Ovarian Tumor Growth Research Articles

Safety Evaluations of Rapamycin Perfluorocarbon Nanoparticles in Ovarian Tumor-Bearing Mice.

Nanomedicine holds great potential for revolutionizing medical treatment. Ongoing research and advancements in nanotechnology are continuously expanding the possibilities, promising significant advancements in healthcare. To fully harness the potential of nanotechnology in medical applications, it is crucial to conduct safety evaluations for the nanomedicines that offer effective benefits in the preclinical stage. Our recent efficacy studies indicated that rapamycin perfluorocarbon (PFC) nanoparticles showed promise in mitigating cisplatin-induced acute kidney injury (AKI). As cisplatin is routinely administered to ovarian cancer patients as their first-line chemotherapy, in this study, we focused on evaluating the safety of rapamycin PFC nanoparticles in mice bearing ovarian tumor xenografts. Specifically, this study evaluated the effects of repeat-dose rapamycin PFC nanoparticle treatment on vital organs, the immune system, and tumor growth and assessed pharmacokinetics and biodistribution. Our results indicated that rapamycin PFC nanoparticle treatment did not cause any detectable adverse effects on cardiac, renal, or hepatic functions or on splenocyte populations, but it reduced the splenocyte secretion of IL-10, TNFα, and IL12p70 upon IgM stimulation. The pharmacokinetics and biodistribution results revealed a significant enhancement in the delivery of rapamycin to tumors by rapamycin PFC nanoparticles, which, in turn, led to a significant reduction in ovarian tumor growth. Therefore, rapamycin PFC nanoparticles have the potential to be clinically beneficial in cisplatin-treated ovarian cancer patients.

SMAC mimetic BV6 acts in synergy with mTOR inhibitor to increase cisplatin sensitivity in ovarian cancer.

The objective of this study is to observe the antitumor efficacy of the second mitochondria-derived activator of caspases (SMAC) mimetic bivalent smac mimetic (BV6) in combination with target of rapamycin (mTOR) inhibitor on DDP (cisplatin) sensitivity. Ovarian cancer cells were exposed to cisplatin, BV6, DDP + BV6, and DDP + BV6 + mTOR inhibitor Rapamycin. Using proteomics and bioinformatics, protein expression profiles in ovarian cancer were determined. Bagg Albino color nude mice were treated with DDP or BV6 alone or in combination, or BV6 + DDP + Rapamycin. The effects of different treatments on ovarian cancer cells and tumor growth were evaluated in vivo and in vitro. Proteomics and bioinformatics analysis revealed significant changes of protein kinase (AKT)/mTOR pathway. Consistently, western blot data indicated that AKT/mTOR axis was gradually activated in BV6-treated ovarian cancer cells and attenuated the cytotoxic effect of BV6. Functional assays showed that DDP or BV6 treatment alone significantly enhanced the sensitivity and inhibited the migration of ovarian cancer cells, but without any synergistic effects. In addition, combination with BV6 and mTOR inhibitor Rapamycin significantly decreased cell viability and inhibited migration of ovarian cancer cells exposed to DDP. Consistently, the xenograft model showed that co-treatment with Rapamycin with BV6 had significantly suppressed tumor growth and metastasis. Our study demonstrated that SMAC analogue BV6 exhibits a strong anticancer effect on ovarian cancer in vitro and in vivo. Combination with Rapamycin overcomes the activation of mTOR pathway by BV6 and increases the chemosensitivity to DDP. These data suggest a potential application of triple combination with DDP + BV6 + Rapamycin in clinical management of ovarian cancer.

CypA/TAF15/STAT5A/miR-514a-3p feedback loop drives ovarian cancer metastasis.

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that participates in multiple cancer events, but the molecular mechanisms of abnormal expression and regulation of CypA in ovarian cancer (OC) have never been considered. This study identifies CypA as a key driver of epithelial-mesenchymal transition (EMT) in ovarian cancer and explores the mechanisms that underly this process. We show that CypA is upregulated in tissues and serum of ovarian cancer patients and that CypA overexpression correlates with poor prognosis. CypA facilitates tumor growth and metastasis in vivo in subcutaneous tumor xenograft and abdominal metastatic models, and in vitro studies suggest a mechanism, showing that CypA accelerates ovarian cancer cell epithelial-mesenchymal transition by activating a PI3K/AKT signaling pathway. Mechanistic studies showed that STAT5A binds pri-miR-514a-3p and inhibits its activity, whereas miR-514a-3p directly binds to the 3'-UTR of CypA to suppress its expression, resulting in STAT5A promoting the expression of CypA, forming the STAT5A/miR-514a-3p/CypA axis. Furthermore, immunoprecipitates and mass spectrometry analysis identifies a CypA interaction with TAF15 that stabilizes TAF15 by suppressing its proteasome degradation and promotes its entry into the nucleus. While STAT5A is positively regulated by TAF15. Our findings identify a novel feedback loop for CypA that drives EMT and ovarian tumor growth and metastasis via a TAF15/STAT5A/miR-514a-3p pathway in ovarian cancer and facilitates the release of CypA into the extracellular, which provides a promising therapeutic target for OC treatment and a diagnostic biomarker.

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils

ABSTRACT High-grade serious ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here, we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ~ 50% of the treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

Hsa_circ_0020093 suppresses ovarian cancer progression by modulating LRPPRC activity and miR-107/LATS2 signaling

A substantive body of evidence has demonstrated the significant roles of circular RNA (circRNA) in cancer. However, the contribution of dysregulated circRNAs to ovarian cancer (OC) remains elusive. We aim to elucidate the critical roles and mechanisms of hsa_circ_0020093, which was demonstrated to be downregulated in OC tissues in our previous study. In this study, we confirmed the decreased expression of hsa_circ_0020093 in OC tissues and cell lines and demonstrated the negative correlation between its expression and FIGO stage, abdominal implantation and CA125 level of OC patients. Through gain and loss of function studies, we confirmed the inhibitory role of hsa_circ_0020093 in ovarian tumor growth in vitro and in vivo. Mechanistically, based on the peri-nuclear accumulation of hsa_circ_0020093, we discovered the interaction between hsa_circ_0020093 and the mitochondrial protein LRPPRC by RNA pull-down, mass spectrometry, RNA Binding Protein Immunoprecipitation. As a result, qRT-PCR and transmission electron microscopy results showed that the mitochondria mRNA expression and mitochondria abundance were decreased upon hsa_circ_0020093-overexpression. Meanwhile, we also unearthed the hsa_circ_0020093/miR-107/LATS2 axis in OC according to RNA-sequencing, RIP and luciferase reporter assay data. Furthermore, LRPPRC and LATS2 are both reported as the upstream regulators of YAP, our study also studied the crosstalk between hsa_circ_0020093, LRPPRC and miR-107/LATS2, and unearthed the up-regulation of phosphorylated YAP in hsa_circ_0020093-overexpressing OC cells and xenograft tumors. Collectively, our study indicated the novel mechanism of hsa_circ_0020093 in suppressing OC progression through both hsa_circ_0020093/LRPPRC and hsa_circ_0020093/miR-107/LATS2 axes, providing a potential therapeutic target for OC patients.

High-grade serous ovarian cancer development and anti-PD-1 resistance is driven by IRE1α activity in neutrophils.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy that remains refractory to current immunotherapies. While advanced stage disease has been extensively studied, the cellular and molecular mechanisms that promote early immune escape in HGSOC remain largely unexplored. Here we report that primary HGSO tumors program neutrophils to inhibit T cell anti-tumor function by activating the endoplasmic reticulum (ER) stress sensor IRE1α. We found that intratumoral neutrophils exhibited overactivation of ER stress response markers compared with their counterparts at non-tumor sites. Selective deletion of IRE1α in neutrophils delayed primary ovarian tumor growth and extended the survival of mice with HGSOC by enabling early T cell-mediated tumor control. Notably, loss of IRE1α in neutrophils sensitized tumor-bearing mice to PD-1 blockade, inducing HGSOC regression and long-term survival in ∼50% of treated hosts. Hence, neutrophil-intrinsic IRE1α facilitates early adaptive immune escape in HGSOC and targeting this ER stress sensor might be used to unleash endogenous and immunotherapy-elicited immunity that controls metastatic disease.

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.

12-O-deacetyl-phomoxanthone A inhibits ovarian tumor growth and metastasis by downregulating PDK4

AimsThe xanthone dimer 12-O-deacetyl-phomoxanthone A (12-ODPXA) was extracted from the secondary metabolites of the endophytic fungus Diaporthe goulteri. The 12-ODPXA compound exhibited anticancer properties in murine lymphoma; however, the anti-ovarian cancer (OC) mechanism has not yet been explored. Therefore, the present study evaluated whether 12-ODPXA reduces OC cell proliferation, metastasis, and invasion by downregulating pyruvate dehydrogenase kinase (PDK)4 expression. MethodsCell counting kit-8, colony formation, flow cytometry, wound healing, and transwell assays were performed to examine the effects of 12-ODPXA on OC cell proliferation, apoptosis, migration, and invasion. Transcriptome analysis was used to predict the changes in gene expression. Protein expression was determined using western blotting. Glucose, lactate, and adenosine triphosphate (ATP) test kits were used to measure glucose consumption and lactate and ATP production, respectively. Zebrafish xenograft models were constructed to elucidate the anti-OC effects of 12-ODPXA. ResultsThe 12-ODPXA compound inhibited OC cell proliferation, migration, invasion, and glycolysis while inducing cell apoptosis via downregulation of PDK4. In vivo experiments showed that 12-ODPXA suppressed tumor growth and migration in zebrafish. ConclusionOur data demonstrate that 12-ODPXA inhibits ovarian tumor growth and metastasis by downregulating PDK4, revealing the underlying mechanisms of action of 12-ODPXA in OC.

Stromal cartilage oligomeric matrix protein as a tumorigenic driver in ovarian cancer via Notch3 signaling and epithelial-to-mesenchymal transition

BackgroundCartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP’s role in ovarian cancer, exploring clinicopathological links and mechanistic insights.MethodsTo study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed.ResultsBased on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-β. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active β-catenin in ovarian cancer cells.ConclusionThis study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Abstract 2368: Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth

Abstract Her-2 is a well-known driver of carcinogenesis that is overexpressed in breast, ovarian and other tumors. Immunotherapy with bispecific antibodies is a promising cancer therapy approach. Her-2 (Herceptin) antibody was used to test bispecific antibody against ovarian cancer cells. First, Her-2-CAR-T cells with Her-2 (4D5) ScFv and 4-1BB costimulatory domain were generated and tested using in vitro killing and IFN-gamma assays, and in vivo efficacy SKOV-3 xenograft NSG mouse model. Her-2-CAR-T cells killed SKBR-3 breast and SKOV-3 ovarian cancer cells and stopped SKOV-3 xenograft tumor growth. This Her-2-ScFv was used to generate bispecific Her-2 ScFv-CD3-ScFv human Fc antibody using in vitro transcribed mRNA. The 5’capped Her-2-CD3-hFc mRNA with 5’UTR, 3’UTR and poly A tail was generated by in vitro transcription using DNA template. The mRNA was embedded into lipid nanoparticles (LNP) using microfluidic PreciGenome Flex S NanoGenerator. Western blotting detected >160 kDa bispecific antibody secreted from A1847 and 293 cells transfected with mRNA-LNPs. The bispecific antibody was binding Her-2-positive A1847 ovarian cancer cell lines and T cells by FACS analysis. The Her-2-CD3-hFc bispecific antibody with T cells effectively killed Her-2-positive target cells in a dose-dependent manner in RTCA assay and produced high level of IFN-gamma. Moreover, intratumoral delivery of Her-2-CD3-hFc mRNA-LNP with intravenous injection of T cells completely blocked A1847 xenograft tumor growth. In conclusion. intratumoral delivery of Her-2-CD3 mRNA-LNP blocks ovarian xenograft tumor growth and provides a solid basis for future pre-clinical and clinical studies. Citation Format: Vita M. Golubovskaya, John Sienkiewicz, Jinying Sun, Shiming Zhang, Yanwei Huang, Liang Hu, Hua Zhou, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth [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 2368.

Abstract 4779: The effects of glutamine deprivation on ovarian cancer initiation and progression

Abstract High-grade serous ovarian cancer (HGSC) is the most common subtype of ovarian cancer. Most patients with HGSC are diagnosed at advanced stage diseases. Despite the high response rate to platinum/taxane-based chemotherapy, most of these patients will develop recurrent chemoresistant disease. Recent studies showed the cancer associated fibroblasts (CAFs) in the tumor microenvironment (TME) play an important role in modulating the malignant phenotypes of HGSC. CAFs are primarily responsible for producing factors such as cytokines and growth factors to modify the TME niche to promote the malignant phenotypes of cancer cells. Recently, we have identified a previously unrecognized mechanism in which metabolism of reactive CAFs is reprogrammed through an upregulated glutamine anabolic pathway in HGSC. This dysfunctional CAF metabolism confers atypical metabolic flexibility and adaptive mechanisms in CAFs, allowing them to harness carbon and nitrogen from noncanonical sources to synthesize glutamine in nutrient-deprived conditions existing in the TME. Our preliminary data show that tumors developed from syngeneic fallopian epithelial cell-derived cancer cells injected IP into C57BL/6 mice, which started to be fed with normal diet two weeks before tumor cells injection had significantly higher tumor burden than those fed with the glutamine-deprived diet. Moreover, tumor-bearing mice treated with glutaminase inhibitor for 3 weeks demonstrated significantly lower tumor burden than those treated with the control vehicle. These findings suggest that the interplay between CAFs and HGSC cells enriches glutamine in the ovarian TME; and glutamine depletion can not only suppress ovarian tumor growth but also prevent the development of advanced progressive diseases. In addition to the direct promoting effect of glutamine on the glutamine addicted HGSC cells, increased CAF-derived glutamine can also affect the activity of other stromal cell types such as immune cells in the TME, which subsequently modulate HGSC cell growth. Imaging mass cytometry (IMC) and Mass Spectrometry Imaging (MSI) were performed on serial tissue sections of tissue microarrays generated from the abovementioned murine samples. Mice fed with a glutamine-deprived diet or treated with the glutaminase inhibitor not only have a significantly lower tumor burden but also induced dramatic changes in the cellular and molecular composition within the TME. In particular, mice fed with a glutamine-deprived diet had greater B-cell-related immune response, reduced stemness, and EMT of ovarian cancer cells, and more activated CAFs, which may result in increased stiffness of the ECM and subsequently enhance the malignant phenotype of HGSC cells. Further studies to delineate the molecular mechanisms by which spatial cellular and metabolic profiles modulate HGSC pathogenesis and progression are warranted. Citation Format: Sammy Ferri-Borgogno, Kathryn Lu, Basant T. Gamal, Chi Lam Au Yeung, Erin Seeley, Javier A. Gomez, Akshay Basi, Jared K. Burks, Olamide Animasahun, Deepak Nagrath, Samuel C. Mok. The effects of glutamine deprivation on ovarian cancer initiation and progression [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 4779.

Abstract A090: An inflammatory adipose microenvironment promotes ovarian cancer tumor growth and cisplatin resistance

Abstract Epithelial ovarian cancer (EOC) commonly targets the omentum, an adipocyte rich organ of the peritoneum. Omental adipocytes have been shown to promote EOC development and chemoresistance via transfer of free fatty acids (FFAs) and the induction of bi-directional metabolic changes. Furthermore, obesity and adiposity are risk factors for EOC incidence and prognosis. The metastatic peritoneal tumor microenvironment (TME) is highly inflamed, while obesity is often characterized by chronic inflammation of adipose tissue and hyperlipidemia. Given that inflammation of adipocytes is associated with increased release of FFAs, we hypothesized that acute inflammation in the ovarian metastatic TME exacerbates pro-tumorigenic effects of cancer-associated adipocytes. Using a three-dimensional transwell co-culture model, we explore the impact of inflamed adipocytes on EOC activity. Adipocytes generated from human adipose-derived stem cells were inflamed with 100ng/ml TNFα and cultured alongside tumor spheroids generated from EOC cell lines. Cytokine abundance in serum samples from ovarian cancer patients undergoing cisplatin treatment were also analyzed. We found that inflammation of adipocytes increased cellular lipolysis and release of FFAs, as well as inducing de-differentiation and loss of lipid droplets. Upon co-culture with inflamed adipocytes, EOC cells displayed increased proliferation in 2D monolayer and 3D spheroid cultures. Transfer of FFAs from adipocytes to cancer cells was visualized and quantified using a fluorescent fatty acid analog via confocal microscopy and flow cytometry. Co-culture induced expression of fatty acid chaperone protein FABP4 in EOC cell lines, while blocking fatty acid metabolism in cancer cells reversed pro-tumor effects of adipocytes. To understand signaling mechanisms generating bi-directional phenotypic changes, cytokine content of secretome of co-cultures was assessed by Luminex. IL-6 and IL-8 were highly abundant in co-cultures, and increased expression was confirmed by qPCR. Treatment of EOC cell lines with recombinant IL-6 and IL-8 induced increased uptake of FFAs and blocking of IL-6 and IL-8 signaling reversed pro-cancer effects of adipocytes. Serum samples from patients undergoing cisplatin therapy were analyzed and IL-6 and IL-8 levels were found to be associated with resistance to therapy. To further investigate the impact of inflamed adipocyte signaling on therapeutic resistance, co-culture were treated with cisplatin and tumor spheroids were found to be highly resistant in the presence of inflamed adipocytes. STAT3 activity is known to be upregulated by both IL-6 and IL-8 and we found that STAT3 expression was increased in co-cultures. Knockdown of STAT3 in EOC reversed the chemoprotective effects of inflamed adipocytes. These data suggest an important role for inflammation of adipose tissue in the development of therapeutic resistance in EOC and highlight a potential mechanism linking clinically observed associations between obesity and ovarian cancer risk. Citation Format: Michael E. Williams, David Howard, Deyarina Gonzalez, Francesca Taraballi. An inflammatory adipose microenvironment promotes ovarian cancer tumor growth and cisplatin resistance [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 A090.

Abstract B084: Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer

Abstract Background: Over 20,000 women are diagnosed with ovarian cancer annually, and more than half will die within 5 years. This rate has changed little in the last 30 years, highlighting the need for therapy innovation. Although immunotherapy has revolutionized cancer treatment, efforts to harness endogenous patient immune responses have yielded limited therapeutic activity in ovarian cancer patients. T cells engineered to express a T cell receptor (TCR) targeting proteins uniquely overexpressed in tumors have the potential to control tumor growth without toxicity. Mesothelin (Msln) is over-expressed in ovarian cancer, contributes to the malignant and invasive phenotype, and has limited expression in healthy cells, making it a candidate immunotherapy target in these tumors. Methods: The ID8VEGF mouse cell line was used to evaluate if T cells engineered to express a mouse Msln-specific high-affinity T cell receptor (TCRMsln) can kill ovarian cancer. Tumor-bearing mice were treated with TCRMsln T cells plus anti-PD-1, anti-Tim-3 or anti-Lag-3 checkpoint-blocking antibodies alone or in combination, ultimately targeting up to three inhibitory receptors simultaneously. Single-cell RNA-sequencing (scRNAseq) was used to profile the impact of combination checkpoint blockade on engineered T cells and the tumor microenvironment (TME). Results: In a disseminated ID8 tumor model, adoptively transferred TCRMsln T cells preferentially accumulated in established tumors, delayed ovarian tumor growth, and prolonged mouse survival. However, elements in the TME limited engineered T cell persistence and cytolytic function. Triple checkpoint blockade, but not single- or double-agent treatment, dramatically increased antitumor function by intratumoral TCRMsln T cells. scRNAseq of tumor-infiltrating cells revealed distinct transcriptome changes in engineered and endogenous T cells and myeloid-derived cells. Engineered T cells, when combined with triple checkpoint blockade, increased expression of genes associated with effector and memory gene signatures, including proliferation and metabolic function, and reduced expression of genes associated with exhaustion. Moreover, combining adoptive immunotherapy with triple checkpoint blockade significantly prolonged survival in the cohort of treated tumor-bearing mice, relative to mice that received TCRMsln T cells alone or with anti-PD1 or double-agent treatments. Conclusions: Inhibitory receptor/ligand interactions within the TME can dramatically reduce T cell function, suggesting tumor cells may upregulate the ligands for PD-1, Tim-3 and Lag-3 for protection from tumor-infiltrating lymphocytes. In an advanced ovarian cancer model, triple checkpoint blockade significantly improved engineered T cell function and outcomes in mice in a setting where single checkpoint blockade had no significant activity. These results suggest that disrupting multiple inhibitory pathways simultaneously, which can be more safely pursed in a cell intrinsic form through genetic engineering, may be necessary for improved efficacy in patients. Citation Format: Kristin G. Anderson, Yapeng Su, Madison G. Burnett, Breanna M. Bates, Magdalia L. Rodgers Suarez, Susan L. Ruskin, Valentin Voillet, Raphael Gottardo, Philip D. Greenberg. Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced 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 B084.

Methylene Blue Metabolic Therapy Restrains In Vivo Ovarian Tumor Growth.

Ovarian cancer remains a significant challenge, especially in platinum-resistant cases where treatment options are limited. In this study, we investigated the potential of methylene blue (MB) as a metabolic therapy and complementary treatment approach for ovarian cancer. Our findings demonstrated a significant in vivo reduction in the proliferation of TOV112D-based ovarian-cell-line xenografts. In this preclinical study, which used a carboplatin-resistant ovarian cancer tumor model implanted into mice, MB-mediated metabolic therapy exhibited superior tumor slowdown compared to carboplatin treatment alone. This indicates, for the first time, MB's potential as an alternative or adjuvant treatment, especially for resistant cases. Our in vitro study on TOV112D and ARPE-19 sheds light on the impact of such an MB-based metabolic therapy on mitochondrial energetics (respiration and membrane potential). MB showed a modulatory role in the oxygen consumption rate and the mitochondrial membrane potential. These results revealed, for the first time, that MB specifically targets TOV112D mitochondria and probably induces cell apoptosis. The differential response of normal (ARPE-19) and cancer (TOV112D) cells to the MB treatment suggests potential alterations in cancer cell mitochondria, opening avenues for therapeutic approaches that target the mitochondria. Overall, our findings suggest the efficacy of MB as a possible treatment for ovarian cancer and provide valuable insights into the mechanisms underlying the efficacy of methylene blue metabolic therapy in ovarian cancer treatment.

Abstract C147: The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments

Abstract Previous studies have demonstrated WEE1i and ATR inhibitors (ATRi and WEE1i) to be promising cancer therapeutics through synthetic lethality with various cancer associated mutations. However, a key limitation to the use of these inhibitors as cancer therapies in prior clinical trials has been the occurrence of adverse hematological effects, including anemia and thrombocytopenia. Herein, we describe two novel inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi) that are potentially both effective in tumor suppression in animal models and well tolerated. ATRN-1051 was developed to be both a potent WEE1i and selective for WEE1 over other kinases (PLK1, PLK2 and PLK3). ATRN-1051 has an IC50 of 2.2 nM for WEE1 and limits the proliferation of various cancer cell lines in culture in the 100 nM to 200 nM range. Notably, ATRN-1051 is particularly effective in suppressing the growth of Cyclin E overexpressing cells lines, pinpointing Cyclin E as a potential biomarker for ATRN-1051. In addition, ATRN-1051 has potentially favorable pharmacokinetic properties that permits 3-8 times lower dosing than other clinical WEE1 inhibitors to achieve similar exposure (AUC, 0-24) levels (1). Consistent with the increase selectivity of ATRN-1051 fostering increased tolerability, dose-range finding studies indicate that doses potentially expected to cause significant tumor suppression are hematological well tolerated in mice, with red blood cell and platelet counts remaining in a non-pathogenic range. Importantly, daily oral dosing of ATRN-1051 suppresses the growth of CCNE1-amplified high-grade serous ovarian xenografted tumors over the course of 28 days, providing further evidence of the role of CCNE1-overexpression as a biomarker for ATRN-1051 treatment. Based on these data, ATRN-1051 has entered and is now progressing through IND-enabling studies. As a distinct upstream DNA replication checkpoint inhibitor, ATRN-119 is a macrocyclic ATRi that is highly specific for inhibition of ATR over other phosphatidylinositol kinase-related kinases (PIKKs), such as ATM, DNA-PK, and MTOR, implying the potential for increased tolerability. Supporting this implication, daily dosing of ATRN-119 suppresses tumor growth in xenograft mouse models of colon, pancreatic, and prostate cancers and causes no appreciable loss of body weight or hematologic toxicity. Daily dosing of ATRN-119 in combination with PARP inhibition causes significant tumor reduction in a BRCA2-deficient PDX model of high-grade serous ovarian cancer, again with no appreciable loss of body weight. These findings have led to a biomarker-driven Phase 1/2a clinical trial of ATRN-119 with daily dosing (Simpkins, PI). Together, these findings underscore the promise of ATRN-1051 and ATRN-119 as DNA replication checkpoint inhibitors for the treatment of a variety of cancers. (1) Data from study in A-427 NSCLC xenograft model as reported in Zentalis Corporate Overview, March 2022 Citation Format: Joseph Vacca, Stephen Rocca, Molly Hansbarger, Sonia Ritzmann, Justin Frye, Fiona Simpkins, Eric J. Brown, Oren Gilad. The DNA replication checkpoint inhibitors, ATRN-1051 (WEE1i) and ATRN-119 (ATRi), are potentially well tolerated and effective cancer treatments [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C147.

Triptolide inhibits epithelial ovarian tumor growth by blocking the hedgehog/Gli pathway.

Epithelial ovarian cancer (EOC), the most predominant subtype of ovarian cancer (OC), involves poor prognosis and exhibits high aggression. Triptolide (TPL), like other Chinese herbs, has historically played a significant role in modern medicine. The screening system based on Gli-dependent luciferase reporter activity assessed the effects of over 800 natural medicinal materials on hedgehog (Hh) signaling pathway activity and discovered that TPL had an excellent inhibitory effect on Hh signaling pathway activity. However, the significance and mechanism of TPL involvement in regulating the Hh pathway have not been well explored. Thus, this work aimed to understand better how TPL affects the Hh pathway activity, which, in turn, influences the biological behavior of EOC. Our findings observed that Smo agonist SAG-induced EOC cell proliferation, migration, and invasion were drastically reversed by TPL in a concentration-dependent pattern. Further evidence suggested that TPL promotes the degradation of Gli1 and Gli2 to inhibit the activity of the Hh signaling pathway by relying on Gli1 and Gli2 ubiquitination. Our in vivo studies also confirmed that TPL could significantly inhibit the tumor growth of EOC. Taken together, our results revealed that one of the antitumor mechanisms of TPL was the targeted inhibition of the Hh/Gli pathway.

MiR-146a inhibits ovarian tumor growth in vivo via targeting immunosuppressive neutrophils and enhancing CD8+ T cell infiltration

Immunotherapies have emerged as promising strategies for cancer treatment. However, existing immunotherapies have poor activity in high-grade serous ovarian cancer (HGSC) due to the immunosuppressive tumor microenvironment and the associated low tumoral CD8+ Tcell (CTL) infiltration. Through multiple lines of evidence, including integrative analyses of human HGSC tumors, we have identified miR-146a as a master regulator of CTL infiltration in HGSC. Tumoral miR-146a expression is positively correlated with anti-cancer immune signatures in human HGSC tumors, and delivery of miR-146a to tumors resulted in significant reduction in tumor growth in both ID8-p53-/- and IG10 murine HGSC models. Increasing miR-146a expression in tumors improved anti-tumor immune responses by decreasing immune suppressive neutrophils and increasing CTL infiltration. Mechanistically, miR-146a targets IL-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 adaptor molecules of the transcription factor nuclear factor κB signaling pathway in ID8-p53-/- cells and decreases production of the downstream neutrophil chemoattractant, C-X-C motif chemokine ligand 1. In addition to HGSC, tumoral miR-146a expression also correlates strongly with CTL infiltration in other cancer types including thyroid, prostate, breast, and adrenocortical cancers. Altogether, our findings highlight the ability of miR-146a to overcome immune suppression and improve CTL infiltration in tumors.

Mast Cells Retard Tumor Growth in Ovarian Cancer: Insights from a Mouse Model.

Ovarian cancer has the highest mortality rate among female reproductive tract malignancies. A complex network, including the interaction between tumor and immune cells, regulates the tumor microenvironment, survival, and growth. The role of mast cells (MCs) in ovarian tumor pathophysiology is poorly understood. We aimed to understand the effect of MCs on tumor cell migration and growth using in vitro and in vivo approaches. Wound healing assays using human tumor cell lines (SK-OV-3, OVCAR-3) and human MCs (HMC-1) were conducted. Murine ID8 tumor cells were injected into C57BL6/J wildtype (WT) and MC-deficient C57BL/6-KitW-sh/W-sh (KitW-sh) mice. Reconstitution of KitW-sh was performed by the transfer of WT bone marrow-derived MCs (BMMCs). Tumor development was recorded by high-frequency ultrasonography. In vitro, we observed a diminished migration of human ovarian tumor cells upon direct or indirect MC contact. In vivo, application of ID8 cells into KitW-sh mice resulted in significantly increased tumor growth compared to C57BL6/J mice. Injection of BMMCs into KitW-sh mice reconstituted MCs and restored tumor growth. Our data show that MCs have a suppressive effect on ovarian tumor growth and may serve as a new therapeutic target.

A pilot in vivo study: potential ovarian cancer therapeutic by placental extracellular vesicles.

The biological links between cancer and pregnancy are of interest due to parallel proliferative, immunosuppressive, and invasive mechanisms between tumour and placental cells. However, the proliferation and invasion of placental cells are strictly regulated. The understanding of this regulation is largely unknown. Placental extracellular vesicles (EVs) may play an important role in this regulation, as placental EVs are known to contribute to maternal adaptation, including adaptation of the vascular and immune systems. We have previously reported that placental EVs significantly inhibited ovarian cancer cell proliferation by delaying the progression of the cell cycle. We therefore performed this pilot in vivo study to investigate whether placental EVs can also inhibit ovarian tumour growth in a SKOV-3 human tumour xenograft model. A single intraperitoneal injection of placental EVs at 15 days post tumour implantation, significantly inhibited the growth of the tumours in our in vivo model. Signs of cellular necrosis were observed in the ovarian tumour tissues, but not in other organs collected from mice that had been treated with placental EVs. Expression of receptor-interacting kinase 1 (RIPK1) and mixed linkage kinase domain-like (MLKL), which are mediators of necroptosis were not observed in our xenografted tumors. However, extensive infiltration of CD169+ macrophages and NK cells in ovarian tumour tissues collected from placental micro-EVs treated mice were observed. We demonstrate here that inhibition of ovarian tumour growth in our xenograft model by placental EVs involves cellular necrosis and infiltration of CD169+ macrophages and NK cells into the tumour tissues.