Ovarian Cancer Xenograft Model Research Articles

Abstract 1772: Development of a novel L1CAM-targeted CAR-T, CX804, and its therapeutic efficacy in ovarian and gastric cancer

Abstract L1 cell adhesion molecule (L1CAM) is involved in cancer development and aberrantly expressed in various tumor. It is also associated with aggressive progression, metastasis, and poor prognosis. Public database, a Kaplan-Meier plotter (http://www.kmplot.com) shows that L1CAM high-expression significantly reduced survival of ovarian and gastric cancer patients. We developed a novel chimeric antigen receptor (CAR)-T cell targeted to L1CAM. A novel scFV candidate (Clone H8) to L1CAM was obtained by bio-panning of phage displaying human synthetic scFv fragments to human and mouse L1CAM proteins. H8 scFV was linked to IgD-IgG1 hinge, CD28 transmembrane domain, CD28 and ICOS co-stimulation domain, and CD3ζ domain, resulting in CX804 CAR-T. The efficacy of L1CAM CAR-T cells, CX804 showed correlation with the level of L1CAM in human ovarian and gastric cancer cells. CX804 exerted cytotoxicity toward 293T cells expressing human or mouse L1CAM protein. CX804 also reacted with L1CAM-positive ovarian SKOV3 cancer cell and gastric MKN-28 and AGS cancer cells and secreted T-cell activation cytokines, IFN-γ and TNF-α, in response to these cells. However, CX804 rarely lysed L1CAM-negative normal 293 cells and SNU-719 gastric cancer cells. Furthermore, L1CAM CAR-T cells dramatically lysed high L1CAM expressing patient derived cell (PDC) of gastric cancer compared with low L1CAM expressing PDC cell. Xenograft model of ovarian cancer was developed by intraperitoneal (IP) injecting SKOV3-Fluc cells into NOD/SCID or NSG mice. CAR-T cells were administrated via intravenous (IV) or IP route on 5 days after tumor inoculation. Administration of CX804 significantly decreased tumor burden and increased survival in mouse model of ovarian cancer. Route of administration significantly affected bio-distribution and antitumor effect of CX804 where antitumor activity of CX804 was only observed when CX804 were given IP, but not the IV route. Taken together, our results suggest that CX804 exerts potent and specific effect against L1CAM positive cancer cells in vitro and in vivo. Furthermore, we propose that newly established CAR-T cells targeting L1CAM facilitate therapy against L1CAM-positive ovarian and gastric cancer patients Citation Format: Xiumei Che, Un-Jung Yun, Seokwon Lee, Youngyoub Kim, Junshub Lee, Jina Chae, Jehee Suh, Eun-Ji Nam, Gun Min Kim, Hyoyoung Kim, Minkyu Jung. Development of a novel L1CAM-targeted CAR-T, CX804, and its therapeutic efficacy in ovarian and gastric cancer [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 1772.

Abstract 3189: Pre-clinical evaluation of novel folate receptor 1 directed CAR T cells for high grade serous ovarian cancer

Abstract Purpose: Treatment of ovarian cancer remains challenging. A high unmet clinical need for targeted, efficient, and persisting drugs still is a major issue for clinicians and patients diagnosed with ovarian cancer, particularly platinum-resistant and-refractory, recurrent disease. Genetically modified T cells expressing chimeric antigen receptors (CARs) have shown efficient, as well as persisting anti-tumor functionality in hematological malignancies. However, solid cancers have not yet been as efficiently treated with CAR T cells, potentially due to the lack of appropriate targets. Therefore, we thoroughly characterized FOLR1 as CAR T cell target for ovarian cancer, designed novel CAR T cells, and assessed these drug candidates preclinically in vitro and in vivo. Experimental Design: We performed ultra-high content imaging on high-grade serous epithelial ovarian cancer as well as healthy tissue samples to profile the expression of the tumor-associated antigen FOLR1. Subsequently, we designed a series of CAR T cell candidates based on different monoclonal antibodies targeting FOLR1, in order to leverage on clinical experience with these binders, particularly safety aspects. The CAR designs differed in the VL-VH orientation and the spacer domain of the CAR construct. The preclinical assessment of various FOLR1-directed CAR T cell constructs was evaluated against various ovarian cancer cell lines in vitro and in vivo. Results: In this study we show high and consistent expression of the tumor-associated antigen FOLR1 on primary ovarian cancer samples, namely high-grade serous ovarian cancer. Moreover, FOLR1 expression is low, restricted, and polarized in healthy tissues. We generated diverse FOLR1-specific CAR T cells and characterized their efficacy and specificity in vitro as well as in an ovarian cancer xenograft model. CAR T cells rapidly and efficiently eradicate xenograft tumors. This anti-tumor response is accompanied by CAR T cell expansion, FOLR1-dependent cytokine secretion, and infiltration of CAR T cells into the tumor. Conclusions: These findings demonstrate the feasibility of a preclinical CAR T cell approach targeting FOLR1 as treatment option for ovarian cancer and potentially other FOLR1-expressing tumors. Citation Format: Christoph Herbel, Julie Daigre, Manuel Martinez-Osuna, Maria Bethke, Larissa Steiner, Janina Brauner, Jens Kopatz, Paurush Praveen, Dominik Eckardt, Andreas Bosio. Pre-clinical evaluation of novel folate receptor 1 directed CAR T cells for high grade serous ovarian cancer [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 3189.

Abstract 2904: CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice

Abstract While chimeric antigen receptor (CAR)-engineered immune cell therapies have been at the forefront of cancer immunotherapy for hematological malignancies, patients with solid tumors have yet to benefit from such therapies. Engineered, off-the-shelf, allogeneic natural killer (NK) cells are particularly attractive as cell therapies for solid tumors given their clinical safety, efficacy, and multimodal recognition of tumor cells. We describe here the pre-clinical pharmacokinetics, efficacy, biodistribution and safety of CAT-179, a novel allogeneic, cryopreserved CAR-NK cell therapy, in naïve animals as well as multiple xenograft models of HER2-amplified ovarian and gastric cancer. CAT-179 cells are engineered to express an optimized HER2-directed CAR to effectively target tumor cells, a transforming growth factor b (TGFβ) dominant negative receptor (DNR) to protect against TGFβ-mediated immunosuppression, and interleukin-15 (IL-15) to enhance NK cell persistence. A single intravenous (IV) dose of CAT-179 resulted in IL-15-dependent (p<0.0001) expansion and persistence of CAT-179 cells for at least 140 days in NOD-scid IL2Rgnull (NSG) mice, peaking at approximately 15,000 cells/µL of blood (by day 60). Persisting CAT-179 NK cell levels at day 60 were functionally active and significantly reduced tumor burden when challenged intraperitoneally (IP) with HER2+ SKOV3-luc tumor cells. No significant changes in body weight or condition were observed during this study. The therapeutic anti-tumor activity of CAT-179 against established tumors was assayed using two different xenograft models. In the first model, NSG mice were given an IP dose of 1 million SKOV3-luc tumor cells (derived from ovarian cancer) followed by either 4 million CAT-179 or control NK cells on days 4, 11 and 18. CAT-179 dosed animals showed a rapid and sustained 95% decline in tumor burden (p<0.0001) and a significant extension in survival relative to animals dosed with control NK cells (p<0.0001). In the second model, 1 million HER2+ N87 cancer cells (derived from gastric cancer) were implanted subcutaneously into the right flank of NSG mice. When tumors reached 70mm3, a single IV dose of 2M CAT-179 or control NK cells was administered. CAT-179 dosed animals showed a 96% durable tumor regression and significant survival benefit relative to animals dosed with control NK cells (p<0.0001). Efficacy strongly correlated with the circulating levels of CAT-179, which significantly infiltrated the tumor xenograft. Our pre-clinical results demonstrate the potential of CAT-179 as a novel, durable, and off-the-shelf cell therapy to overcome the challenges associated with solid tumors and provide quantitative insights into pharmacokinetics, pharmacodynamics and anti-tumor activity of engineered NK cells expressing CAR, TGFb DNR and IL-15. Citation Format: Bashar Hamza, Angela Nunez, Marilyn Marques, Alexia Barandiaran, Henry Moreno, Finola Moore, Meghan Walsh, Eugene Choi, Kisha Pradhan, Krista Daniel, Jennifer Johnson, Charlotte Franco, Andres Alvarez, Karl Malakian, Keith H. Wong, Joseph Gold, Vipin Suri, Dominic Picarella. CAT-179, an allogeneic NK cell product expressing HER2-CAR, IL-15 and TGFβ dominant negative receptor, durably regresses HER2-expressing xenograft tumors in mice [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 2904.

Abstract 5731: Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK

Abstract Many of the most effective anti-cancer therapies induce apoptosis in cancer cells by damaging DNA. Even when effective at eradicating primary cancers, DNA-damaging anti-cancer agents cause mutations that can lead to the development of secondary cancers. Furthermore, DNA damage responses are dependent on wild-type p53, which is mutated in over 50% of cancers and causes resistance to therapies. Fortunately, many p53 mutant cancers continue to express high levels of the pro-apoptotic, pore-forming proteins BAX and BAK and are consequently “primed for apoptosis.” Primed cells are highly sensitive to pro-apoptotic signals and this vulnerability can be exploited using inhibitors of pro-survival BCL-2 family proteins such as the BCL-2 inhibitor ABT-199, which has been successful as therapy for chronic lymphocytic and acute myeloid leukemias (AML). Furthermore, most irreplaceable cells within healthy tissues are apoptosis resistant and express low levels of BAX and BAK. We therefore hypothesized that direct activators of BAX or BAK could be effective single-agent therapies for primed cancers and chemo-/radio-sensitizers for unprimed cancers. Using WT versus BAX -/- BAK -/- HeLa cells, we performed a high-throughput screen of nearly 100,000 compounds to identify small molecules that could directly activate BAX or BAK. 196 compounds were identified as hits and multiple orthogonal validation studies identified Apoptosis Inducing Agent 1 (AIA1) as the most specific and potent putative activator of BAX and BAK. Further validation studies demonstrated that AIA1 directly induces cytochrome c release from mitochondria in a BAX/BAK dependent manner and strongly enhances BIM-mediated activation of BAX and BAK and permeabilization of liposomes. Single-agent AIA treatment triggers apoptotic cell death in a broad panel of cancer cell lines and also sensitizes cancer cells to chemotherapeutic agents and especially BH3 mimetics targeting pro-survival BCL-2 family proteins. Importantly, AIA1 sensitizes ovarian cancer cells to BCL-XL inhibitors and AML cells to BCL-2 inhibitors regardless of p53 status. In vivo, AIA1 suppresses tumor growth and prolongs overall survival in both ovarian cancer and AML xenograft models without causing weight loss, thrombocytopenia or leukopenia. Notably, unprimed cancer cells that don’t immediately undergo apoptosis in response to single-agent AIA1 treatment upregulate expression of BAX and BAK, pro-survival proteins (BCL-XL, BCL-2, MCL-1) and pro-apoptotic proteins (BIM, BID, PUMA, Noxa) to produce a more highly primed apoptosis pathway and setting the stage for increased sensitivity to BH3 mimetics. Based on these findings, AIA1 may exploit and induce apoptotic vulnerabilities in cancers in a p53-independent manner and represent a safer strategy to target primed cancer cells while eliminating the potential for secondary malignancies and p53-mediated resistance. Citation Format: Xingping Qin, Cameron Fraser, Adam Presser, Johan KE Spetz, Stacey J. Yu, Gary A. Bradshaw, Marian Kalocsay, Bo R. Rueda, Tudor Moldoveanu, Kristopher A. Sarosiek. Apoptosis Inducing Agent 1 enhances cancer therapy-induced apoptosis by direct interaction with BAX and BAK. [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 5731.

Abstract 654: Ovarian cancer-cell glucocorticoid receptor activity modulates cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment

Abstract Elevated levels of immunosuppressive myeloid-derived suppressor cells (MDSCs) circulate in the peripheral blood or infiltrate the tumors as well as ascites in ovarian cancer patients, however the cause for this observed phenotype is unknown. MDSCs contribute to tumor immune tolerance primarily via inhibition of cytotoxic T-cells or by activation of immunosuppressive T-regulatory cells. We previously showed that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. Additionally, using primary tumor data retrieved from the ovarian cancer TCGA dataset, we show high tumor GR expression is associated with significantly higher expression of Foxp3, a marker of immunosuppressive T-regulatory cells, as well as CD33, a marker of immunosuppressive myeloid cells, further suggesting an immunosuppressive phenotype in ovarian cancer patients. We thus hypothesize that tumor-intrinsic GR activation in ovarian cancer is associated with increased MDSC infiltration into the tumor microenvironment in part via modulating tumor cell cytokine secretion. To determine whether tumor-cell GR activation modulates the cytokine secretome, we previously showed that GR activation in ovarian cancer cells upregulated secretion of immunomodulatory factors including G-CSF, TGFb and CXCL5. Moreover, co-treatment of cells with a selective GR modulator (SGRM), reverses this secretome effect. Considering that elevated levels of these cytokines are necessary for the production and recruitment of MDSCs, these data highlight the relevance of this evident glucocorticoid receptor-cytokine circuit in ovarian cancer tumor cells. Furthermore, we show that this tumor-cell cytokine secretome has the capacity to promote differentiation of MDSCs from precursor myeloid cells in healthy PBMCs. Importantly, measuring expression of activation markers such as Arginase-1 and NOS2 illustrated that these MDSCs are in their functional state. To our knowledge this is the first evidence that a nuclear receptor in ovarian cancer tumor cells is actively driving MDSC differentiation. Finally, employing ovarian cancer xenograft models as well as a syngeneic ID8 tumor model, treatment with SGRMs demonstrated reduced levels of circulating MDSCs in the peripheral blood as well as tumor infiltrated MDSCs. Using neutralizing antibodies for cytokines, we will now identify key mechanisms, specifically, identify which targetable immunomodulatory factors are contributing to immune suppression. Based on the insight provided by these data, we propose to determine whether targeting ovarian tumor cell-intrinsic GR activation and resulting MDSC infiltration contributes to improved anti-tumor immune response. Citation Format: Manisha Taya, Eleanore LeBlanc, Lynda Bennett, Suzanne D. Conzen. Ovarian cancer-cell glucocorticoid receptor activity modulates cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment [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 654.

Abstract 2499: Complement activation at the interface between cancer cells and adipocytes

Abstract Introduction: Ovarian cancer (OC) preferred site of metastasis is the fat-rich omentum. The interaction between cancer cells and adipocytes induces genotypical and phenotypical changes favoring increased invasiveness and metastatic potential of OC cells. In this study we stablish a novel in-vitro direct co-culture model by using immortalized human visceral pre-adipocytes and OC cells to understand the genotypical and phenotypical changes that adipocytes coculture generates over OC cells. Materials and Methods: OVCAR5 or OVCAR8 OC cells expressing either GFP or RFP were co-cultured with matured primary immortalized human visceral pre-adipocyte (VNPAD). The cells were seeded together in direct monolayer co-culture. After co-culture, OC cells were sorted by FACS and used to evaluate cell proliferation, resistance to cisplatin and transcriptomic changes. Results: Direct co-culture (3 days) of adipocytes and OC cells increased proliferation (p < 0.001), invasiveness (p < 0.005), and induced epithelial to mesenchymal transition (EMT) of OC cells relative to cells cultured alone. Co-culture with adipocytes also increased resistance to platinum (cisplatin IC50 from 3.90 µM to 7.07 µM for OVCAR5, and from 7.54 µM to 9.35 µM for OVCAR8 cells). RNAseq of co-cultured OVCAR5 cells detected 7783 differentially expressed genes (FDR<0.05, fold change >2). The most affected pathways included the PI3K/AKT pathway, inflammatory response, complement activation, and lipid localization. Activation of the complement (C) system in cells co-cultured with adipocytes was associated with upregulation of both C3 and C5 which was confirmed by quantitative RT-PCR, western blot and ELISA. Treatment of primary OC cells with media conditioned by adipocytes increased the expression of C3 and C5 (P<0.05). Similarly, treatment of OVCAR5 cells with a lipid mixture increased expression of C3 and C5 (mRNA and protein). The co-culture of OC cells with adipocytes and the treatment with lipids activated the PI3K pathway and the integrated stress response (ISR) pathway mediated by ATF4. shRNA knockdown of C3 and a chemical inhibitor for the C3 and C5 receptor (sb290157) reverted the increased proliferative phenotype and activation of PI3K in cocultured OC cells. Stable knockdown of C3 with shRNA in OVCAR5 cells reduced tumor volume and tumor weight in an intraperitoneal OC xenograft model (p<0.05). IHC of paired primary tumors and metastatic implants in the omentum showed increased C3 expression adjacent to fat (p<0.05). Conclusion: Co-culture of adipocytes and cancer cells drives changes in proliferation and cisplatin responsiveness in OC cells. These changes are induced by transfer of lipids from adipocytes to cancer cells and subsequent activation of the complement proteins C3 and C5 through the ISR pathway and ATF4. Citation Format: Andres Felipe Valdivia, Hao Huang, Horacio Cardenas, Aloysius Klingelhutz, Daniela Matei. Complement activation at the interface between cancer cells and adipocytes [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 2499.

Depleting the 19S proteasome regulatory PSMD1 subunit as a cancer therapy strategy.

Proteasome inhibitors are in use in treating certain types of cancers. These drugs inhibit the catalytic activity of the 20S proteasome, shared by all the different proteasome complexes. Inhibitors of the 26S-associated deubiquitinating activity explicitly inhibit the 26S proteasomal degradation of ubiquitinylated substrates. We have previously reported an alternative strategy that is based on reducing the 26S/20S ratio by depleting PSMD1, 6, and 11, the subunits of the 19S proteasome regulatory complex. Given the addiction of the many cancer types to a high 26S/20S ratio, the depletion strategy is highly effective in killing many aggressive cancer cell lines but not mouse and human immortalized and normal cells. We used two aggressive cell lines, MDA-MB-231, a triple-negative breast tumor cell line, and OVCAR8, a high-grade ovary adenocarcinoma. Cell culture, mouse MDA-MB-231, OVCAR8 xenografts, and patient-derived ovarian cancer xenograft (PDX) models were transduced with lentivectors expressing PSMD1 shRNA. Tumor size was measured to follow treatment efficacy. Using different experimental strategies of expressing shRNA, we found that PSMD1 depletion, either by expressing PSMD1 shRNA in an inducible manner or in a constitutive manner, robustly inhibited MDA-MB-231, and OVCAR8 xenograft tumor growth. Furthermore, the PSMD1 depletion strategy compromised the growth of the PDX of primary ovarian cancer. Our results suggest that reducing the 26S/20S ratio might be a valuable strategy for treating drug-resistant aggressive types of cancers.

Engineering Bifunctional Calcium Alendronate Gene‐Delivery Nanoneedle for Synergistic Chemo/Immuno‐Therapy Against HER2 Positive Ovarian Cancer

Ovarian cancer is the most lethal gynecological malignancy. Most patients are diagnosed at an advanced stage with widespread peritoneal dissemination and ascites. Bispecific T-cell engagers (BiTEs) have demonstrated impressive antitumor efficacy in hematological malignancies, but the clinical potency is limited by their short half-life, inconvenient continuous intravenous infusion, and severe toxicity at relevant therapeutic levels in solid tumors. To address these critical issues, the design and engineering of alendronate calcium (CaALN) based gene-delivery system is reported to express therapeutic level of BiTE (HER2×CD3) for efficient ovarian cancer immunotherapy. Controllable construction of CaALN nanosphere and nanoneedle is achieved by the simple and green coordination reactions that the distinct nanoneedle-like alendronate calcium (CaALN-N) with a high aspect ratio enabled efficient gene delivery to the peritoneum without system in vivo toxicity. Especially, CaALN-N induced apoptosis of SKOV3-luc cell via down-regulation of HER2 signaling pathway and synergized with HER2×CD3 to generate high antitumor response. In vivo administration of CaALN-N/minicircle DNA encoding HER2×CD3 (MC-HER2×CD3) produces sustained therapeutic levels of BiTE and suppresses tumor growth in a human ovarian cancer xenograft model. Collectively, the engineered alendronate calcium nanoneedle represents a bifunctional gene delivery platform for the efficient and synergistic treatment of ovarian cancer.

Abstract P6-10-01: The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4

Abstract Immunotherapy has dramatically impacted cancer therapy, but it has been challenging to apply immunotherapy to estrogen receptor (ER) positive breast cancer and many other solid tumors that do not display neoantigens. One way to target these tumors is to induce necrosis, which robustly activates immune cells, inducing immunogenic cell death. However, anticancer therapy-induced necrosis was primarily characterized by morphological changes, and the molecular drivers of necrosis were largely obscure. To probe necrosis, we used our necrosis inducing anticancer agents, the small molecules BHPI and second-generation ErSO, which kill cancer cells by hyperactivating the anticipatory unfolded protein response (a-UPR). In orthotopic mouse xenografts, ErSO induces complete regression without recurrence of large, therapy-resistant primary ER positive breast tumors, of most lung, bone, and liver metastases, near complete regression of challenging breast cancer brain metastases and robust responses in PDX and patient derived organoids (PDOs) models. ErSO also induces complete or near complete regression in mouse xenograft models of ER positive ovarian and endometrial cancer. Using genome wide CRISPR-Cas9 screens with negative selection against our necrosis-inducing a-UPR hyperactivators, BHPI and ErSO, we identified the calcium-activated, ATP-inhibited, plasma membrane sodium channel, Transient Receptor Potential Melastatin Member 4 (TRPM4) as critical for anticancer therapy induced necrosis. TRPM4 knockout in multiple models abolished ErSO-induced ATP depletion, sustained UPR activation, cell swelling, necrotic cell death and increased migration of immune cells. Notably, knockout of TRPM4 completely abolished the ability of ErSO to induce regression of ER positive breast tumors in mice. Supporting a broad role for the TRPM4 pathway in anticancer therapy induced necrosis, rapid cancer cell death induced by four necrosis-inducing cancer therapies unrelated to ErSO, that range from FDA-approved to preclinical, is strongly reversed by TRPM4 knockout. ErSO treatment induces migration of macrophage into regressing tumors. Medium from cancer cells killed by necrosis-inducing ErSO, but not by an apoptosis inducer, dramatically increases macrophage migration and activation, as shown by induction of pro-inflammatory cytokines. This work identifies a protein that plays a pivotal role in the action of diverse anticancer therapies inducing immunogenic necrosis. Since increasing levels of TRPM4 increase sensitivity of breast cancer cells to killing by ErSO, TRPM4 is a novel biomarker whose levels can be used to identify patients most likely to benefit from ErSO and other necrosis-inducing cancer therapies. Citation Format: Santanu Ghosh, Rachel Yang, Darjan Duraki, Ji Eun Kim, Junyao Zhu, Mara Livezey, Matthew Boudreau, Ben H. Park, TImothy Fan, Erik R. Nelson, Paul J. Hergenrother, David J. Shapiro. The Executioner Protein for Immunogenic Anticancer Drug-induced Necrosis in ER Positive Breast Cancer is Transient Receptor Potential Melastatin Member 4 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-01.

Abstract OT3-28-01: A Dose-escalation Study of the Safety and Pharmacology of DAN-222 in Subjects with Metastatic Breast Cancer NCT05261269

Abstract Clinical Study: This clinical study is designed to evaluate DAN-222 as a monotherapy and in combination with a PARP inhibitor which is expected to increase efficacy without significant increase in toxicity. DAN-222 is a novel therapeutic nanoparticle with a complimentary mechanism for combination with a PARP inhibitor anticipated for patients with HRD+ and HRD- tumors, and not restricted to BRCAm. This is an ongoing phase 1/2 study to evaluate the safety and pharmacology of DAN-222 in patients with metastatic breast cancer and initial evaluation of efficacy. Here we report on the initial pharmacology. The results in the patients show the expected characteristics of the designed product, including consistency with preclinical models (e.g. T1/2= 28 hours), which is a feature of this platform given the non-enzymatic release mechanism of the payload that allows for improved translation from preclinical species to clinical patients as well low variability between patients (e.g. CV%=16.5). Study Rationale: DAN-222 is a topoisomerase-1 inhibitor (Camptothecin) nanoparticle therapeutic that has been optimized for tumor biodistribution and pharmacokinetics. DAN-222 has a broad therapeutic index in preclinical evaluation and the complementary mechanism of action with PARP inhibitors provides significantly enhanced efficacy while also sparing bone marrow. Importantly, the complementary enhanced efficacy is independent of tumor homologous repair deficiency (HRD) status, including BRCA status. The efficacy of DAN-222 was evaluated alone and in combination with a PARP inhibitor (niraparib) in HRD+ breast cancer (MDA-MB-436) and HRD- ovarian cancer (OVCAR-3) xenograft models. Table 1 highlights the endpoints of the study as measured by partial response, complete response, tumor free survival and median tumor volume at end of study (Day 60). DAN-222 alone had PR effects and reduced median tumor volume compared to niraparib alone. The combination demonstrated enhancement of response as evidenced by an increase in partial response, a shift from partial to complete response, an increase in tumor-free survivors, and significant further reduction in median tumor volume. Conclusion: The clinical pharmacology will be presented and demonstrates the designed behavior of the nanoparticle. A design feature of the nanoparticle is that the payload and linker are sequestered in the core, protecting them from circulatory components. Moreover, the covalent attachment of the payload allows for tunable release kinetics via a hydrolytic linker, preventing burst release and associated toxicities common to physical encapsulation-based nanoparticle systems (e.g., liposomes, micelles). The preliminary PK profile in patients supports the translatability across species and consistency of exposure across patients. Table 1: Efficacy of DAN-222 Alone or Combination with Niraparib Citation Format: Ashley P. Wright, Emily A. Wyatt, Timothy Hagerty. A Dose-escalation Study of the Safety and Pharmacology of DAN-222 in Subjects with Metastatic Breast Cancer NCT05261269 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT3-28-01.

Exploiting the Receptor-Binding Domains of R-Spondin 1 to Target Leucine-Rich Repeat-Containin G-Coupled Protein Receptor 5-Expressing Stem Cells in Ovarian Cancer.

Leucine-rich repeat-containing G-protein-coupled receptor (LGR5) and LGR6 mark epithelial stem cells in normal tissues and tumors. They are expressed by stem cells in the ovarian surface and fallopian tube epithelia from which ovarian cancer arises. High-grade serous ovarian cancer is unique in expressing unusually high levels of LGR5 and LGR6 mRNA. R-spondins are the natural ligands for LGR5 and LGR6 to which they bind with nanomolar affinity. To target stem cells in ovarian cancer, we used the sortase reaction to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) via a protease sensitive linker to the two furin-like domains of RSPO1 (Fu1-Fu2) that mediate its binding to LGR5 and LGR6 and their co-receptors Zinc And Ring Finger 3 and Ring Finger Protein 43 via a protease-cleavable linker. An immunoglobulin Fc domain added to the N-terminal end served to dimerize the receptor-binding domains so that each molecule carries two MMAE. The resulting molecule, FcF2-MMAE, demonstrated: 1) selective LGR5-dependent low nanomolar cytotoxicity against ovarian cancer cells in vitro; 2) selectivity that was dependent on binding to both the LGR receptors and ubiquitin ligase co-receptors; 3) favorable stability and plasma pharmacokinetic properties when administered intravenously with an elimination half-life of 29.7 hours; 4) selective inhibition of LGR5-rich as opposed to isogenic LGR5-poor tumors in vivo; and, 5) therapeutic efficacy in three aggressive wild-type human ovarian cancer xenograft models. These results demonstrate the successful use of the Fu1-Fu2 domain of RSPO1 as a drug carrier and the ability of FcF2-MMAE to target cells in tumors that express stem cell markers. SIGNIFICANCE STATEMENT: FcF2-MMAE is a novel cancer therapeutic that exploits the high-affinity binding domains of RSPO1 to target monomethyl auristatin E to tumor stem cells that express LGR5. FcF2-MMAE has low nanomolar LGR5-dependent cytotoxicity in vitro, favorable pharmacokinetics, and differential efficacy in an isogenic LGR5-poor versus LGR5-rich ovarian cancer xenograft model when given on a weekly schedule.

Accelerated Discovery of Macrocyclic CDK2 Inhibitor QR-6401 by Generative Models and Structure-Based Drug Design.

Selective CDK2 inhibitors have the potential to provide effective therapeutics for CDK2-dependent cancers and for combating drug resistance due to high cyclin E1 (CCNE1) expression intrinsically or CCNE1 amplification induced by treatment of CDK4/6 inhibitors. Generative models that take advantage of deep learning are being increasingly integrated into early drug discovery for hit identification and lead optimization. Here we report the discovery of a highly potent and selective macrocyclic CDK2 inhibitor QR-6401 (23) accelerated by the application of generative models and structure-based drug design (SBDD). QR-6401 (23) demonstrated robust antitumor efficacy in an OVCAR3 ovarian cancer xenograft model via oral administration.

Human macrophage-engineered vesicles for utilization in ovarian cancer treatment.

Ovarian cancer is a deadly female malignancy with a high rate of recurrent and chemotherapy-resistant disease. Tumor-associated macrophages (TAMs) are a significant component of the tumor microenvironment and include high levels of M2-protumor macrophages that promote chemoresistance and metastatic spread. M2 macrophages can be converted to M1 anti-tumor macrophages, representing a novel therapeutic approach. Vesicles engineered from M1 macrophages (MEVs) are a novel method for converting M2 macrophages to M1 phenotype-like macrophages. Macrophages were isolated and cultured from human peripheral blood mononuclear cells. Macrophages were stimulated to M1 or M2 phenotypes utilizing LPS/IFN-γ and IL-4/IL-13, respectively. M1 MEVs were generated with nitrogen cavitation and ultracentrifugation. Co-culture of ovarian cancer cells with macrophages and M1 MEVs was followed by cytokine, PCR, and cell viability analysis. Murine macrophage cell line, RAW264.7 cells were cultured and used to generate M1 MEVs for use in ovarian cancer xenograft models. M1 MEVs can effectively convert M2 macrophages to an M1-like state both in isolation and when co-cultured with ovarian cancer cells in vitro, resulting in a reduced ovarian cancer cell viability. Additionally, RAW264.7 M1 MEVs can localize to ovarian cancer tumor xenografts in mice. Human M1 MEVs can repolarize M2 macrophages to a M1 state and have anti-cancer activity against ovarian cancer cell lines. RAW264.7 M1 MEVs localize to tumor xenografts in vivo murine models.

Drug repositioning of disulfiram induces endometrioid epithelial ovarian cancer cell death via the both apoptosis and cuproptosis pathways.

Various therapeutic strategies have been developed to overcome ovarian cancer. However, the prognoses resulting from these strategies are still unclear. In the present work, we screened 54 small molecule compounds approved by the FDA to identify novel agents that could inhibit the viability of human epithelial ovarian cancer cells. Among these, we identified disulfiram (DSF), an old alcohol-abuse drug, as a potential inducer of cell death in ovarian cancer. Mechanistically, DSF treatment significantly reduced the expression of the anti-apoptosis marker B-cell lymphoma/leukemia-2 (Bcl-2) and increase the expression of the apoptotic molecules Bcl2 associated X (Bax) and cleaved caspase-3 to promote human epithelial ovarian cancer cell apoptosis. Furthermore, DSF is a newly identified effective copper ionophore, thus the combination of DSF and copper was used to reduce ovarian cancer viability than DSF single treatment. Combination treatment with DSF and copper also led to the reduced expression of ferredoxin 1 and loss of Fe-S cluster proteins (biomarkers of cuproptosis). In vivo, DSF and copper gluconate significantly decreased the tumor volume and increased the survival rate in a murine ovarian cancer xenograft model. Thus, the role of DSF revealed its potential for used as a viable therapeutic agent for the ovarian cancer.

Kinase Inhibitors in the Treatment of Ovarian Cancer: Current State and Future Promises.

Ovarian cancer is the deadliest gynecological cancer, the high-grade serous ovarian carcinoma (HGSC) being its most common and most aggressive form. Despite the latest therapeutical advancements following the introduction of vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors and poly-ADP-ribose-polymerase (PARP) inhibitors to supplement the standard platinum- and taxane-based chemotherapy, the expected overall survival of HGSC patients has not improved significantly from the five-year rate of 42%. This calls for the development and testing of more efficient treatment options. Many oncogenic kinase-signaling pathways are dysregulated in HGSC. Since small-molecule kinase inhibitors have revolutionized the treatment of many solid cancers due to the generality of the increased activation of protein kinases in carcinomas, it is reasonable to evaluate their potential against HGSC. Here, we present the latest concluded and on-going clinical trials on kinase inhibitors in HGSC, as well as the recent work concerning ovarian cancer patient organoids and xenograft models. We discuss the potential of kinase inhibitors as personalized treatments, which would require comprehensive assessment of the biological mechanisms underlying tumor spread and chemoresistance in individual patients, and their connection to tumor genome and transcriptome to establish identifiable subgroups of patients who are most likely to benefit from a given therapy.

The Impact of Eribulin on Stathmin Dynamics and Paclitaxel Sensitivity in Ovarian Cancer Cells.

Eribulin, an inhibitor of microtubule dynamics, is used for treating breast cancers and sarcomas. The microtubule-destabilizing protein stathmin may modulate the antiproliferative activity of eribulin on breast cancer cells and leiomyosarcoma cells. The antitumor activity of eribulin in ovarian cancers has not been fully explored, so the present study aimed to determine the antitumor efficacy of eribulin and the involvement of stathmin in ovarian cancers. In a xenograft model of ovarian cancer, eribulin treatment reduced the tumor weight, which was accompanied by an increased level of phosphorylated stathmin. Eribulin stimulated the phosphorylation of stathmin in cultured cancer cell lines. The eribulin-induced phosphorylation of stathmin was inhibited by treatment with FTY720, an activator of protein phosphatase 2A (PP2A), and eribulin downregulated the expression of PP2A subunits. Furthermore, stathmin knockdown abrogated the inhibitory effects of eribulin on cell viability. Eribulin enhanced the antiproliferative effects of paclitaxel and concomitantly decreased stathmin expression. These results suggest that eribulin-induced phosphorylation of stathmin, mediated in part by PP2A downregulation, reduces stathmin activity and enhances the antiproliferative effects of paclitaxel in ovarian cancer. Collectively, the results of this study indicate that eribulin may suppress the proliferation of ovarian cancer cells partly by regulating the activity of stathmin.

SN-38 Sensitizes BRCA-Proficient Ovarian Cancers to PARP Inhibitors through Inhibiting Homologous Recombination Repair.

As a multifunctional protein posttranslational modification enzyme in eukaryotic cells, Poly-ADP-ribose polymerase (PARP) acts as a DNA damage sensor, which helps to repair DNA damage through recruiting repair proteins to the DNA break sites. PARP inhibitors offer a significant clinical benefit for ovarian cancer with BRCA1/2 mutations. However, the majority of ovarian cancer patients harbor wild-type (WT) BRCA1/2 status, which narrows its clinical application. Here, we identified a small compound, SN-38, a CPT analog, which sensitizes BRCA-proficient ovarian cancer cells to PARP inhibitor treatment by inhibiting homologous recombination (HR) repair. SN-38 treatment greatly enhanced PARP inhibitor olaparib induced DNA double-strand breaks (DSBs) and DNA replication stress. Meanwhile, the combination of SN-38 and olaparib synergistically induced apoptosis in ovarian cancer. Furthermore, combination administration of SN-38 and olaparib induced synergistic antitumor efficacy in an ovarian cancer xenograft model in vivo. Therefore, our study provides a novel therapeutic strategy to optimize PARP inhibitor therapy for patients with BRCA-proficient ovarian cancer.

Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed invitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing invivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

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

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

Abstract 1763: Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail

Abstract Antibody Drug Conjugates (ADCs) are failing due to 3 critical limitations: Low potency, ineffective solid-tumor penetration and poor tolerability. The industry is full of approaches where full-length Immunoglobulins have been engineered to carry defined numbers of payloads with higher-loadings of less potent payloads appearing to be well-tolerated. However, antibody fragments (e.g. single-chain Fcs-scFvs), which have many advantages including rapid tumor penetration, faster clearance, inexpensive manufacture, have been technologically challenging to apply in oncology. Our novel approach enables scFvs to have a high Drug:Antibody loading ratio (DAR) whilst retaining effective binding and other favorable biophysical properties, leading to a new product class tailored for solid tumors. Antikor has two FDC products in development for solid tumors, notably gastric: anti-HER2 FDC (ANT-043) and a second target (ANT-045) which will be disclosed during this presentation. ANT-043 has pM potencies in a range of HER2-expressing cell-lines, including trastuzumab-resistant models, excellent tumor ablation effects in breast, ovarian and gastric cancer xenograft models and superior tolerability compared to an ADC in rat toxicology studies at a dose of 1mg/kg/weekly. Quantitative payload tumor uptake and fluorescent immuno-histological studies demonstrate superior solid tumor penetration across the entire tumor and diffusion from blood vessels. In collaboration with our partners, Essex Biotechnology, Antikor is taking ANT-043 into IND-enabling studies for clinical development. ANT-045, which emerged from Antikor’s proprietary FDC ‘discovery engine’, is progressing towards IND-filing and updated data will illustrate how ANT-045 could have a broader patient benefit in gastro-intestinal cancers. ANT-045 has excellent in vitro cell-kill potency (pM IC50s) and excellent stability and superior in vivo tumor cure efficacy, compared to a leading clinical stage benchmark ADC. This presentation will focus on Antikor’s FDC discovery platform (stable high-DAR scFv-display libraries, tailored linker-payloads and design features) that has the potential to generate first-in-class products for difficult to treat solid tumors for patient benefit and promising to succeed where ADCs have failed to deliver. Citation Format: Mahendra P. Deonarain, Gokhan Yahioglu, Ioanna Stamati, Bryan Edwards, Soraya Diez-Posada, Isabel Perez-Castro, Anja Pomowski, Laura Bouche, Ashleigh Stewart, Monika Maciuszek, Sam Ness, Malcolm Ngiam, Quinn Xue. Gastric cancer antibody fragment drug-conjugates (FDCs): Succeeding in solid tumors where ADCs fail [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1763.