Preclinical Models Of Ovarian Cancer Research Articles

PARG inhibitor sensitivity correlates with accumulation of single-stranded DNA gaps in preclinical models of ovarian cancer

Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA-damaging agents. PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as cancer patients with germline or somatic BRCA1/2-mutations. PARP inhibitors kill BRCA-deficient cancer cells by increasing single-stranded DNA gaps (ssGAPs) during replication. Here, we report that, like PARP inhibitor (PARPi), PARG inhibitor (PARGi) treatment also causes an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-phase-specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands and induced ssGAPs, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication forks and at the ssDNA sites in sensitive cells. Additionally, PARGi exhibited monotherapy activity in specific HR-deficient, as well as HR-proficient, patient-derived, or patient-derived xenograft (PDX)-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssGAPs. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssGAPs due to OFP defects during replication. Regardless of the BRCA/HRD-status, the induction of ssGAPs in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids (PDOs) may be a useful model system for testing PARGi sensitivity and functional biomarkers.

Inhibiting mitochondria potentiates PARPi-triggered, STING-dependent immune response in pre-clinical models of epithelial ovarian cancer

Inhibiting mitochondria potentiates PARPi-triggered, STING-dependent immune response in pre-clinical models of epithelial ovarian cancer

Trastuzumab-Mediated Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Enhances Natural Killer Cell Cytotoxicity in HER2-Overexpressing Ovarian Cancer.

Ovarian cancer is the deadliest gynecologic cancer. Although human epidermal growth factor receptor-2 (HER2) overexpression, a poor prognostic molecular marker in ovarian cancer, is found in almost 30% of ovarian cancer cases, there are no established therapies for HER2-overexpressing ovarian cancer. In this study, we investigated the efficacy of combined samfenet, a biosimilar compound of trastuzumab, and natural killer (NK) cells in preclinical model of HER2-overexpressing ovarian cancer. Firstly, we screened the HER2 expression in three ovarian cancer cell lines and eight ovarian cancer patient-derived tumor xenograft (PDTX) samples. Then, immunohistochemistry and silver in situ hybridization (SISH) were performed following clinical criteria. HER2-overexpressing cells exhibited the highest sensitivity to samfenet compared with low-HER2-expressing cells. In addition, the combination of samfenet with natural killer (NK) cells resulted in significantly enhanced sensitivity to HER2-overexpressing cells and showed a significant antitumor effect on PDTX mice compared with monotherapy. It is known that anti-HER2-humanized IgG1 monoclonal antibodies, including trastuzumab, induce antibody-dependent cellular cytotoxicity (ADCC). Consequently, the combination of samfenet with NK cells demonstrated NK cell-mediated ADCC, as confirmed using an in vitro NK cytotoxicity assay and in vivo antitumor efficacy. A transferase dUTP nick end labeling (TUNEL) assay using xenografted tumors further supported the ADCC effects based on the increase in the number of apoptotic cells in the combination group. Furthermore, high HER2 expression was associated with shorter progression-free survival and overall survival based on public mRNA expression data. In this study, we demonstrated that the combination of samfenet and NK cell therapy could be a promising treatment strategy for patients with HER2-overexpressing ovarian cancer, through ADCC effects. Therefore, this study supports a rationale for further clinical studies of the combination of samfenet and NK cells as a therapy for patients with HER2-overexpressing ovarian cancer.

223 (PB211): PHI-101 synergizes with chemotherapy and PARP inhibitors in preclinical models of ovarian cancer

223 (PB211): PHI-101 synergizes with chemotherapy and PARP inhibitors in preclinical models of ovarian cancer

Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs which have demonstrated limited effectiveness in OC patients. We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). In the SKOV-3 Luc Hu-mouse model, we assessed the efficacy of PD-1 blockade with pembrolizumab. We observed the presence of human lymphocyte and myeloid cell subsets within the tumors, lymph nodes, blood, and spleens in these models. Notably, these tumors exhibited a high prevalence of tumor-infiltrating macrophages. Furthermore, we identified HDAC class I target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts in the tumors of Hu-mice treated with pembrolizumab. Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer useful information to explore potential mechanisms associated with unresponsive anti-PD-1 treatment in OC.

Blockade of TGF-β and PD-L1 by bintrafusp alfa promotes survival in preclinical ovarian cancer models by promoting T effector and NK cell responses

BackgroundFailure of immunotherapy in high-grade serous ovarian cancer (HGSC) may be due to high levels of transforming growth factor-β (TGF-β) in ascites or tumour immune microenvironment (TIME). Here, we test whether coordinated blockade of TGF-β and PD-L1 with bintrafusp alfa (BA) can provoke anti-tumour immune responses in preclinical HGSC models.MethodsBA is a first-in-class bifunctional inhibitor of TGF-β and PD-L1, and was tested for effects on overall survival and altered TIME in syngeneic HGSC models.ResultsUsing a mouse ID8-derived HGSC syngeneic model with IFNγ-inducible PD-L1 expression, BA treatments significantly reduced ascites development and tumour burden. BA treatments depleted TGF-β and VEGF in ascites, and skewed the TIME towards cytotoxicity compared to control. In the BR5 HGSC syngeneic model, BA treatments increased tumour-infiltrating CD8 T cells with effector memory and cytotoxic markers, as well as cytolytic NK cells. Extended BA treatments in the BR5 model produced ∼50% BA-cured mice that were protected from re-challenge. These BA-cured mice had increased peritoneal T-effector memory and NK cells compared to controls.ConclusionsOur preclinical studies of BA in advanced ovarian cancer models support further testing of BA as an improved immunotherapy option for patients with advanced ovarian cancer.

Abstract LB406: Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer

Abstract Folate receptor alpha (FRα) is a cell surface GPI-anchored protein overexpressed in several solid tumors with highest prevalence in ovarian cancer and lung adenocarcinoma but restricted expression in normal tissues. AZD5335, an FRα-targeting antibody conjugated to AZ’s proprietary topoisomerase 1 inhibitor (TOP1i) payload, AZ14170132, is currently being investigated in the FONTANA Phase 1/2 clinical trial (NCT05797168). Since homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRRm+) are associated with sensitivity to DNA damaging agents, we evaluated the efficacy of AZD5335 in ovarian cancer patient-derived xenograft (PDX) models assessed for HRRm, as described1. Sixteen ovarian cancer PDX models were each treated with a single dose of AZD5335 (5 mg/kg IV). Of the HRRm- cohort, 8/11 (73%) cohort responded to treatment with a tumor regression >30% from baseline. In the HRRm+ cohort 4/5 (80%) responded to AZD5335. PARP1 can act as a positive regulator of genomic stability by counteracting TOP1-induced DNA damage (Malanga and Althaus, 2004) that led to the hypothesis that the efficacy of TOP1i drugs can be enhanced by combination with PARP inhibitors. To evaluate for potential synergy of AZD5335 with the PARP1 inhibitor, saruparib (AZD5305): Three FRα-expressing cancer cell lines, KB, IGROV-1 and OVCAR-3, were treated with AZD5335 +/- saruparib, revealing evidence of synergistic cytotoxicity. These results encouraged us to investigate this combination in pre-clinical HRRm+ in vivo xenograft models (OVCAR-3 and OV2022F). In OVCAR-3 xenograft models, single agent activity with saruparib (1 mg/kg PO QD x28) was 37% tumor growth inhibition (TGI) and with AZD5335 (0.625 mg/kg IV x1) was 50% TGI while combined administration at the same doses improved TGI to 86%. Similar combinatorial activity was seen in OV2022F with TGI 61%, 54%, and 97% for saruparib, AZD5335, and saruparib +AZD5335, respectively. In conclusion, AZD5335 exhibits a similar degree of single agent antitumor activity in both HRRm+ and HRRm- preclinical models, and the efficacy can be potentiated by combining with the PARP inhibitor, saruparib. 1. Clarke et al. 2022, NEJM Evid 2022;1(9) DOI: 10.1056/EVIDoa22000432. Malanga and Althaus 2004, JBC 2004; 279(7) DOI: 10.1074/jbc.C3004372003. Citation Format: Marco Gymnopoulos, Ravinder Tammali, Ana De Almeida, Jixin Wang, Claire Myers, Mark Albertella, Paula G. Fraenkel, Puja Sapra. Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB406.

Abstract 3909: PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer

Abstract Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair in conjunction with PARP1 by removal of poly (ADP-ribose) (PAR). Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA damaging agents, suggesting it is a potential therapeutic target for cancer therapy. Indeed, PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as ovarian and breast cancer patients with BRCA1/2-mutation. In this study, we investigated PARG inhibitor (PARGi)-PDD00017273 activity in preclinical models of ovarian cancer cell lines and primary patient-derived organoids. PARGi exhibited sensitivity in a subset of ovarian cancer cell lines that was distinct from PARP inhibitor sensitivity. Initially, we evaluated a PARGi-sensitive cell line, RMUGS. By gradually increasing the concentration of PARGi, we generated drug resistant cells. PARGi treatment resulted in increased S phase specific PAR, DNA-PARylation, DNA damage, and replication stress in the sensitive cells but not in cells with de novo or acquired resistance. In the sensitive cells, PARGi exposure also increased accumulation of PAR at the replication fork resulting from unligated Okazaki fragment processing. More importantly, PARGi treatment or PARG loss caused accumulation of single strand DNA (ssDNA) gaps in sensitive cells, as detected by the DNA fiber assays combined with S1 nuclease treatment. Regardless of the BRCA-status, the induction of ssDNA gaps in ovarian cancer cells correlated with PARGi sensitivity. Consistent with an increase in ssDNA gaps, PARGi exposure increased p-RPA, a ssDNA surrogate, at the replication fork in sensitive cells. p-RPA sites also colocalized with PAR suggesting that accumulation of PAR occurs at the ssDNA sites, perhaps, leading to the ssDNA gaps. Collectively, these results suggested that toxic accumulation of PAR at the replication fork leads to accumulation of ssDNA gaps due to Okazaki fragment processing defects and ovarian cancer cell killing after PARG inhibition. We further assessed PARG inhibition in patient-derived organoids (PDOs) and patient-derived xenograft (PDX)-derived organoids (PDXOs) as model systems of ovarian cancer. PARGi exhibited monotherapy activity in specific HR-deficient PDOs and PDXOs with BRCA1 or RAD51C mutation and in a HR-proficient PDXO model. Importantly, PARGi exposure resulted in accumulation of ssDNA gaps in organoids of ovarian cancer and this phenotype directly correlated with drug sensitivity. PDOs can therefore be a useful model system for testing PARGi sensitivity. The detection of ssDNA gaps in PDOs may provide a functional biomarker to predict the response to PARG inhibitors in clinical trials. Citation Format: Ramya Ravindranathan, Ozge Somuncu, Yuqing Jiao, Alexandre André B A da Costa, Benjamin P Lamarre, David B Martignetti, Gabriella A Zambrano, Sirisha Mukkavalli, Joyce Liu, Bose Kochupurakkal, Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I Shapiro, Alan D. D'Andrea. PARG inhibitor sensitivity is correlated with accumulation of single strand DNA gaps in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3909.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p<0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p<0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p<0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p<0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p<0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5909.

Abstract 3997: PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer

Abstract Background: The efficacy of chimeric antigen receptor (CAR) T cell therapy remains modest for the treatment of solid tumors due to restricted T cell infiltration and immunosuppressive tumor microenvironment (TME). Leveraging the window of opportunity between leukapheresis and infusion using bridging therapy might improve TME and potentiate CAR-T cell therapy. Here, we assessed the potential of poly (ADP-ribose) polymerase (PARP) inhibitors plus anlotinib as bridging therapy for CAR-T cells with resistance to TGFβ and capability of rapid expansion by bait-and-switch strategy of dual-targeting mesothelin (MSLN) and CD19 in preclinical models of advanced ovarian cancer. Methods: Immunocompetent mice bearing ID8 tumors were treated with niraparib (21 days) plus anlotinib (14 days) and euthanized at different timepoints (0, 7, or 14 days since discontinuation) to quantify tumor-infiltrating T cells and profile T cell functions. CAR-T cells transfected with dominant-negative TGFβRII dual-targeting MSLN and CD19 were produced by IASO Biotherapeutics. NSG mice bearing MSLN-positive cell- or patient-derived xenografts (CDX or PDX) of ovarian cancer received CAR-T cells alone or after bridging therapy (niraparib plus anlotinib followed by a wash-out period of 7 days). Tumor volume was balanced before CAR-T cell infusion. Replication-deficient NALM6 cells pre-treated with mitomycin C were injected to provide CD19 antigen. A PET scan that visualized granzyme B release was used to track CAR-T cells and assess immune-mediated tumor killing. Results: Niraparib combined with anlotinib increased tumor-infiltrating T cells without impairing T cell functionality. The effects of promoting T cell infiltration remained significant at 14 days after discontinuation, which might associate with lasting activation of cGAS-STING pathway and secretion of downstream chemokines (CXCL10 and CCL5), and normalized tumor vasculature featured by better perfusion and reduced leakage. The engineered CAR-T cells could resist TGFβ and displayed improved proliferation, cytotoxicity, cytokine release, and in vivo antitumor activities upon CD19 stimulation. Importantly, bridging therapy significantly increased CAR-T cell infiltration, curbed tumor growth, and prolonged survival in mice bearing SKOV3 CDX, HRD-negative PDX, and another multidrug-resistant PDX. PET imaging showed that bridging therapy increased tumor-infiltrating CAR-T cells and boosted tumor-killing capability. Conclusions: PARP inhibitors plus anlotinib as bridging therapy facilitated CAR-T cell infiltration and enhanced antitumor activities in multiple preclinical models of advanced ovarian cancer, and an early phase I trial (NCT05141253) is ongoing to evaluate PARP inhibitors plus anlotinib as bridging therapy for CAR-T cell therapy in patients with refractory MSLN-positive ovarian cancer. Citation Format: Huayi Li, Minghua Xiang, Jiahao Liu, Wei Mu, Li Zhu, Xuejiao Zhao, Gordon B. Mills, Qinglei Gao, Yong Fang. PARP inhibitors plus anlotinib as bridging therapy for TGFβ-insensitive CAR-T cell therapy targeting MSLN and CD19 in advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3997.

Abstract 4187: Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice

Abstract Background: Although immunotherapy has not yet been as successful in ovarian cancer (OC), it remains a potential therapeutic strategy. Preclinical models of OC are necessary to evaluate the efficacy of immuno-oncology (IO) drugs targeting human cancer and immune components but have been underutilized. Developing mouse models with a humanized (Hu) immune system can help understand the human immune response to IO drugs, including immune checkpoint inhibitors (ICIs), which have demonstrated limited effectiveness in OC patients. Methods: We established OC xenograft Hu-mouse models by intraperitoneally injecting luciferase-expressing SKOV-3 Luc and OVCAR-3 Luc OC cells into CD34+ Hu-mice. Tumor growth was monitored through bioluminescence imaging (BLI). We assessed the efficacy of PD-1 blockade with pembrolizumab in the SKOV-3 Luc Hu-mouse model. The immune profiles of the tumors were characterized using colorimetric immunostaining and flow cytometry. Additionally, we analyzed RNA-seq data to investigate the gene expression signature of pembrolizumab refractory tumors. Results: We confirmed tumor development in both OC cell lines within CD34+ Hu-mice. In these models, human lymphocyte and myeloid cell subsets were present in the tumors, draining lymph nodes, blood, and spleens. The SKOV-3 Luc tumor-bearing Hu-mice did not respond to pembrolizumab monotherapy. These tumors exhibited a high presence of tumor-infiltrating macrophages. Tumors in Hu-mice unresponsive to pembrolizumab showed a lower abundance of CD8+ T-cells, memory B cells, plasma cells, and a higher proportion of naïve M0 macrophages and mast cells compared to the PBS control. Furthermore, we identified 43 significantly enriched gene sets in these tumors. The differentially expressed genes (DEGs) were predominantly enriched in HDAC class I, RB1, KLF1/3, TCF21, MYD88, SMARCE1 target genes, and genes associated with epithelial-mesenchymal transition (EMT) and fibroblasts. Conclusion: Our xenograft Hu-mouse model of OC provides a valuable tool for investigating the efficacy of IO drugs. The insights gained from this model offer potential avenues to explore mechanisms of resistance to PD-1/PD-L1 blockade in OC. Citation Format: Sung Wan Kang, Ji-young Lee, Ok-Ju Kang, Yong-Man Kim, Eun Kyung Choi, Shin-Wha Lee. Transcriptome profiling and characterization of peritoneal metastasis ovarian cancer xenografts in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4187.

Abstract 1858: Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload

Abstract ROR1 is a pseudo kinase that is involved in the non-canonical Wnt signaling pathway and is highly expressed during early embryonic development. In adult tissues, ROR1 is expressed at very low levels but its increased expression is observed in hematological cancers and a broad range of solid tumors. AT86474 is a highly homogeneous antibody drug conjugate incorporating AxcynCYSTM technology that allows reproducible site-specific conjugation with DAR4 composition greater than 97%. AT86474 utilizes a proprietary payload derived from an FDA approved drug that was optimized in-house for increased potency and with altered PK properties to limit systemic exposure. In vitro, the payload displays broad sub-nanomolar activity across multiple cancer cell lines, including those resistant to paclitaxel, DM1, MMAE and DXd. The payload is conjugated via an enzyme-cleavable linker optimized for solubility, stability, and high drug to antibody ratios. AT86474 uses a humanized and optimized monoclonal antibody that demonstrates selective binding to ROR1-positive cells and subsequent internalization, sub-nanomolar potency in vitro and strong bystander effects. AT86474 was evaluated in a preclinical CDX mouse model of ovarian cancer, where substantial tumor growth inhibition was observed with two weekly doses at as low as 1 mg/kg. As ROR1 over-expression is also observed in many solid tumors, such as pancreatic, gastric, breast and lung cancers, the use of AT86474 can be expanded to benefit a large patient population. Citation Format: Ban Xiong Tan, Xi Yun Zhang, You Xue Wu, Yin Wen Cheng, Shao Jun Liu, Ping Du, Chen Zhong, Bin Zou. Discovery of AT86474, a highly efficacious anti-ROR1 ADC utilizing a proprietary payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1858.

The novel drug candidate S2/IAPinh improves survival in models of pancreatic and ovarian cancer

Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer.

Abstract A082: Targeting sterol regulatory element binding protein 1 (SREBP1) improves response to standard-of-care chemotherapy and alters the tumor microenvironment in pre-clinical models of obesity and ovarian cancer

Abstract Obesity is a recognized non-infectious pandemic that increases ovarian cancer (OvCa) incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity (DIO) mouse model (Liu et al. Can.Res. 2015; 75:5046). DIO also elevated expression and nuclear localization of sterol regulatory element binding protein 1 (SREBP1), a master regulator of de novo lipogenesis and lipid homeostasis. A follow-up study using DIO models showed a significantly diminished response to standard-of-care (SOC) paclitaxel/carboplatin chemotherapy in high fat diet (HFD) mice relative to low fat diet (LFD) controls (Liu et al., J.Exp.Clin.Can.Res. 2023; 42:165). Both studies showed an effect of DIO on tumor fibrosis and immune landscape and these changes were also observed in the ovarian cancer tumors of women with high body mass index. In the current study, we have evaluated the effect of inhibitors of SREBP1 processing, in combination with SOC chemotherapy, in the context of DIO in pre-clinical models of OvCa. Our results show significantly diminished post-treatment tumor recurrence in HFD mice receiving SOC chemotherapy plus an SREBP1 processing inhibitor, with concomitant alterations in the peritoneal immune landscape. Together these data suggest that the reported negative impact of obesity on OvCa patient survival may be due obesity-induced changes in lipid regulatory factors (SREBP1) and the tumor microenvironment (immune landscape, fibrosis) that alter chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting SREBP1, immune cells and/or fibrosis in the obese host. Citation Format: M. Sharon Stack, Yueying Liu, Jing Yang, Tyvette S. Hilliard, Zhikun Wang, Jeff Johnson, Wanrui Wang, Gena Dominique, Elizabeth I. Harper, Nicholas M. Stavrou, Anna Juncker-Jensen. Targeting sterol regulatory element binding protein 1 (SREBP1) improves response to standard-of-care chemotherapy and alters the tumor microenvironment in pre-clinical models of obesity and ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A082.

Tumor Treating Fields (TTFields) induce homologous recombination deficiency in ovarian cancer cells, thus mitigating drug resistance.

Ovarian cancer is the leading cause of mortality among gynecological malignancies. Carboplatin and poly (ADP-ribose) polymerase inhibitors (PARPi) are often implemented in the treatment of ovarian cancer. Homologous recombination deficient (HRD) tumors demonstrate increased sensitivity to these treatments; however, many ovarian cancer patients are homologous recombination proficient (HRP). TTFields are non-invasive electric fields that induce an HRD-like phenotype in various cancer types. The current study aimed to investigate the impact of TTFields applied together with carboplatin or PARPi (olaparib or niraparib) in preclinical ovarian cancer models. A2780 (HRP), OVCAR3 (HRD), and A2780cis (platinum-resistant) human ovarian cancer cells were treated in vitro with TTFields (1 V/cm RMS, 200 kHz, 72 h), alone or with various drug concentrations. Treated cells were measured for cell count, colony formation, apoptosis, DNA damage, expression of DNA repair proteins, and cell cycle. In vivo, ID8-fLuc (HRP) ovarian cancer cells were inoculated intraperitoneally to C57BL/6 mice, which were then treated with either sham, TTFields (200 kHz), olaparib (50 mg/kg), or TTFields plus olaparib; over a period of four weeks. Tumor growth was analyzed using bioluminescent imaging at treatment cessation; and survival analysis was performed. The nature of TTFields-drug interaction was dependent on the drug's underlying mechanism of action and on the genetic background of the cells, with synergistic interactions between TTFields and carboplatin or PARPi seen in HRP and resistant cells. Treated cells demonstrated elevated levels of DNA damage, accompanied by G2/M arrest, and induction of an HRD-like phenotype. In the tumor-bearing mice, TTFields and olaparib co-treatment resulted in reduced tumor volume and a survival benefit relative to olaparib monotherapy and to control. By inducing an HRD-like phenotype, TTFields sensitize HRP and resistant ovarian cancer cells to treatment with carboplatin or PARPi, potentially mitigating a-priori and de novo drug resistance, a major limitation in ovarian cancer treatment.

Functional enhancement of mesothelin-targeted TRuC-T cells by a PD1-CD28 chimeric switch receptor

T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PD-L1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased proinflammatory effector cytokines, decreased anti-inflammatory cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. Anti-MSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PD-L1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Targeting CD47-SIRPa axis shows potent preclinical anti-tumor activity as monotherapy and synergizes with PARP inhibition

The objective was to correlate CD47 gene expression with resistance to immune checkpoint inhibitors (ICI) in tumor tissue of gynecological cancer (GC). Further, we sought to assess the efficacy of targeting CD47 pathway alone and in combination in pre-clinical ovarian cancer (OC) models. We performed transcriptomic analyses in GC treated with ICI. Signaling pathway enrichment analysis was performed using Ingenuity Pathway Analysis. Immune cell abundance was estimated. CD47 expression was correlated with other pathways, objective response, and progression-free survival (PFS). Anti-tumor efficacy of anti-CD47 therapy alone and in combination was investigated both in-vitro and in-vivo using cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) models. High CD47 expression associated with lower response to ICI and trended toward lower PFS in GC patients. Higher CD47 associated negatively with PDL1 and CTLA4 expression, as well as cytotoxic T-cells and dendritic cells but positively with TGF-β, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 significantly enhanced macrophage-mediated phagocytosis of OC cells in-vitro and exhibited potent anti-tumor activity in-vivo in OC CDX and PDX models. In-vitro treatment with PARPi increased CD47 expression. Anti-CD47 led to significantly enhanced in-vitro phagocytosis, enhanced STING pathway and synergized in-vivo when combined with PARP inhibitors in BRCA-deficient OC models. This study provides insight on the potential role of CD47 in mediating immunotherapy resistance and its association with higher TGF-β, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 showed potent anti-tumor activity and synergized with PARPi in OC models. These data support clinical development of anti-CD47 therapy with PARPi in OC.

A phase 1 study of the SYK inhibitor fostamatinib and weekly paclitaxel for recurrent platinum-resistant ovarian cancer.

5574 Background: Response to chemotherapy in patients (pts) with recurrent platinum-resistant ovarian cancer (PROC) is generally < 15%. Spleen tyrosine kinase (SYK) is overexpressed in PROC and is a key mediator of paclitaxel resistance in ovarian cancer cells. Fostamatinib (Fos), an orally available inhibitor of SYK, is currently FDA approved for the treatment of idiopathic thrombocytopenic purpura. The active form of Fos (R406) was shown to synergistically enhance taxane-mediated cytotoxicity in preclinical ovarian cancer models. This phase 1 study aimed to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of Fos when combined with standard weekly paclitaxel (wPac) for pts with PROC. Methods: Pts with PROC, defined as progression ≤6 months of last platinum, were eligible. A modified toxicity probability interval (mTPI) dose escalation design was used to determine the MTD/RP2D and response was assessed using RECISTv1.1 criteria. All pts received paclitaxel (wPac) 80mg/m2 intravenously on Days 1, 8, and 15, of a 28-day cycle. Fos was administered orally twice a day (BID) on a continuous schedule. Three dose levels (DL) were planned: DL1 - 100mg, DL2 - 150mg, and DL3 - 200mg. MTD was determined based on cycle 1 dose-limiting toxicities (DLTs) and responses were assessed every 2 cycles. A dose expansion cohort to further assess adverse events (AEs) and tolerability was performed. Results: 27 eligible pts, median age 62 years (range 35-79 years) with high-grade serous (n = 18), clear cell (n = 4), carcinosarcoma (n = 3), or other (n = 2) histology were treated at 3 centers. Twelve pts were treated in the dose escalation cohort (DL1 = 6, DL2 = 3, DL3 = 3). Common AEs at least possibly attributed to Fos, wPac, or both included diarrhea (70%), fatigue (52%), anemia (44%), neutropenia (33%), nausea (33%), hypertension (30%), and dysgeusia (30%). Treatment-emergent grade 3-4 AEs were neutropenia (37%), anemia (26%), and thromboembolic event (22%). During DL1, updated toxicity management guidelines not consistent with the original protocol were released and 2 of the 3 pts treated prior to the amendment were not considered evaluable for DLT. Thus 3 more pts were enrolled to DL1 after the amendment. Of the 10 pts evaluable for DLT in dose escalation, no DLTs were reported on any of the 3 DLs (DL1 = 4, DL2 = 3, DL3 = 3). DL3 was selected for dose expansion. One of 15 pts in the dose expansion cohort had a DLT (neutropenia) and one patient had a grade 5 infection. Of 18 pts treated at DL3, 7 (39%) had partial or complete response (95% CI: 17.3, 64.3%). Overall, 27 patients received a median of 3 cycles (range 0-10). Analysis of pharmacokinetic and correlative molecular studies of target expression are ongoing. Conclusions: The RP2D of Fos will be 200 mg orally BID when combined with wPac. AE profile of the combination was as expected and the combination demonstrated promising efficacy in pts with recurrent PROC. Clinical trial information: NCT03246074 .

Abstract 2853: TP-6379, an investigational TGFBR1 inhibitor, shows improvement in survival and enhances activity of standard of care in preclinical ovarian cancer models

Abstract Ovarian cancer is still associated with poor prognosis and remains amongst the leading causes of cancer related deaths in females. High recurrence rates, resistance to chemotherapy, and meager outcome highlight the need for improved therapies that stem from understanding the complex and multifactorial etiology of ovarian malignancies. Transforming growth factor-β (TGF-β) signaling can regulate both oncogenesis and metastasis in ovarian cancer (Kumri et al. 2021). This, in part, is done by affecting apoptosis and survival mechanisms, epithelial to mesenchymal transition, immune cell recruitment and response. Inhibition of the TGF-β signaling pathway is a potential pharmaceutical approach for treating ovarian malignancies.TP-6379 is a potent and orally available investigational TGF-β receptor 1 (TGFBR1) inhibitor and is currently in preclinical development at Sumitomo Pharma Oncology, Inc. (SMP Oncology). We hypothesized that TP-6379 may inhibit the TGF-β signaling pathway and show activity in preclinical ovarian cancer models. TP-6379 was observed to inhibit TGFBR1 potently in a biochemical kinase assay. Dose dependent increase in compound concentration was observed in ES-2 tumor burdened animals treated with TP-6379. Preclinical data showed TP-6379 (150 mg/kg, BID) treatment significantly improved overall survival compared to vehicle treatment in an ES-2 mouse xenograft model of clear cell carcinoma. We hypothesize this result was, in part, due to alleviation of ovarian cancer induced cachexia, as animals treated with TP-6379 showed retention of muscle mass in their hind limbs compared to the vehicle treated animals. To investigate a combination effect with the standard of care in ovarian cancer, a SK-OV-3 ovarian cancer adenocarcinoma cell line was treated in vitro with TP-6379 and paclitaxel. Class III β-tubulin (TUBB3), a potential resistance mechanism to paclitaxel, showed induced expression in response to TGF-β, paclitaxel or both, after 24 hr treatment, and this effect was observed to be inhibited by TP-6379 treatment. In a SK-OV-3 xenograft model, TP-6379 (150 mg/kg, BID) treatment showed the trend of tumor growth inhibition (TGI) as a monotherapy and showed enhanced TGI in combination with paclitaxel (7.5 mg/kg, QW) when compared to vehicle treatment. Combination treatment in this xenograft model showed significant reduction of TUBB3 mRNA compared to paclitaxel treatment alone. In conclusion, our preliminary preclinical studies have shown promising activity for TP-6379 in ovarian malignancies as monotherapy and/or in combination with standard of care. TP-6379 may be a viable therapeutic option by targeting the TGF-β pathway in ovarian cancer. Citation Format: Tetyana V. Forostyan, David A. Kircher, Richard E. Heinz, Curtis Allred, Sal Sommakia, Yuta Matsumura, Adam Siddiqui, Jason M. Foulks, Steven L. Warner. TP-6379, an investigational TGFBR1 inhibitor, shows improvement in survival and enhances activity of standard of care in preclinical ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2853.