Mouse Model Of Ovarian Cancer Research Articles

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells

Background: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. Methods: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. Results: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. Conclusion: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

A novel NIR fluorescent probe for enhanced β-galactosidase detection and tumor imaging in ovarian cancer models

The advancement of biological imaging techniques critically depends on the development of novel near-infrared (NIR) fluorescent probes. In this study, we introduce a designed NIR fluorescent probe, NRO-βgal, which exhibits a unique off–on response mechanism to β-galactosidase (β-gal). Emitting a fluorescence peak at a wavelength of 670 nm, NRO-βgal showcases a significant Stokes shift of 85 nm, which is indicative of its efficient energy transfer and minimized background interference. The probe achieves a remarkably low in vitro detection limit of 0.2 U/L and demonstrates a rapid response within 10 min, thereby underscoring its exceptional sensitivity, selectivity, and operational swiftness. Such superior analytical performance broadens the horizon for its application in intricate biological imaging studies. To validate the practical utility of NRO-βgal in bio-imaging, we employed ovarian cancer cell and mouse models, where the probe's efficacy in accurately delineating tumor cells was examined. The results affirm NRO-βgal's capability to provide sharp, high-contrast images of tumor regions, thereby significantly enhancing the precision of surgical tumor resection. Furthermore, the probe's potential for real-time monitoring of enzymatic activity in living tissues underscores its utility as a powerful tool for diagnostics in oncology and beyond.

Abstract 6739: Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model of ovarian cancer

Abstract Ovarian cancer is predominately left undetected and untreated until the later stages of the disease where it becomes incredibly difficult to treat. Preclinical studies demonstrate that immunotherapy can be highly effective. Our lab has been working on an immunotherapy approach to treat women with ovarian cancer that utilizes mesoporous silica nanoparticles(MSN). MSN that present pathogen-associated molecular patterns (PAMPS) are able to significantly reduce tumor burden in mouse models of serous epithelial ovarian cancer when administered by intraperitoneal injection. These microbial mimetic particles are decorated with polyethyleneimine (PEI), CpG oligonucleotide, and monophosphoryl lipid A (MPLA). When in the presence of antigen presenting cells, they bind to Toll-like receptors (TLR) present on the cell surface and within the endosome. To better understand mechanisms underlying their efficacy, we injected fluorescent MSN-PEI-CpG-MPLA (100 or 500 nm) or unmodified MSN into mice with advanced ovarian cancer. The particles, regardless of the presence of TLR-agonists, were rapidly internalized by myeloid cells, with trafficking almost exclusively to immune clusters located in tumor-burdened adipose tissues when administered by intraperitoneal injection. Conversely, intravenous injection of MSN-PEI-CpG-MPLA led to predominate accumulation in liver, with no particles found in tertiary lymphoid clusters 24 hours post injection. Furthermore, unlike intraperitoneal injection, intravenous injection of MSN-PEI-CpG-MPLA was without therapeutic effect. Currently we are studying MSN safety and the impact of administration route and dose on efficacy and cytotoxicity. In summary, MSN, with and without TLR agonists, are trafficked by myeloid cells to tumor-burdened fat-associated lymphoid clusters when administered by intraperitoneal injection. Intraperitoneal injection of TLR-agonist modified MSN effectively reduces tumor burden in mice with ovarian cancer. Citation Format: Lorel Y. Medina, Rita E. Serda, Benjamin Marwedel, Melanie S. Jun, Josh Adogola. Efficacy and toxicity of microbial mimetic mesoporous silica nanoparticles in a mouse model 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 6739.

Abstract 115: Developing uPAR CAR T Cells for High-Grade Serous Ovarian Cancer

Abstract Chimeric Antigen Receptor (CAR) T cells are a modality of immunotherapy that act to eliminate cancer cells by redirecting the cytolytic activity through targeting a protein overexpressed on the surface of the cancer cell. In contrast to move conventional cancer therapies, their anti-cancer activity does not depend on a cancer-specific molecular vulnerability but rather the differential expression of the target antigen on tumor cells compared to normal tissues. We have developed CAR T cells targeting the urokinase plasminogen activator receptor (uPAR), which is overexpressed on senescent cells but not expressed highly in vital organs, to selectively target senescent cells in a range of tissue damage pathologies where they are known to be pathogenic. uPAR is also highly overexpressed in a wide range of cancer types, including pancreatic, kidney, bladder, brain and ovarian carcinomas, respectively, raising the possibility that CAR T cells targeting uPAR may also be effective against cancer. Indeed, leveraging an electroporation-based genetically-engineered mouse model of ovarian cancer, we demonstrate the robust anti-tumor efficacy of murine uPAR CAR T cells in immunocompetent syngeneic mice. Moreover, through an exhaustive functional screening of 37 distinct human uPAR single-chain fragment variants (scFVs), we have successfully identified lead scFVs exhibiting subnanomolar affinity to membrane-anchored uPAR. Significantly, these human uPAR CAR T cells exhibit the capability to eliminate both orthotopic and metastatic human HGSOC xenograft tumors without inducing severe adverse effects. Ongoing efforts encompass the assessment of the long-term safety and toxicity profiles of uPAR CAR T cells utilizing a humanized mouse model platform. Our results provide a strong rationale for developing uPAR CAR T cells as an anticancer strategy relevant to a broad range of tumor types, and provide an avenue for safety profiling of uPAR CAR T cells prior to clinical testing in non-cancer patients harboring senescence-related pathologies. Citation Format: Zeda Zhang, Xin Fang, Yu-jui Ho, Sascha Haubner, Friederike Kogel, Clemens Hinterleitner, Stella Paffenholz, Kevin Chen, Wei Luan, Amanda Kulick, Gertrude Gunset, Andreina Garcia Angus, Jing Zhang, Zijian Xu, Adam Wang, Qingwen Jiang, Elisa de Stanchina, Britta Weigelt, Dmitriy Zamarin, Aveline Filliol, Judith Feucht, Jorge Mansilla-Soto, Corina Amor, Michel Sadelain, Scott Lowe. Developing uPAR CAR T Cells for High-Grade Serous 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 115.

Abstract 2035: A Benzenesulfornamide with potential to improve chemoresponse of ovarian cancer through activation of AMPK and immunogenic cell death

Abstract Introduction: Ovarian cancer is a leading cause of death among women globally often characterized by poor prognosis and aggressive tumor growth. The therapeutic outcomes of ovarian cancer patients are limited by the development of acquired chemoresistance and the lack of targeted therapies. This study aimed to understand the mechanism in which a benzenesulfornamide named Y3 enhances the efficacy of chemotherapeutic agents. Y3 was previously identified by our team as an activator of AMPK with the activity to induce immunogenic cell death of ovarian cancer cells. Material and Methods: Patient-derived ovarian cancer cell lines were pretreated with Y3 for 3 h before treatment of carboplatin or paclitaxel. Celltiter Glo assay was performed to determine the impact of Y3 pretreatment on the IC50 values of carboplatin or paclitaxel after 48 h. Apoptosis was evaluated using Annexin VI/PI staining and Caspase 3 activity assays. The role of AMPK pathway was assessed using AMPK siRNAs and western blot. The potential of Y3 to enhance immunogenic cell death induced by carboplatin or paclitaxel was evaluated in a syngeneic mouse model of ovarian cancer using a vaccination assay. The control group mice were vaccinated by subcutaneously injecting mouse ovarian cancer cells treated by carboplatin in vitro for 24 h into the left flank. The test group mice were vaccinated by injecting Y3-carboplatin co-treated cells. One week after vaccination, untreated live cancer cells were injected into the right flank of both groups. Tumor growth was monitored. Results: Ovarian cancer cell lines pretreated with Y3 showed decreased IC50 values for carboplatin and paclitaxel treatments. The apoptotic cell populations stained by Annexin VI were increased in the Y3 pretreated in comparisons with the single treatment groups. Caspase-3 activity validated the results of Annexin VI/PI staining assay. AMPK knockdown by siRNAs effectively reduced the levels of phospho-AMPK that was induced by Y3, which also dampened the Y3-promoted chemotherapeutic effects on ovarian cancer cells. In the in vivo vaccination assay, tumor growth in the right flank was suppressed in the mice vaccinated with Y3-carboplatin co-treated cells to an extend that is more significant than the control group. Conclusion: Y3 pretreatment increased the sensitivity of ovarian cancer cell lines to carboplatin and paclitaxel. The effects of Y3 on ovarian cancer cells depend on the activation of AMPK. Y3 enhances the immunogenicity of carboplatin-treated ovarian cancer cells. Citation Format: Juhyun Kim, Nia Montero, Shefali Sharma, Miranda Mansolf, Kevin Yang, Tobias Hartwich, Yang Yang-Hartwich. A Benzenesulfornamide with potential to improve chemoresponse of ovarian cancer through activation of AMPK and immunogenic cell death [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 2035.

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.

Abstract 6974: Spatial exosome analysis reveals the location heterogeneity of exosomal miRNAs in ovarian cancer patients

Abstract Extracellular vesicles (EVs), including exosomes, are recognized as promising functional tools involved in cancer progression. Although evidence of the functions of EV in cancer biology has been accumulating, optimizing EV isolation is still a major challenge. In ovarian cancer, peritoneal dissemination is a crucial prognostic factor due to the lack of effective treatment, and EVs in malignant ascites play a key role for progression. In this study, we invented cellulose nanofiber (CNF) sheets which enable to capture EVs from approximately 10 μL of biofluids and enabled the analysis of the bioactive molecules included in the EVs (Nature Communications 2023). By attaching CNF sheets to moistened organs, the EVs in trace amounts of ascites were collected, which was sufficient to perform small RNA sequence analyses. In the ovarian cancer mouse model, CNF sheets enabled the detection of cancer-associated miRNAs from the very early phase when the mice did not have apparent ascites, and the EVs from different locations, such as pelvic walls or liver surfaces, had unique miRNA profiles. To confirm the utility of the CNF sheets in cancer patients, we directly collected the EVs on the organ surfaces by the EV attachment method during surgery under appropriate ethical institutional approval with individual consent. EVs were collected from the pelvic peritoneum, omentum, liver surface, or tumor surface, and the miRNA profiling demonstrated each location formed unique clusters. Notably, several tumor-suppressive miRNAs have been identified in liver surface EVs, such as miR-122-3p, and this is consistent with actual ovarian cancer cell progression, which tends to metastasize to the diaphragm rather than to the liver surface. In addition, the trajectory analysis revealed that the pattern of connection in each site was different between patients with localized and advanced disease. We identified ten miRNAs were high in tumors and in EVs from tumor rupture sites, and the level of each miRNA decreased with distance from the primary tumors. Among ten miRNAs, hsa-miR-200b-3p and hsa-miR-429 were confirmed as highly expressed in tumor tissues compared to adjacent normal tissues or benign tumors, and three miRNAs in serum, urine and saliva decreased post-surgery. In summary, spatial CNF sheet analyses in patient bodies identified location-based heterogeneity in EV-miRNA profiles, and the profiles reflected disease conditions. This new method could provide unveiled biological aspects of EVs, and suggest a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning. Citation Format: Akira Yokoi, Kosuke Yoshida, Yusuke Yamamoto, Takao Yasui, Hiroaki Kajiyama. Spatial exosome analysis reveals the location heterogeneity of exosomal miRNAs in ovarian cancer patients [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 6974.

Abstract 4114: AST-201 (pUMVC3-hIGFBP2 N-terminus) demonstrates anti-tumor effect in an ovarian cancer mouse model

Abstract Background: IGFBP2, known to enhance the invasion capacity of ovarian cancer cells, has been suggested that its inhibition could be potentially a treatment strategy of ovarian cancer. AST-201 (pUMVC3-hIGFBP2) is a therapeutic cancer vaccine using a plasmid DNA encoding IGFBP2 N-terminus. In a phase 1 study (NCT01322802) completed, 100 μg AST-201 (intradermal immunization, id) showed not only a significant efficacy by inducing the Th1-cell immunity against IGFBP2 but also safety and tolerability profiles in ovarian cancer patients. The primary objective of this study is to evaluate whether the administration of AST-201 alone and the combination with pembrolizumab could show anti-tumor efficacy and/or synergic effect in the ID8-Luc2 ovarian cancer mouse model. Also, immunological responses were observed as explorative endpoint. Methods: AST-201 (100 μg/animal, id, mixed with mGM-CSF as an immune adjuvant) was immunized to mice (C57BL/6) once a week for a total of 4 times on different days, either alongside pembrolizumab (10 mg/kg, intraperitoneal injection) twice a week for a total of 3 times on different days. Also, AST-201 was immunized was a mono treatment. The efficacy was evaluated by a tumor growth inhibition (TGI) rate at the last day, and immune cell profiling via FACS analysis was conducted with splenocytes and tumor tissues collected at 8 weeks after the first injection. Results: As a primary endpoint, a TGI rate at Day 55 of AST-201 mono treatment was 67%, comparing to a control group (p<0.05). The anti-tumor effect of AST-201 combining with pembrolizumab was better than standard dose pembrolizumab, based on a TGI rate at Day 55 (78% vs 66%, not significant). Immune profiling showed that AST-201 and pembrolizumab combination regimen could inhibit a tumor growth by transforming TME into inflamed-type (‘hot’) form the low immunoreactivity, which was supported by increased CD4+TEM, and CD8+TEM and T helper cells in splenocyte and TIL analysis. Conclusion: A study demonstrated that AST-201 (IGFBP2 cancer vaccine) showed an anti-tumor effect as mono treatment and would be potentially leading to a synergistic effect with a combination regimen of pembrolizumab. Phase 2 randomized-controlled study of AST-201 in ovarian cancer will be initiated under the MRCT strategy (CornerStone-004 study, NCT05794659). Citation Format: Jinback Lim, Jinho Kang, Hyo-Hyun Park, Jin Kyeong Choi, Jee Hyun Choi, Seong-Yong Jang, Min-Ah Kim, Myeong-Kyu Park, Mary L Disis, Eunkyo Joung, Ashley Yongmin Kim, Hun Jung. AST-201 (pUMVC3-hIGFBP2 N-terminus) demonstrates anti-tumor effect in an ovarian cancer mouse model [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 4114.

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 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [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 1321.

Abstract 7549: Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1

Abstract DNA methylation inhibitors such as 5-azacytidine (Aza) and 5-aza-2′-deoxycytidine have been extensively used in patients with hematologic disorders and other neoplasms since their approval by the FDA. However, global DNA demethylating agents can result in intolerable toxicities; therefore, targeted epigenetic modulation is needed. Here, we searched for epigenetic changes with adaptive resistance to anti-VEGF antibody (AVA) in the TME and a novel strategy for targeted demethylation. Methods: We examined methylation and transcriptional changes in orthotopic ovarian cancer models with emergence of AVA resistance. We developed a targeted demethylation strategy for BARD1 to enhance the effectiveness of AVA. We used a novel dCas9-CRISPR-based system for targeted BARD1 CpG demethylation (derived by fusing the deactivated Cas9 [dCas9] with the TET1 catalytic domain) delivered in vivo using a nanoliposomal carrier. Results: We identified BARD1 methylation and reduced expression as lead candidate with AVA resistance; multiple CpG island sites in the BARD1 gene from adaptive AVA-resistant tumors were methylated. Next, we investigated the biological effects of global and targeted DNA methylation. Azacytidine along with AVA therapy was tested in a series of models (fibrosarcoma, PDX and orthotopic ovarian cancer models) and resulted in reduced tumor burden compared with the control and monotherapy groups (p<0.01). Next, to determine whether targeted demethylated BARD1 would have antitumor effects in vivo, we used an orthotopic SKOV3ip1luc ovarian cancer mouse model. Mice were treated with DOPC-dCas9-TET1-sgcontrol- (control) and DOPC-dCas9-TET1-sgBARD1 (sgBARD1). Tumor weight in the sgBARD1-3 group was significantly (p<0.01) reduced compared with the control group with no significant change in mouse body weight. We found that BARD1 expression was significantly increased in sgBARD1-treated group compared with control (p<0.001). Next, to determine whether demethylated BARD1 would combine well with AVA therapy in the SKOV3ip1 model, we combined sgBARD1-3 with bevacizumab (bev); sgBARD1-3 alone led to a significant decrease in tumor burden compared with control groups. Moreover, tumor burden was significantly reduced in the sgBARD1-3 and bevacizumab combination group compared with the sgcontrol group and bev alone group. BARD1 expression was significantly increased in sgBARD1-3-treated and in combination tumor samples compared with controls. To check for potential off-target effects, we tested the efficacy of another sgBARD1 and found that tumor burden in the sgBARD1-1 group was significantly reduced compared with control group. Conclusion: Our novel targeted approach results in robust antitumor effects when combined with AVA therapy in multiple cancer models. Collectively, our findings provide a novel approach for gene targeted demethylation and could have implications for epigenetic therapies. Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Elaine Stur, Sudhir Kumar, Lingegowda S. Mangala, Nazende Nur Bayram, Pahul Hanjra, Sara Corvigno, Stephen Baylin, Gabriel Lopez-Berestein, Anil K. Sood. Overcoming resistance to anti-VEGF therapy via epigenetic regulation of BARD1 [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 7549.

Abstract 4742: Targeting the RNA-binding protein LARP1 overcomes therapeutic resistance in ovarian cancer

Abstract La-related protein 1 (LARP1) is an RNA-binding protein that plays a pivotal role in regulating mRNA on a post-transcriptional level. Overexpression of LARP1 has been detected in multiple cancers substantiating the critical role of LARP1 as a driver for cancer progression. This study aims to investigate the downstream target pathways regulated by LARP1 and to determine the impact of LARP1 targeting on the resistance to cisplatin and olaparib therapy in ovarian cancer (OC). Analysis of The Cancer Genome Atlas OC dataset, reverse phase protein array, in vitro functional and mechanistic experiments, and a xenograft OC mouse model using OVCAR8 cell line were executed. Our results revealed that overexpression of LARP1 is negatively correlated with progression-free survival in OC patients. Furthermore, LARP1 knockdown using two independent small interfering RNAs (siRNAs) suppressed cell growth, invasion, and migration, and ameliorated cisplatin resistance in vitro. Mechanistic analyses identified AXL as a target of LARP1 and this finding was further supported clinically in which co-overexpression of LARP1 and AXL is associated with poor prognosis in OC. Furthermore, LARP1 knockdown resulted in reduced phosphorylation of mTOR, AKT, and SRC with no significant effect on FAK. In vivo, administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoparticles loaded with LARP1-siRNA suppressed tumor growth and improved the response to cisplatin and olaparib in a xenograft OC mouse model. In conclusion, LARP1 acts as an oncogenic RNA-binding protein in OC, and targeting LARP1 may represent an innovative therapeutic approach for ameliorating resistance to chemotherapy and targeted therapy in OC. Keywords: RNA-binding proteins; cisplatin resistance; olaparib resistance; chemotherapy resistance; LARP1; ovarian cancer. Citation Format: Abdelrahman M. Elsayed, Paola Amero, Emine Bayraktar, Anil K. Sood, Gabriel Lopez-Berestein, Cristian Rodriguez-Aguayo. Targeting the RNA-binding protein LARP1 overcomes therapeutic resistance in 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 4742.

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.

Interrogating the Theranostic Capacity of a MUC16-Targeted Antibody for Ovarian Cancer.

Aberrantly expressed glycans on mucins such as mucin-16 (MUC16) are implicated in the biology that promotes ovarian cancer (OC) malignancy. Here, we investigated the theranostic potential of a humanized antibody, huAR9.6, targeting fully glycosylated and hypoglycosylated MUC16 isoforms. Methods: In vitro and invivo targeting of the diagnostic radiotracer [89Zr]Zr-DFO-huAR9.6 was investigated via binding experiments, immuno-PET imaging, and biodistribution studies on OC mouse models. Ovarian xenografts were used to determine the safety and efficacy of the therapeutic version, [177Lu]Lu-CHX-A″-DTPA-huAR9.6. Results: In vivo uptake of [89Zr]Zr-DFO-huAR9.6 supported invitro-determined expression levels: high uptake in OVCAR3 and OVCAR4 tumors, low uptake in OVCAR5 tumors, and no uptake in OVCAR8 tumors. Accordingly, [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in the OVCAR3 model and improved overall survival in the OVCAR3 and OVCAR5 models in comparison to the saline control. Hematologic toxicity was transient in both models. Conclusion: PET imaging of OC xenografts showed that [89Zr]Zr-DFO-huAR9.6 delineated MUC16 expression levels, which correlated with invitro results. Additionally, we showed that [177Lu]Lu-CHX-A″-DTPA-huAR9.6 displayed strong antitumor effects in highly MUC16-expressing tumors. These findings demonstrate great potential for 89Zr- and 177Lu-labeled huAR9.6 as theranostic tools for the diagnosis and treatment of OC.

Kynurenine in IDO1high cancer cell-derived extracellular vesicles promotes angiogenesis by inducing endothelial mitophagy in ovarian cancer

BackgroundMitophagy, a prominent cellular homeostasis process, has been implicated in modulating endothelial cell function. Emerging evidence suggests that extracellular vesicles (EVs) participate in intercellular communication, which could modulate tumor angiogenesis, a hallmark of ovarian cancer (OC) progression. However, the underlying mechanisms through how EVs regulate endothelial mitophagy associated with tumor angiogenesis during OC development remain obscure.MethodsThe effect of cancer cell-derived EVs on endothelial mitophagy and its correlation with tumor angiogenesis and OC development were explored by in vitro and in vivo experiments. Multi-omics integration analysis was employed to identify potential regulatory mechanisms of cancer cell-derived EVs on endothelial mitophagy, which is involved in tumor angiogenesis associated with OC development. These insights were then further corroborated through additional experiments. An orthotopic OC mouse model was constructed to assess the antiangiogenic and therapeutic potential of the Indoleamine 2,3 dioxygenase-1 (IDO1) inhibitor.ResultsCancer cell-derived EVs promoted tumor angiogenesis via the activation of endothelial mitophagy, contributing to the growth and metastasis of OC. The aberrantly high expression of IDO1 mediated abnormal tryptophan metabolism in cancer cells and promoted the secretion of l-kynurenine (L-kyn)-enriched EVs, with associated high levels of L-kyn in EVs isolated from both the tumor tissues and patient plasma in OC. EVs derived from IDO1high ovarian cancer cells elevated nicotinamide adenine dinucleotide (NAD +) levels in endothelial cells via delivering L-kyn. Besides, IDO1high ovarian cancer cell-derived EVs upregulated sirt3 expression in endothelial cells by increasing acetylation modification. These findings are crucial for promoting endothelial mitophagy correlated with tumor angiogenesis. Notably, both endothelial mitophagy and tumor angiogenesis could be suppressed by the IDO1 inhibitor in the orthotopic OC mouse model.ConclusionsTogether, our findings unveil a mechanism of mitophagy in OC angiogenesis and indicate the clinical relevance of EV enriched L-kyn as a potential biomarker for tumorigenesis and progression. Additionally, IDO1 inhibitors might become an alternative option for OC adjuvant therapy.Graphical

Abstract IA020: Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion

Abstract Transposable elements (TEs) comprise the majority of the human genome. In most somatic tissues, TEs are silenced by DNA methylation and other epigenetic modifications. Tumors exhibit changes in DNA methylation including global loss of methylation at regions that are silenced for genome stability, like TEs, and gain of methylation at the promoter regions of tumor suppressor genes. We showed that low doses of DNMT inhibitors (DNMTis) upregulate immune signaling in solid tumor cell lines and patient samples. DNMTis activate type I interferon signaling by reducing methylation and increasing expression of endogenous retroviruses (ERVs), a type of TE, that activate dsRNA sensors. DNMTis plus HDACis (histone deacetylase inhibitors, which increase histone acetylation) increase TEs in a mouse model of ovarian cancer, activating interferon signaling and recruiting CD8+ T cells to kill cancer cells and sensitize to immune checkpoint blockade therapy. We performed a genome-wide analysis of the response to DNMTi, HDACi, and combination HDACi/DNMTi in four ovarian cancer cell lines (RNA-Seq, global methylome profiling, ATAC-Seq, and P53 chIP-Seq). The addition of HDACis to DNMTis augments the upregulation of specific ERVs and the resulting downstream interferon response in human ovarian cancer cell lines. Other TE species, including LINE1 and Alu elements, are increased by DNMTi and HDACi and may contribute to the interferon response. Alu elements, which can directly bind to the dsRNA sensor MDA5, are the most significantly demethylated TEs after DNMTi treatment. Importantly, we find considerable overlap between TEs upregulated by DNMTi in cell lines and TEs upregulated by DNMTi in ovarian cancer patients from a DNMTi clinical trial. We observed that ovarian cancer cell lines with TP53 mutations exhibited significantly fewer upregulated TEs with epigenetic therapy than wild-type TP53 cell lines. This observation was validated using isogenic cell lines; the CRISPR-edited TP53-mutant cell line had significantly higher baseline expression of TEs but upregulated fewer upon epigenetic treatment. p53 bound directly to TE DNA by chIP-Seq analysis and upregulated TEs in wild type but not mutant cell lines. In addition, our RNA-sequencing analysis indicated that A-to-I RNA editing of TEs by ADAR1 was increased after DNMTi treatment. This A-to-I editing makes TE RNA less immunogenic and inhibits the immune response to DNMTi. We find that combining ADAR1 knockdown and DNMTi treatment significantly increases type I interferon signaling, recruitment and activation of host immune cells, and survival in a murine model of ovarian cancer. These data thus give a comprehensive, genome-wide picture of TE chromatin and transcription-related changes in ovarian cancer after epigenetic treatment and implicate novel regulators (p53 and ADAR1) in response to epigenetic therapies for cancer. Citation Format: Katherine B. Chiappinelli. Targeting transposable elements with epigenetic modulators to reverse ovarian cancer immune evasion [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 IA020.

Abstract A102: ROR2/Wnt5a signaling regulates directional cell migration and early tumor cell invasion in ovarian cancer

Abstract Adhesion to and clearance of the mesothelial monolayer are key events in the early metastatic seeding of ovarian cancer. To identify mediators of ovarian cancer cell adhesion and invasion, we developed and conducted a cellular, high-throughput genetic screen using a human kinome siRNA library. Through this screen, we identified ROR2 as a driver of ovarian cancer adhesion and invasion. ROR2 is a receptor tyrosine kinase that interacts with Wnt5a ligand to activate non-canonical Wnt signaling and has been previously shown to be upregulated in ovarian cancer tissue. However, no prior study has evaluated the mechanistic role of ROR2 in ovarian cancer. Using in vitro adhesion and mesothelial clearance assays, we show that ROR2 promotes ovarian cancer cell adhesion and clearance of the mesothelial monolayer, key early steps in ovarian cancer metastasis. Further, we show that depletion of ROR2 reduces metastatic tumor burden in a syngeneic mouse model of ovarian cancer. Additionally, using a microfluidic device capable of producing stable fluid gradients, we demonstrate that ROR2 expression in ovarian cancer cells serves to drive directed cell migration of tumor cells, preferentially to areas of high Wnt5a ligand. As Wnt5a has been previously shown to be produced and upregulated in the mesothelial cells of the ovarian cancer microenvironment, these findings represent a novel role for ROR2 to promote the homing of ovarian tumor cells to the mesothelial lined metastatic niche by using Wnt5a as a chemotactic cue. These findings support the role of ROR2 as a critical factor contributing to the steps of ovarian cancer metastasis. Therapeutic targeting of the ROR2/Wnt5a signaling axis could provide a means of improving treatment for patients with advanced ovarian cancer. Citation Format: Whitney R. Grither, Breanna Baker, Vasilios A. Morikis, Katherine C. Fuh, Gregoery D Longmore. ROR2/Wnt5a signaling regulates directional cell migration and early tumor cell invasion in 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 A102.

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract B106: A minimal driver pair for a spontaneous high-grade serous ovarian cancer mouse model: Myc and p53-R270H

Abstract Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment can be understood. One challenge of the last decade has been to provide evidence for which cells are responsible for the origin of epithelial ovarian cancer. Different promoters, including Pax8, Lgr5, Ogvp1, and others have allowed for expression of transgenes in specific epithelial compartments in mice. Ovgp1 has been shown to be specific for fallopian tube epithelium (FTE), which most HGSOC is now thought to originate from. Pairing Ovgp1 with multiple homozygous deletions of powerful tumor suppressors like Brca1, Trp53, Rb1, and Nf1 yields murine disease after a year of age. However, human tumors do not contain homozygous deletions of these many tumor suppressors. Others have utilized Pax8 to knock out Trp53 and Pten to yield tumors at 3-8 months of age, but human tumors rarely have PTEN knockouts and Pax8 drives kidney and thyroid expression as well. We postulated that it is possible to strictly mimic human HGSOC genetics in a mouse model and nonetheless drive oncogenesis within the lifespan of a mouse. Here, we describe the OvTrpMyc model, Trp53em1Jdel_Tg(Ovgp1-Trp53*R270H-Myc), in which p53-R270H and Myc expression are driven by the Ovgp1 promoter. The transgene was inserted at the endogenous Trp53 locus, predicting that this design would allow for loss of the opposing wild-type chromosome 11 (syntenic to chromosome 17 in humans) to better mimic human HGSOC genetics. This model generated lethal disease at a median of 15.6 months of age. Dissected metastatic tumors were peritoneal and expressed Pax8, consistent with human disease. Immunohistochemical staining of the fallopian tube, ovary, and uterus indicated clear nuclear staining of p53 and Myc on the FTE. Notably, pockets of intraepithelial metastases expressing p53 and Myc were also found on the uterine epithelium. Cells grown ex vivo from uterus or fallopian tube dissections were analyzed for transcriptomes. RNA-seq revealed a clustering of these extracted cancer cells with human HGSOC and p53 mutant serous endometrial cancers. Loss of heterozygosity at the p53 locus was universal in these ex vivo grown cancer cells, as was extensive aneuploidy. The OvTrpMyc model requires only a single transgene for its breeding strategy, better enabling cancer detection, prevention, and treatment experiments. Taken together, the OvTrpMyc model of p53-R270H mutation and Myc expression driven by Ovgp1 is sufficient to recapitulate cancer hallmarks of HGSOC and, surprisingly, p53 mutant serous uterine cancer as well. Citation Format: Joe R Delaney. A minimal driver pair for a spontaneous high-grade serous ovarian cancer mouse model: Myc and p53-R270H [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 B106.