Weroha, S. John

Abstract Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. Significance: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Abstract A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant patients with ovarian cancer is forthcoming. ARID1A-mutated ovarian clear cell carcinoma (OCCC) is a relatively indolent and chemoresistant ovarian cancer histotype. In OCCC-derived cells ARID1A simultaneously drives GLS1 expression and metabolism reprograming. In ARID1A-mutated OCCC-derived mouse models, loss of ARID1A corresponds to GLS1 upregulation and increases sensitivity to GLS1 inhibition. Thus, targeting of GLS1 with CB839 has been suggested as a targeted approach for patients with OCCC with tumors harboring ARID1A mutations. Here, we investigated whether GLS1 is differentially expressed between patients with OCCC whose tumors are ARID1A positive and patients whose tumors are ARID1A negative. In clinical specimens of OCCC, we found that GLS1 overexpression was not correlated with ARID1A loss. In addition, GLS1 overexpression was associated with better clinical outcomes. Our findings have implications for human trials using experimental therapeutics targeting GLS1. Significance: GLS1 differential expression in patients with OCCC with or without ARID1A mutations is significant because a clinical trial with a GLS1 inhibitor is forthcoming. Tumors without ARID1A have low levels of GLS1 and GLS1 expression is associated to better outcome. Thus, blockade of GLS1 could be counterproductive for patients with OCCC.

Abstract Ovarian cancer is the 7th most common cancer in women. It is the most fatal of all female reproductive cancers, due to the 5-year overall survival of 10-30% and over half of patients being diagnosed with late-stage disease. Despite initial response to front line platinum- and taxol-based chemotherapies, over 70% of patients develop recurrent and chemotherapy resistant disease and nearly all of these women die of their disease. Recently, the use of targeted therapies such as bevacizumab and PARP inhibitors have been shown to improve progression-free survival. However, these regimens have thus far failed to improve overall survival in patients without specific genetic signatures, highlighting the urgent need for alternative therapies. We have identified lestaurtinib as a potent inhibitor of many ovarian cancer cell lines, including platinum and PARP inhibitor resistant models, and patient derived organoid models. RNA-sequencing following lestaurtinib treatment identified JAK/STAT signaling as one of the most profoundly downregulated pathways. Additionally, it was recently reported via single cell RNA sequencing of patient tumors that downstream mediators of the JAK/STAT pathway are among the most highly expressed genes in ovarian tumor cells. Further, we have discovered that STAT1 and STAT3 are constitutively activated in chemotherapy and PARP inhibitor resistant cell lines as reflected by high levels of Tyr701/705 and Ser727 phosphorylation suggesting induction of this pathway may drive resistance. Selective siRNA-mediated knockdown of STAT1/3 resulted in significant growth inhibition confirming their importance in maintaining cell viability. Surprisingly, we have identified profound differences in the ability of various JAK/STAT inhibitors to suppress ovarian cancer cell growth. For example, ruxolitinib, the only JAK/STAT inhibitor currently in clinical trials for ovarian cancer, failed to inhibit the growth sensitive, platinum resistant or PARP inhibitor resistant cell lines. Our preliminary data indicate that JAK/STAT inhibitor efficacy in preventing ovarian cancer cell growth is related to their ability to block specific phosphorylation events on STAT1/3 proteins. We believe that precise and selective inhibition of STAT1 and STAT3 signaling represents a robust, durable, and novel approach for the treatment of highly aggressive and resistant forms of ovarian cancer. Further we plan to elucidate the molecular mechanisms by which STAT1 and STAT3 function to support ovarian cancer cell viability and growth, in order to substantially advance our understanding of JAK/STAT signaling in ovarian cancer and to identify the most effective ways to pharmacologically inhibit JAK/STAT signaling in ovarian cancer cells. Outcomes from this work will have both immediate and long-term impacts for our patients and will lay the foundation for future biomarker-driven clinical trials. Citation Format: Esther Rodman, Michael Emch, Elizabeth Bruinsma, Xiaonan Hou, John Weroha, John Hawse. Interrogating JAK/STAT signaling in ovarian cancer as a potential oncogenic driver and therapeutic target [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 1129.

The poly(ADP-ribose) binding protein CHFR regulates cellular responses to mitotic stress. The deubiquitinase UBC13, which regulates CHFR levels, has been associated with better overall survival in paclitaxel-treated ovarian cancer. Despite the extensive use of taxanes in the treatment of ovarian cancer, little is known about expression of CHFR itself in this disease. In the present study, tissue microarrays containing ovarian carcinoma samples from 417 women who underwent initial surgical debulking were stained with anti-CHFR antibody and scored in a blinded fashion. CHFR levels, expressed as a modified H-score, were examined for association with histology, grade, time to progression (TTP) and overall survival (OS). In addition, patient-derived xenografts from 69 ovarian carcinoma patients were examined for CHFR expression and sensitivity to paclitaxel monotherapy. In clinical ovarian cancer specimens, CHFR expression was positively associated with serous histology (p = 0.0048), higher grade (p = 0.000014) and higher stage (p = 0.016). After correction for stage and debulking, there was no significant association between CHFR staining and overall survival (p = 0.62) or time to progression (p = 0.91) in patients with high grade serous cancers treated with platinum/taxane chemotherapy (N = 249). Likewise, no association between CHFR expression and paclitaxel sensitivity was observed in ovarian cancer PDXs treated with paclitaxel monotherapy. Accordingly, differences in CHFR expression are unlikely to play a major role in paclitaxel sensitivity of high grade serous ovarian cancer.

Abstract PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. Significance: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Abstract In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved. In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages. In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic “scarring,” indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy. As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy. Significance: Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.