or secondary mutations.MethodsKINOMEscan® and LANCE TR-FRET were used to characterize targets and selectivity of APG-2449. Water-soluble tetrazolium salt (WST-8) viability assay and xenograft tumorigenicity were employed to evaluate therapeutic efficacy of monotherapy or drug combination in preclinical models of solid tumors. Western blot, pharmacokinetic, and flow cytometry analyses, as well as RNA sequencing were used to explore pharmacokinetic–pharmacodynamic correlations and the mechanism of actions driving drug combination synergy.ResultsIn mice bearing wild-type or ALK/ROS1-mutant non-small-cell lung cancer (NSCLC), APG-2449 demonstrates potent antitumor activity, with correlations between pharmacokinetics and pharmacodynamics in vivo. Through FAK inhibition, APG-2449 sensitizes ovarian xenograft tumors to paclitaxel by reducing CD44+ and aldehyde dehydrogenase 1-positive (ALDH1+) cancer stem cell populations, including ovarian tumors insensitive to carboplatin. In epidermal growth factor receptor (EGFR)-mutated NSCLC xenograft models, APG-2449 enhances EGFR TKI-induced tumor growth inhibition, while the ternary combination of APG-2449 with EGFR (osimertinib) and mitogen-activated extracellular signal-regulated kinase (MEK; trametinib) inhibitors overcomes osimertinib resistance. Mechanistically, phosphorylation of ALK, ROS1, and FAK, as well as their downstream components, is effectively inhibited by APG-2449.ConclusionsTaken together, our studies demonstrate that APG-2449 exerts potent and durable antitumor activity in human NSCLC and ovarian tumor models when administered alone or in combination with other therapies. A phase 1 clinical trial has been initiated to evaluate the safety and preliminary efficacy of APG-2449 in patients with advanced solid tumors, including ALK+ NSCLC refractory to earlier-generation ALK inhibitors.Trial registrationClinicaltrial.gov registration: NCT03917043 (date of first registration, 16/04/2019) and Chinese clinical trial registration: CTR20190468 (date of first registration, 09/04/2019).

Abstract 3457: Rebastinib, a TIE2 antagonist improves chemotherapy response in homologous recombination proficient epithelial ovarian cancer murine models

Abstract Objectives: The Angiopoietin (ANGPT)/TIE2 kinase axis plays a critical role in cancer angiogenesis. Rebastinib, an investigational TIE2 inhibitor inhibits Tie2 receptor in endothelial cells and macrophages. M2-like Tie2+ macrophages, being pro-tumorigenic, are an attractive target for cancer therapy. Current clinical trials are testing rebastinib with chemotherapy in platinum-resistant ovarian cancer. However, the mechanism is not fully understood. The objective of this study was to determine the effects of rebastinib alone and combined with chemotherapy in preclinical models of ovarian cancer. Methods: RNA-Seq was carried out to determine rebastinib-induced gene expression in ID8 cells and PMJ2R murine peritoneal macrophages. To determine the pharmacodynamics of immune cells in ascites, syngeneic ID8 mice were pre-treated with 10 mg/kg/day rebastinib/control for a week, followed by rebastinib with or without 20 mg/kg carboplatin + 12 mg/kg paclitaxel (chemo) for two weeks. Mice were sacrificed 24h after the last treatment to harvest ascites for flow cytometry. Cohorts of mice from syngeneic ID8 murine model and from a patient derived xenograft (PDX) model of ovarian cancer were used for survival analysis. Results: RNA-Seq showed that in ID8 cells, 1528 genes were upregulated and 3115 genes were downregulated by rebastinib. In macrophages, 2302 genes were upregulated and 2970 genes were downregulated. Several ANGPT-like genes (ANGPTL2, ANGPTL4, ANGPTL6) involved in tumorigenesis, angiogenesis, proliferation were downregulated 2X-10X (p&amp;lt;0.001) in ID8 cells and macrophages. ANGPTL1, an anti-angiogenic and anti-apoptotic gene, was increased 10X in ID8 cells (p&amp;lt;0.001) but not altered in macrophages. ANGPT2, a context-dependent agonist/antagonist of TIE2 pathway, was increased 1.42X in ID8 (P&amp;lt;0.05) cells and 4.6X in macrophages (p&amp;lt;0.001). Flow studies showed that rebastinib significantly increased CD45+ macrophage (p&amp;lt;0.03 vs. chemo; p&amp;lt;0.02 vs. rebastinib+chemo), maintained Th leukocytes (p&amp;lt;0.03, vs. chemo) and significantly increased cytotoxic T cells (p&amp;lt;0.02 vs. control; p&amp;lt;0.0004, p&amp;lt;0.0001 vs. chemo and rebastinib+chemo, respectively). Rebastinib had no significant effect on regulatory T cells, Tie2+ macrophages or Tie2+ M2 macrophages. Rebastinib combined with chemotherapy improved median survival significantly in murine models of ovarian cancer - ID8 (p&amp;lt;0.01) and PDX (p&amp;lt;0.0001). Conclusions: Rebastinib exerts differential effects on tumor cells and macrophages that could contribute to its mechanism of action. Rebastinib alters immune cells and increases cytotoxic T cells in ascites. Rebastinib combined with chemotherapy extends survival in PDX and syngeneic ID8 murine models of ovarian cancer. Further investigation using rational combinations of rebastinib and chemotherapy are underway. Citation Format: Vijayalaxmi G. Gupta, Katherine F. Roby, Harsh B. Pathak, Andrew K. Godwin, Sumedha Gunewardena, Dineo Khabele. Rebastinib, a TIE2 antagonist improves chemotherapy response in homologous recombination proficient epithelial ovarian cancer murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3457.

Abstract 5288: Development of lead mutant-p53 reactivating peptides towards clinical application

Abstract p53 is the most extensively studied tumor suppressor. It serves as a major barrier against malignant transformation and the p53 pathway is inactivated in almost all human tumors. A large percentage of tumors inactivate p53 by point mutations in the DNA binding domain (DBD) of the protein. Most mutations destabilize p53 protein folding, causing its partial denaturation at physiological temperature. Thus a high proportion of human tumors overexpress a potential potent tumor suppressor in a non-functional, improperly folded form. Previously, we reported the use of phage display technology to select for peptides that reactivate mutant p53 by stabilizing a wt protein conformation, taking advantage of a unique combination of selection steps (Tal P, at al Oncotarget. 2017 Jan 3;8(1):164-178). We now describe further progress in the development of one lead peptide, pCAP-250, which contains 9 amino acids and a myristic fatty acid at its N-terminus. The pCAP-250 sequence bears 100% homology to RAD9, a known p53 interacting protein. We show physical interaction between pCAP-250 and the p53DBD. Characterization of this interaction by NMR reveals a significant chemical shift of amino acids located in the p53DBD L1 loop and Helix-2, two regions critical for p53 transcriptional activity and interaction with DNA. We also report the optimization of our lead sequence by rational design to yield more potent derivatives with higher efficacy in cancer cell elimination and longer in vivo half-life. RNA-seq analysis shows that our lead peptides induce robust activation of p53 target genes. Furthermore, we report that in addition to p53 activation, pCAP-250 also has a p53-independent anti-cancer activity. Overall, our lead peptides cause specific cell death in a wide spectrum of mutant p53 expressing cell lines, with an IC50 of 0.1-12µM, while having no effect on non-transformed cells at these concentrations. The pharmacokinetic profile of pCAP-250 and its derivatives shows that they are completely stable in human serum and have plasma half-life of 1.5-10h in vivo when administered IV or subcutaneously, presumably due to interaction with albumin through the fatty acid moiety. Remarkably, our lead peptides elicit remarkable regression of very aggressive tumors in several preclinical mouse models of renal, colon, pancreatic and ovarian cancer. In vivo efficacy can be achieved through several routes of administration, including intra-tumor injection as well as systemic subcutaneous continuous slow release and subcutaneous bi-daily injections. Thus, such peptides might serve as novel therapeutic agents for p53-mutated human cancer. Citation Format: Perry Tal, Moshe Oren, Orna Palgi, Varda Rotter, KWANGIL JEONG. Development of lead mutant-p53 reactivating peptides towards clinical application [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5288.

Abstract 3591: First in class drug ZB131 shows efficacy in cholangiocarcinoma models

Abstract Cholangiocarcinoma (CCA) is a lethal cancer with a high unmet need. The survival rate is low (5-year survival &amp;lt;20%), most non-palliative patients relapse within two years, and treatments in the advanced setting are limited. While FGFR2 and IDH1 inhibitors are effective in patients with select genomic alterations, they address only a small fraction (&amp;lt;20%) of biliary tract cancers. We now present the discovery and characterization of ZB131, a first in class humanized monoclonal antibody targeted to cancer specific plectin (CSP). CSP is a pro-tumorigenic protein exclusively expressed on the cancer cell surface. Previously, we demonstrated remarkable tumor regressions in preclinical murine models of pancreatic and ovarian cancer using a tool anti-CSP murine antibody. Here, we show that intra- and extra-hepatic CCA are 92% and 86% positive for membranous plectin staining by IHC, respectively, suggesting that anti-CSP therapy could be broadly effective against CCA. Indeed, in CCA cell lines in vitro, ZB131 (0.4nM binding affinity) arrests cell cycle, inhibits cell migration, and triggers intracellular ROS accumulation. In addition, both ZB131 and its murine version significantly suppressed tumor growth in 83% of the mice in a xenograft model and caused complete regression in the remaining 17%. Previous data from our group and others suggesting that CSP plays a role in chemoresistance led us to evaluate the efficacy of ZB131 in combination with gemcitabine. In vitro, we treated CSP-positive WITT cells with combinations of 0-500nM gemcitabine plus 0-1000nM ZB131 and found pockets of synergy or additivity. In a CCA xenograft model, the combination of 1mg/kg ZB131 and 100mg/kg gemcitabine showed 91% suppression in tumor growth compared with 74% in gemcitabine alone and 77% in huIgG plus gemcitabine at day 32. Collectively, our results suggest that ZB131 renders CCA tumors more chemosensitive and enhances the antitumor effects of gemcitabine. In contrast to the standard-of-care CCA treatment regimen, ZB131 showed remarkably low toxicity, likely because the antibody bypasses any non-malignant cells where plectin remains strictly cytoplasmic. Fc effector functional studies revealed a lack of antibody-dependent cell-mediated and complement-dependent cytotoxicity. Hemolysis and cytokine release studies suggested that ZB131 is compatible with human blood and unlikely to cause adverse cytokine release. Furthermore, in GLP toxicology studies, no signs of toxicity were observed at the highest dose evaluated of 100mg/kg/week, providing a &amp;gt;30-fold safety margin from the estimated human efficacious dose of 3mg/kg/week. In summary, ZB131 is an exciting new avenue for the treatment of CCA and other CSP-positive cancers with an excellent safety profile and strong antitumor activity in vitro and in murine models. Phase I clinical trials of ZB131 have been initiated. Citation Format: Samantha M. Perez, Randall Burton, Denise Krawitz, Christopher Sheth, Pina Cardarelli, Molly Owens, Brian Murphy, Matthew J. Reilley, Lindsey T. Brinton, Kimberly A. Kelly. First in class drug ZB131 shows efficacy in cholangiocarcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3591.

G6PD inhibition sensitizes ovarian cancer cells to oxidative stress in the metastatic omental microenvironment.

SUMMARYOvarian cancer (OC) is the most lethal gynecological malignancy, with aggressive metastatic disease responsible for the majority of OC-related deaths. In particular, OC tumors preferentially metastasize to and proliferate rapidly in the omentum. Here, we show that metastatic OC cells experience increased oxidative stress in the omental microenvironment. Metabolic reprogramming, including upregulation of the pentose phosphate pathway (PPP), a key cellular redox homeostasis mechanism, allows OC cells to compensate for this challenge. Inhibition of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, reduces tumor burden in pre-clinical models of OC, suggesting that this adaptive metabolic dependency is important for OC omental metastasis.

The PARP inhibitor ABT-888 impairs the suppressive capacity of regulatory T cells through modulation of FoxP3 and CTLA-4.

Abstract Immune checkpoint blockade (ICB) has achieved long-term survival in melanoma but results remain limited in other solid malignancies including ovarian cancer. Combination treatments are a promising strategy to optimize responses to ICB. We have previously shown that the PARP inhibitor (PARPi) ABT-888 synergizes with CTLA-4 ICB to achieve long-term survival in a preclinical model of ovarian cancer. While the role of PARPi on DNA repair is well established, the impact on T cells is unclear. Published data using PARP1-KO mice suggests that PARP impairs regulatory T cell (Treg) function through modulation of FoxP3. Tregs suppress conventional T cell responses using multiple mechanisms including consumption of co-stimulatory molecules via the inhibitory receptor CTLA-4. Here we sought to define the impact of ABT-888 on the suppressive function of Tregs using FoxP3-eGFP transgenic mice. Using flow cytometry, we examined the levels of FoxP3 and CTLA-4 in Tregs stimulated in the presence of increasing doses of ABT-888 (0 – 50μM). Independent of stimulation, the GMFI of FoxP3 and CTLA-4 were significantly reduced by ABT-888 in a dose-dependent manner (P &amp;lt; 0.05). To test suppressive function, we pre-treated Tregs with increasing doses of ABT-888 prior to use in a standard proliferation assay. Similarly, ABT-888 pre-treatment significantly reduced the suppressive capacity of Tregs in a dose-dependent manner (P &amp;lt; 0.05). Taken together, these data identify a potentially novel area of synergy between PARPi and ICB. Furthermore, these data highlight the immunomodulatory effects of PARPi which are independent of their role in DNA repair. Future studies will focus on the impact of ABT-888 on tumor-associated Tregs and optimization of ICB therapy. This research was supported by NCI R37CA229221 (PI: Adams) and UNM Comprehensive Cancer Center Support Grant NCI P30CA11810. D.F. was supported by the Academic Science Education and Research Training program at the University of New Mexico Health Sciences Center (NIGMS Institutional Research and Academic Career Development Award; K12-GM088021).

The targeted SMAC mimetic SW IV-134 augments platinum-based chemotherapy in pre-clinical models of ovarian cancer

BackgroundOvarian cancer is initially responsive to frontline chemotherapy. Unfortunately, it often recurs and becomes resistant to available therapies and the survival rate for advanced and recurrent ovarian cancer is unacceptably low. We thus hypothesized that it would be possible to achieve more durable treatment responses by combining cisplatin chemotherapy with SW IV-134, a cancer-targeted peptide mimetic and inducer of cell death. SW IV-134 is a recently developed small molecule conjugate linking a sigma-2 ligand with a peptide analog (mimetic) of the intrinsic death pathway activator SMAC (second-mitochondria activator of caspases). The sigma-2 receptor is overexpressed in ovarian cancer and the sigma-2 ligand portion of the conjugate facilitates cancer selectivity. The effector portion of the conjugate is expected to synergize with cisplatin chemotherapy and the cancer selectivity is expected to reduce putative off-target toxicities.MethodsOvarian cancer cell lines were treated with cisplatin alone, SW IV-134 alone and a combination of the two drugs. Treatment efficacy was determined using luminescent cell viability assays. Caspase-3/7, − 8 and − 9 activities were measured as complementary indicators of death pathway activation. Syngeneic mouse models and patient-derived xenograft (PDX) models of human ovarian cancer were studied for response to SW IV-134 and cisplatin monotherapy as well as combination therapy. Efficacy of the therapy was measured by tumor growth rate and survival as the primary readouts. Potential drug related toxicities were assessed at necropsy.ResultsThe combination treatment was consistently superior in multiple cell lines when compared to the single agents in vitro. The expected mechanism of tumor cell death, such as caspase activation, was confirmed using luminescent and flow cytometry-based assay systems. Combination therapy proved to be superior in both syngeneic and PDX-based murine models of ovarian cancer. Most notably, combination therapy resulted in a complete resolution of established tumors in all study animals in a patient-derived xenograft model of ovarian cancer.ConclusionsThe addition of SW IV-134 in combination with cisplatin chemotherapy represents a promising treatment option that warrants further pre-clinical development and evaluation as a therapy for women with advanced ovarian cancer.

Fast and Durable Intraoperative Near-infrared Imaging of Ovarian Cancer Using Ultrabright Squaraine Fluorophores.

The residual tumor after surgery is the most significant prognostic factor of patients with epithelial ovarian cancer. Near-infrared (NIR) fluorescence-guided surgery is actively utilized for tumor localization and complete resection during surgery. However, currently available contrast-enhancing agents display low on-target binding, unfavorable pharmacokinetics, and toxicity, thus not ideal for clinical use. Here we report ultrabright and stable squaraine fluorophores with optimal pharmacokinetics by introducing an asymmetric molecular conformation and surface charges for rapid transporter-mediated cellular uptake. Among the tested, OCTL14 shows low serum binding and rapid distribution into cancer tissue via organic cation transporters (OCTs). Additionally, the charged squaraine fluorophores are retained in lysosomes, providing durable intraoperative imaging in a preclinical murine model of ovarian cancer up to 24 h post-injection. OCTL14 represents a significant departure from the current bioconjugation approach of using a non-targeted fluorophore and would provide surgeons with an indispensable tool to achieve optimal resection.

Fast and Durable Intraoperative Near‐infrared Imaging of Ovarian Cancer Using Ultrabright Squaraine Fluorophores

AbstractThe residual tumor after surgery is the most significant prognostic factor of patients with epithelial ovarian cancer. Near‐infrared (NIR) fluorescence‐guided surgery is actively utilized for tumor localization and complete resection during surgery. However, currently available contrast‐enhancing agents display low on‐target binding, unfavorable pharmacokinetics, and toxicity, thus not ideal for clinical use. Here we report ultrabright and stable squaraine fluorophores with optimal pharmacokinetics by introducing an asymmetric molecular conformation and surface charges for rapid transporter‐mediated cellular uptake. Among the tested, OCTL14 shows low serum binding and rapid distribution into cancer tissue via organic cation transporters (OCTs). Additionally, the charged squaraine fluorophores are retained in lysosomes, providing durable intraoperative imaging in a preclinical murine model of ovarian cancer up to 24 h post‐injection. OCTL14 represents a significant departure from the current bioconjugation approach of using a non‐targeted fluorophore and would provide surgeons with an indispensable tool to achieve optimal resection.

Ovarian Cancer: Applications of Chickens to Humans.

The lack of preclinical models of spontaneous ovarian cancer (OVCA), a fatal gynecological malignancy, is a significant barrier to generating information on early changes indicative of OVCA. In contrast to rodents, laying hens develop OVCA spontaneously, with remarkable similarities to OVCA in women regarding tumor histology, OVCA dissemination, immune responses, and risk factors. These important features of OVCA will be useful to develop an early detection test for OVCA, which would significantly reduce mortality rates; preventive strategies; immunotherapeutics; prevention of resistance to chemotherapeutics; and exploration of gene therapies. A transvaginal ultrasound (TVUS) imaging method for imaging of hen ovarian tumors has been developed. Hens can be monitored prospectively by using serum markers, together with TVUS imaging, to detect early-stage OVCA, provided that a panel of serum markers can be established and imaging agents developed. Recent sequencing of the chicken genome will further facilitate the hen model to explore gene therapies against OVCA.

Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer

Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise; however, current clinical trials are limited by modest response rates. Radiotherapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. Ultrahigh-dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, however, the immunomodulatory properties of FLASH irradiation remain unknown. Here, we demonstrate that single high-dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH-treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8+ T cells. Compared with conventional irradiation, FLASH irradiation increased intratumoral T-cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8+ T-cell infiltration and enhance the efficacy of αPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer.

Senescence induction dictates response to chemo- and immunotherapy in preclinical models of ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is a cancer with dismal prognosis due to the limited effectiveness of existing chemo- and immunotherapies. To elucidate mechanisms mediating sensitivity or resistance to these therapies, we developed a fast and flexible autochthonous mouse model based on somatic introduction of HGSOC-associated genetic alterations into the ovary of immunocompetent mice using tissue electroporation. Tumors arising in these mice recapitulate the metastatic patterns and histological, molecular, and treatment response features of the human disease. By leveraging these models, we show that the ability to undergo senescence underlies the clinically observed increase in sensitivity of homologous recombination (HR)-deficient HGSOC tumors to platinum-based chemotherapy. Further, cGas/STING-mediated activation of a restricted senescence-associated secretory phenotype (SASP) was sufficient to induce immune infiltration and sensitize HR-deficient tumors to immune checkpoint blockade. In sum, our study identifies senescence propensity as a predictor of therapy response and defines a limited SASP profile that appears sufficient to confer added vulnerability to concurrent immunotherapy and, more broadly, provides a blueprint for the implementation of electroporation-based mouse models to reveal mechanisms of oncogenesis and therapy response in HGSOC.

Onvansertib and paclitaxel combined in platinum-resistant ovarian carcinomas.

Background:Ovarian carcinoma is extremely sensitive to (platinum-based) chemotherapy; however, most patients will relapse with platinum-resistant disease, badly affecting their prognosis. Effective therapies for relapsing resistant tumors are urgently needed.Methods:We used patient-derived xenografts (PDXs) of ovarian carcinoma resistant to cisplatin (DDP) to test in vivo the combination of paclitaxel (15 mg/kg i.v. once a week for 3 weeks) and onvansertib, a plk1 inhibitor, (50 mg/kg orally 4 days a week for 3 weeks). The PDX models were subcutaneously (s.c.) or orthotopically transplanted in nude mice and antitumor efficacy was evaluated as tumor growth inhibition and survival advantages of the combination over untreated and single agent treatment.Results:The combination of onvansertib and paclitaxel was very well tolerated with weight loss no greater than 15% in the combination group compared with the control group. In the orthotopically transplanted PDXs, single onvansertib and paclitaxel treatments prolonged survival; however, the combined treatment was much more active, with median survival from three- to six-fold times that of untreated mice. Findings were similar with the s.c. transplanted PDX, though there was greater heterogeneity in tumor response. Ex vivo tumors treated with the combination showed greater induction of γH2AX, marker of apoptosis and DNA damage, and pSer10H3, a marker of mitotic block.Conclusion:The efficacy of onvansertib and paclitaxel combination in these preclinical ovarian cancer models supports the clinical translatability of this combination as an effective therapeutic approach for platinum-resistant high-grade ovarian carcinoma.

Abstract P217: The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer

Abstract Since the approval of poly (ADP-ribose) polymerase inhibitors (PARPi) for BRCA-mutated ovarian cancer patients, different PARPi have been developed and have shown efficacy in homologous recombination deficient (HRD) tumours. These first-generation drugs inhibit both PARP1 and PARP2 (as well as other PARP family members) and present undesirable adverse effects, such as haematological and intestinal toxicity, that restricted their use especially in combination with already poorly tolerated chemotherapeutic agents. Here, patient-derived ovarian cancer xenografts (OC-PDXs) were used i) to evaluate the dose response efficacy of AZD5305, a next generation, potent and selective PARP1 inhibitor and ii) to investigate the effect of the combination with carboplatin, a standard-of-care treatment for ovarian cancer patients. AZD5305 was administered orally (po) once daily (QD) for 8 weeks as monotherapy (0.1, 1 and 10 mg/kg) and in combination with carboplatin (CPT) dosed intravenously (35 mg/kg; Q7x4). Two BRCA1 mutated (HOC106 and HOC107) and a BRCA wild-type (HOC84) OC-PDXs (all TP53 mutated) were implanted subcutis and the effect of treatments on tumour growth evaluated. AZD5305 dosed at 0.1, 1 and 10 mg/kg to mice bearing HOC106 tumours, inhibited tumours growth in a dose dependent manner. Administered in combination at 0.1 and 1 mg/kg, AZD5305 potentiated the effect of carboplatin, causing a significant and sustained regression of HOC106 tumours. The anti-tumour efficacy was also evident against the PARPi-resistant OC-PDX HOC107, whereby the tumour growth was inhibited by AZD5305 at 1 and 10 mg/kg dose. Combined with carboplatin, AZD5305 stabilized the growth of HOC107 tumours at doses as low as 0.1 mg/kg that on its own was not effective. As expected, none of the treatments affected the growth of the BRCA wild-type HOC84 (PARPi and carboplatin resistant OC-PDX). Overall, AZD5305 a next generation PARP1-selective inhibitor and trapper, shows improved efficacy and tolerability in combination with chemotherapy compared to first generation, PARP1/2 inhibitors, making it a promising clinical candidate for the treatment of ovarian cancer. Citation Format: Giulia Dellavedova, Alessandra Decio, Anna Staniszewska, Elisabetta Leo, Raffaella Giavazzi, Maria Rosa Bani. The next generation PARP inhibitor AZD5305 is active in a broad range of pre-clinical models of ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P217.

Protein kinase RNA-activated controls mitotic progression and determines paclitaxel chemosensitivity through B-cell lymphoma 2 in ovarian cancer.

Anti-tubulin agents, such as paclitaxel, have been used extensively for treatment of several types of cancer, including ovarian, lung, breast, and pancreatic cancers. Despite their wide use in cancer treatment, however, patient response is highly variable and drug resistance remains a major clinical issue. Protein kinase RNA-activated (PKR) plays a critical role in immune response to viral infection. We identified PKR as a phospho-protein in response to anti-tubulin agents and this phosphorylation occurs independent of its own kinase activity. PKR is phosphorylated by cyclin-dependent kinase 1 (CDK1) during anti-tubulin treatment and unperturbed mitosis and that PKR regulates mitotic progression in a phosphorylation-dependent manner. Furthermore, inactivation of PKR confers resistance to paclitaxel in ovarian and breast cancer cells in vitro and in vivo. PKR expression levels and activity are decreased in chemotherapeutic recurrent ovarian cancer patients. Mechanistically, our findings suggest that PKR controls paclitaxel chemosensitivity through repressing Bcl2 expression. Pharmacological inhibition of Bcl2 with FDA-approved agent venetoclax overcomes paclitaxel resistance in preclinical animal models of ovarian cancer. Our results suggest that PKR is a critical determinant of paclitaxel cytotoxicity and that PKR-Bcl2 axis as a potential therapeutic target for the treatment of recurrent drug-resistant ovarian tumors.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.