Rational design of immunogenic nanoparticles as a platform to reduce ovarian tumor burden in mice.

We have recently reported that multiple immune checkpoints are expressed in the TILs (tumor infiltrating T lymphocytes) and TALs (tumor associated T lymphocytes) from a murine ovarian cancer model and that blockade of one checkpoint pathway induces an upregulation of the other unblocked checkpoints. We hypothesized that resistance to single agent blockade (e.g. anti-PD1) is resulted from the compensatory upregulation of the other pathways (e.g. CTLA4 or LAG-3). To test this hypothesis in human ovarian cancer, we first examined the level of several immune checkpoints and their ligands in TILs or TALs from ovarian cancer patients. In addition to the highly expressed PD-1 and CTLA4, other checkpoint molecules such as TIM3, LAG3, VISTA, and TIGIT are also elevated in TALs as compared with control PBMC. The levels of multiple ligands for the immune receptors in the tumor samples were also analyzed. To translate the results from mice to humans, we developed several aggressive human ovarian cancer cell lines from patient derived xenografts to test the combinatorial checkpoint blockade strategies. Using a humanized mouse model of ovarian cancer we observed that adaptive transfer of autologous T cells in combination with checkpoint blockade of PD1 mildly delays ovarian tumor growth. The effect of blocking PD-1 and CTLA-4 pathways on tumor growth and the upregulation of other checkpoints are currently underway. Citation Format: Ruea-Yea Huang, Kunle Odunsi. Targeting multiple immune checkpoints and their ligands using a humanized mouse model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 606. doi:10.1158/1538-7445.AM2017-606

The five-year survival rate for ovarian cancer patients remains below 50%, underscoring the need for innovative therapies. One promising approach involves engineering T cells to specifically target proteins uniquely overexpressed in tumors, thereby controlling tumor growth without toxicity to healthy tissues. Mesothelin (MSLN) contributes to the malignant and invasive phenotype in ovarian cancer and has limited expression in healthy cells, making it a candidate immunotherapy target. Our previous results in a mouse model of ovarian cancer demonstrated that T cells engineered to express a T cell receptor (TCR) targeting MSLN (TCRMSLN) mediated therapeutic activity, delaying tumor growth and prolonging mouse survival. However, inhibitory ligands expressed in the tumor microenvironment (TME) interacted with inhibitory receptors on activated T cells, suppressing antitumor function. We hypothesized combining engineered T cells with checkpoint blockade would enhance T cell function and improve therapeutic efficacy, but administration of monospecific antibodies targeting individual inhibitory pathways had no significant impact on T cell efficacy. By contrast, the combination of PD-1, Tim-3, and Lag-3 blockade with engineered T cells significantly improved T cell function and overall animal survival relative to treatment with antibody alone or TCRMSLN with singlet or doublet antibody combinations. Single-cell RNA sequencing revealed TCRMSLN T cells treated with the triplet antibody combination increased expression of genes involved in interferon responses and metabolic function, and reduced expression of genes associated with exhaustion. These results suggest that strategies to disrupt multiple inhibitory pathways simultaneously may be necessary for improved adoptive T cell therapy efficacy in patients.

Camptothecin derivatives topotecan and irinotecan are currently the only FDA-approved topoisomerase I (Top1) inhibitors. Despite their wide usage they have many drawbacks: chemical instability, short half-life, dose-limiting bone marrow and gastrointestinal toxicity, and they are drug efflux substrates. The novel non-camptothecin Top1 inhibitors, indenoisoquinolines (LMP400, LMP776 and LMP744), have been developed to overcome these limitations. We explored whether treatment with LMPs could induce “synthetic lethality” in cells with impaired homologous recombination (HR) repair mechanism and whether it could be enhanced by combining them with PARP inhibitor, olaparib. Using the DT40 cell line, we assessed in vitro the role of HR in the cellular responses to the LMPs. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the LMP compounds than wild type cells. Moreover, combination treatments showed a significant synergy between each of the three LMPs and olaparib. To confirm the differential response of Brca1 wild type or Brca1 deficient cells to LMP treatment we assessed compound potency in primary ovarian cancer cell lines derived from a genetically engineered mouse (GEM) model for serous epithelial ovarian cancer (SEOC). As in DT40 cells, we observed that murine Brca1 deficient cells were more sensitive to all 3 LMP compounds than Brca1 wild type cells. Our results suggest that Brca1 deficiency renders cells more sensitive to treatment with LMPs or combination of LMPs and olaparib, potentially directing selection of patients with impaired HR repair mechanism in the phase 2 clinical trials that are in preparation. To confirm this observation in vivo, we are performing efficacy studies using LMP compounds alone or in combination with olaparib in orthotopic allograft mouse models for ovarian cancer derived from the GEM model. In Brca1 deficient tumors, treatment with each of the 3 LMP compounds led to significant increase of survival compared to vehicle treated group. LMP400 had the greatest beneficial impact on survival and tumor regression. To confirm synergy between LMPs and olaparib we selected LMP400 to represent the LMPs and repeated the efficacy study using Brca1 deficient tumor model. We observed that suboptimal doses of LMP400 or olaparib used as single treatments resulted in some increase in survival, however, when combined they significantly increased survival due to tumor regression. Our data demonstrate that the LMPs synergize with olaparib and provide a rationale for personalized treatment and Phase 2 clinical trials with the indenoisoquinolines in combination with PARP inhibitors in HR-deficient ovarian cancers. Citation Format: Ludmila Szabova, Melanie Gordon, Laetitia Matzi, Yves Pommier, Zoe Weaver Ohler. EFFICACY OF NOVEL TOPOISOMERASE 1 INHIBITORS AND THEIR SYNERGY WITH PARP INHIBITORS IN A MURINE MODEL FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-110.

Objectives: Given the heterogeneity of ovarian cancer, it is imperative to identify subtype-directed treatments. The novel antibody NEO-201 targets a specific tumor-associated antigen (TAA) expressed on some ovarian and uterine malignancies, providing a tumor-directed approach. Here, we aimed to identify the cancer subtypes which express the NEO-201 target and demonstrate its cytotoxic effects in vitro and in mouse models of ovarian cancer. Methods: NEO-201 is a genetically humanized novel monoclonal antibody developed through vaccines with TAAs. This antibody targets malignant tissues that express tumor-specific epitopes in membrane-anchored protein CEACAM-6. We performed immunohistochemistry (IHC) on tissue microarrays from formalin-fixed paraffin-embedded subtyped ovarian and uterine cancers to estimate the incidence of cancers expressing the NEO-201 target. Ovarian and uterine cell line pellet arrays were stained by IHC to identify in vitro models. We examined the cytotoxicity of NEO-201 in two high- and three low-expressing cell lines using Calcein AM cell viability assays alone and with purified natural killer (NK) cells with and without IL-2 stimulation. Studies of NEO-201 in ovarian cancer mouse models are ongoing. Results: IHC of NEO-201 in tissue microarrays demonstrated 51% and 12% reactivity in uterine and ovarian samples, respectively. Similar expression patterns were identified in representative cell lines by IHC and Western blot. NEO-201 killed cell lines expressing its target in the range of 0.5-20μg/mL. Over 3 weeks, NEO-201 treatment of tumor-bearing mice demonstrated control of tumor growth with 3 doses of 250μg NEO-201 and tumor regression with 100μg in combination with IL2-stimulated PBMC. Studies are ongoing to further investigate NEO-201 cytotoxicity in ovarian cancer mouse models as well as identify the mechanism of tumor cell death and the target epitope of NEO-201. Conclusions: NEO-201, a novel monoclonal antibody, specifically targets epithelial malignancies including ovarian and uterine cancer. This potentially therapeutic antibody demonstrates tumor-specific cytotoxicity in cancer cell lines expressing its target in vitro and in xenografts with IL2-stimulated PBMC, suggesting both antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity mechanisms are involved in tumor inhibition and regression. These studies lay the groundwork for future examination of TAA-directed therapy for ovarian or uterine malignancies. Citation Format: Monica K. Neuman, Lidia Hernandez, Xue­-Ping Wang, Olga Saric, Alex Dubeykovskiy, Philip Arlen, Christina M. Annunziata. Identification of target and cytotoxicity of novel monoclonal antibody NEO-201 in ovarian and uterine cancer subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1496.

<div>Abstract<p>Although ovarian cancer has a low incidence rate, it remains the most deadly gynecologic malignancy. Previous work has demonstrated that the DNMTi 5-Azacytidine (5AZA-C) activates type I interferon signaling to increase IFNγ<sup>+</sup> T cells and natural killer (NK) cells and reduce the percentage of macrophages in the tumor microenvironment. To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, may further decrease immunosuppressive cell populations improving outcome. We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that <i>in vivo</i>, 5AZA-C and DFMO, either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ<sup>+</sup>) CD4<sup>+</sup> T cells, CD8<sup>+</sup> T cells, and NK cells. The combination therapy had a striking increase in survival when compared with single-agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of 5AZA-C + DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival.</p>Significance:<p>Combined epigenetic and polyamine-reducing therapy stimulates M1 macrophage polarization in the tumor microenvironment of an ovarian cancer mouse model, resulting in decreased tumor burden and prolonged survival.</p></div>

Ovarian cancer (OC) is the fifth leading cause of cancer-related deaths among women in the U.S. Studies show that cancer patients have elevated stress hormones due to various stressors, which can disrupt cancer progression and immune response in the tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) are immature, immunosuppressive cells that play a key role in the TME and are linked to poor prognoses in cancer patients by disrupting several pathways, including the Notch signaling pathway (NSP). In various cancers, NSP dysregulation contributes to cancer progression. Increasing evidence suggests that chronic stress enhances MDSC infiltration, promoting immunosuppression in the TME. However, the role of the NSP in stress-mediated MDSC infiltration and OC progression remains poorly understood. This study explores how chronic stress influences NSP and MDSC biology in the OC TME and its role in disease progression. We hypothesize that chronic stress increases MDSC infiltration in the OC TME, enhancing NSP activity in OC cells. To test this, we inoculated female C57BL/6 mice with ID8Luc or IG10Luc OC cells and subjected them to daily restraint stress. Mice were sacrificed weekly for tumor and bone marrow collection, followed by immunofluorescence and flow cytometry analyses to characterize MDSCs and immune cells based on surface markers. We also isolated bone marrow from naïve C57BL/6 mice to differentiate myeloid cell precursors into MDSCs. These cells were then exposed to stress hormones (norepinephrine (NE), epinephrine (EPI), and corticosterone (CC)) for 72 hours to analyze MDSC populations. Our results showed that chronic restraint stress increased MDSC infiltration and enriched polymorphonuclear-MDSCs (PMN-MDSCs) in the TME and bone marrow of both OC mouse models. Also, results showed that chronic restraint stress decreased the CD8+ and CD4+ T cells in the TME of both OC mouse models. Ex vivo experiments indicated increased PMN-MDSC enrichment alongside a depletion of monocytic-MDSCs in groups treated with stress hormones. To further examine how stress hormones impact NSP dysregulation, we treated OC cells (ID8 and IG10) in vitro with NE, EPI, or CC. Our findings revealed elevated levels of Notch1, Jagged2, and Hes1 mRNA and protein in the stress hormone groups mediated by GSK-3β phosphorylation. Additionally, we analyzed NSP member expression in tumors from the in vivo experiments. The results indicated higher levels of Notch1, Jagged2, NICD, and HES1 in tumors from mice subjected to restraint stress. In addition, Milliplex analysis of cytocytokine/chemokine profiles from ID8Luc or IG10Luc OC-bearing mice serum samples subjected to chronic restraint stress showed higher levels of IL-1β, IL-6, GM-CSF, G-CSF, MCP-1, RANTES, TNF-α, and low levels of IFN-γ associated with MDSC biology and function. These data suggest that chronic stress may induce MDSC infiltration and activity, possibly mediated by NSP activation, thereby contributing to an immunosuppressive TME and OC progression. Citation Format: Yadiel A. Rivera-Lopez, Luinet L. Meléndez-Rodríguez, Alanis P. Torres-Rosado, Jaydiel A. Casiano-Martínez, Melanie Ortiz-León, Luis M. Rivera-Pérez, Orlando I. Torres-Rodríguez, Sofia M. Hernández-Carrasquillo, Raian Imad-Hamad, Zuliann S. Galarza-Ruiz, Paulo C. Rodríguez, Guillermo N. Armaiz-Pena. Chronic stress impairs anti-tumor immunity in ovarian cancer via MDSCs and notch pathway activation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr A014.

We previously discovered that BM-derived Mesenchymal stromal cells (BM-MSCs) are migrating to the stroma of numerous cancers and their metastasis, forming TAFs and secrete proteins intra-tumorally. MSCs have not been used in cancer therapy because of their immune suppressive properties, limited replicative capacity, source dependent quality and controversial roles in cancer biology. Here, we report the characterization of novel MSCs (iMSCs), which were generated from adult skin fibroblast-derived induced pluripotent stem cells (iPSC). These iPSCs were generated via a unique, transient mRNA transfection technique and subsequently differentiated into iMSCs. Notably, iMSCs displayed contact inhibition and significantly enhanced proliferative capacity under both normoxic and hypoxic conditions, outperforming bone marrow-derived MSCs (BM-MSCs) while fully retaining trilineage differentiation potential. Comprehensive molecular profiling, including RNA sequencing, single-cell mass cytometry (CyTOF), and Luminex assays, revealed strong phenotypic and functional resemblance between iMSCs and BM-MSCs. Crucially, no sarcoma formation was detected in NSGS mice following intraperitoneal, subcutaneous, or intravenous administration of iMSCs, underscoring their safety profile. We further engineered a DNA cassette into these cells to enable IL-7 and IL-15 cytokine production, expressed as either individual molecules (P2A) or a single fused molecule (FUS). Both P2A and FUS cells demonstrated the capacity to drive T cell proliferation autonomously in co-culture. Cytokine secretion from these modified iMSCs enhanced tumor cell death in a triple co-culture system comprising iMSCs, the ovarian cancer cell line ID8, and human PBMCs. In a syngeneic mouse model of ovarian cancer (ID8 in C57BL/6 mice), administration of P2A or FUS MSCs resulted in reduced tumor growth and extended survival. Immunohistochemical and flow cytometric analyses revealed massive infiltration of T cells, macrophages, NK cells, and other immune cells into the tumor microenvironment (TME) in both FUS or P2A groups, but not in control, unmodified MSC, or PBS injected animals. Moreover, the TME in P2A- and FUS-treated mice showed enrichment in M1-type macrophages, with no detection of any of the T cell exhaustion markers tested, in contrast to the control groups (PBS or GFP-iMSC). In conclusion, our findings indicate that IL7-IL15-secreting iMSCs migrate into solid tumors, induce massive immune cell infiltration into the TME and enhance antitumor immunity in a syngeneic mouse model of cancer. These cytokine-producing iMSCs represent a promising therapeutic strategy for modulating the immune response against tumors and advancing anticancer immunotherapy by converting “cold” into “hot” tumor microenvironments. Citation Format: Sandeep Singh, Andrea Bedoy, Dipmoy Nath, Kyung Hee Chang, Lauren B. Ostermann, Ivo Veletic, Christopher D. Pacheco, Po Yee Mak, Edward Ayoub, Muharrem Muftuoglu, Mahesh Basyal, Taeyun Kim, Raven Dance Hinkel, Kyle M. Garland, Matthew Angel, Sanjeev Luther, Christopher B. Rohde, Michael Andreeff. iPS-derived MSC secreting IL7 and IL15 exert immune mediated anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3473.

BackgroundGenetically engineered mice are ideal models to advance our understanding the tumorigenesis of ovarian cancer. Our original objective was to establish an ovarian cancer model induced by Kras activation and Pten deletion. However, proficiently establishing the model remains a technical problem, which limits its application.MethodsWe established the Kras activation/Pten deletion-induced mouse model of ovarian cancer by injecting Cre recombinase-expressing adenovirus in the ovarian bursa. PCR analysis, Western blotting, and immunohistochemistry staining were performed to verify the alteration of conditional genes. We detected expression of canonical molecular markers in order to examine the origin of the tumors.ResultsSubcutaneous lumps developed accidentally in mice with ovarian cancer, as early as 2 weeks post in vivo genetic manipulation, far before the destructive growth of ovarian cancer. PCR analysis confirmed the efficient Cre-mediated recombination of Kras and Pten in tumor tissues, which are consistent with the activation of the MAPK and PI3K/Akt/mTOR pathways. Histomorphological and histological analysis showed that the lumps were actually rhabdomyosarcoma (RMS). We confirmed that the leakage of adenovirus transformed healthy adjacent tissues into RMS.ConclusionsAvoiding accidental exposure of non-target tissues to adenovirus is crucial to successfully establish the ovarian cancer mouse model. Moreover, non-specific genetic manipulations can induce the development of RMS.

Studies have shown enhanced survival of ovarian cancer patients in which the tumors are infiltrated with tumor infiltrating lymphocytes and natural killer cells showing the importance of immune surveillance and recognition in ovarian cancer. Therefore, in this study, we tested cellular immunotherapy and varying combinations of cytokines (IL-2 and/or pegylated-IFNα-2b) in a xenograft mouse model of ovarian cancer. SKOV3-AF2 ovarian cancer cells were injected intra-peritoneally (IP) into athymic nude mice. On day 7 post-tumor cell injection, mice were injected IP with peripheral blood mononuclear cells (PBMC; 5×10(6) PBMC) and cytokine combinations [IL-2±pegylated-IFNα-2b (IFN)]. Cytokine injections were continued weekly for IFN (12,000 U/injection) and thrice weekly for IL-2 (4000 U/injection). Mice were euthanized when they became moribund due to tumor burden at which time tumor and ascitic fluid were measured and collected. Treatment efficacy was measured by improved survival at 8 weeks and overall survival by Kaplan-Meier analysis. We observed that the mice tolerated all treatment combinations without significant weight loss or other apparent illness. Mice receiving PBMC plus IL-2 showed improved median survival (7.3weeks) compared to mice with no treatment (4.2weeks), IL-2 (3.5weeks), PBMC (4.0weeks), or PBMC plus IL-2 and IFN (4.3weeks), although PBMC plus IL-2 was not statistically different than PBMC plus IFN (5.5weeks, p>0.05). We demonstrate that cytokine-stimulated cellular immune therapy with PBMC and IL-2 was well tolerated and resulted in survival advantage compared to untreated controls and other cytokine combinations in the nude-mouse model.

The modulation of adaptive immune response by oncogene K-ras; mouse model of ovarian cancer

Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-alpha is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-alpha was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-alpha-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25- cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-alpha signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-kappaB system, TGF-beta1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-alpha in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.

Purpose: Despite high initial response rates with cytoreductive surgery, conventional chemotherapy and the incorporation of biologic agents, ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed.Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry, respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1, 4, and 8. Human PBMCs were injected IP simultaneously as effector cells.Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon, pancreatic, and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian, colon, and pancreatic cancer cell lines. From these screening, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition, NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly, NEO-201 prolonged survival of tumor-bearing mice.Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors, but not in normal tissues, allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center.

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance

Novel therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. In addition, therapies that target unique vulnerabilities in the tumor microenvironment (TME) of EOC have largely been unrealized. One strategy to achieve selective drug delivery for EOC therapy involves use of targeted antifolates via their uptake by folate receptor (FR) proteins, resulting in inhibition of essential one-carbon (C1) metabolic pathways. FRα is highly expressed in EOCs, along with the proton-coupled folate transporter (PCFT); FRβ is expressed on activated macrophages, a major infiltrating immune population in EOC. Thus, there is great potential for targeting both the tumor and the TME with agents delivered via selective transport by FRs and PCFT. In this report, we investigated the therapeutic potential of a novel cytosolic C1 6-substituted pyrrolo[2,3-d]pyrimidine inhibitor AGF94, with selectivity for uptake by FRs and PCFT and inhibition of de novo purine nucleotide biosynthesis, against a syngeneic model of ovarian cancer (BR-Luc) which recapitulates high-grade serous ovarian cancer in patients. In vitro activity of AGF94 was extended in vivo against orthotopic BR-Luc tumors. With late-stage subcutaneous BR-Luc xenografts, AGF94 treatment resulted in substantial anti-tumor efficacy, accompanied by significantly decreased M2-like FRβ-expressing macrophages and increased CD3+ T cells, whereas CD4+ and CD8+ T cells were unaffected. Our studies demonstrate potent anti-tumor efficacy of AGF94 in the therapy of EOC in the context of an intact immune system, and provide a framework for targeting the immunosuppressive TME as an essential component of therapy.

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.