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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.

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10–CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1–005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1–005 improved the survival of mice bearing Trp53−/− null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Supplementary Figure from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Table from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary Data from Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma

Supplementary methods and figures

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.

Supplementary Table from The Genomic Landscape of Early-Stage Ovarian High-Grade Serous Carcinoma

Purpose:Ovarian high-grade serous carcinoma (HGSC) is usually diagnosed at late stage. We investigated whether late-stage HGSC has unique genomic characteristics consistent with acquisition of evolutionary advantage compared with early-stage tumors.Experimental Design:We performed targeted next-generation sequencing and shallow whole-genome sequencing (sWGS) on pretreatment samples from 43 patients with FIGO stage I–IIA HGSC to investigate somatic mutations and copy-number (CN) alterations (SCNA). We compared results to pretreatment samples from 52 patients with stage IIIC/IV HGSC from the BriTROC-1 study.Results:Age of diagnosis did not differ between early-stage and late-stage patients (median 61.3 years vs. 62.3 years, respectively). TP53 mutations were near-universal in both cohorts (89% early-stage, 100% late-stage), and there were no significant differences in the rates of other somatic mutations, including BRCA1 and BRCA2. We also did not observe cohort-specific focal SCNA that could explain biological behavior. However, ploidy was higher in late-stage (median, 3.0) than early-stage (median, 1.9) samples. CN signature exposures were significantly different between cohorts, with greater relative signature 3 exposure in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic.Conclusions:Early-stage and late-stage HGSCs have highly similar patterns of mutation and focal SCNA. However, CN signature analysis showed that late-stage disease has distinct signature exposures consistent with whole-genome duplication. Further analyses will be required to ascertain whether these differences reflect genuine biological differences between early-stage and late-stage or simply time-related markers of evolutionary fitness.See related commentary by Yang et al., p. 2730

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10–CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1–005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1–005 improved the survival of mice bearing Trp53−/− null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Background: High grade serous carcinoma (HGSC) is the most common and poorest prognosis subtype of ovarian cancer. The large majority of patients present with advanced (stage IIIC or IV) disease with a median survival of only 3-4 years. By contrast, approximately 10% patients are diagnosed with early stage disease, confined to the ovary/fallopian tube, of whom 90% are cured with surgery and platinum-based chemotherapy. Beyond TP53 mutation, classic activating oncogene mutations are rare in HGSC. Rather, HGSC is marked by extreme copy number (CN) abnormalities, and this complexity has prevented detailed understanding of the mutational processes underpinning outcomes in HGSC. We have used shallow whole genome sequencing (sWGS) to develop novel CN signatures that are able to deconvolute the complexity of HGSC genomes. However, nearly all genomics studies in HGSC have examined advanced stage disease, and little is known about the genomics of early stage HGSC - specifically, it is unclear whether these tumors are identified purely by chance or whether they represent a specific subset that does not metastasize. Hypotheses: We hypothesized that early stage HGSC has a distinct copy number landscape compared to stage IIIC/IV disease. Methods and results: We have identified 43 cases of FIGO stage I-IIA HGSC from the pathology archives of three large London Gynaecological Cancer Centres and 52 late-stage (stage IIIC-IV) cases from BriTROC-1 cohort. Median age at diagnosis was 61.3 years vs 62.3 years respectively. There were no significant differences in mutation rates of TP53 and BRCA1/2, and TP53 mutations were near-universal in both cohorts. We also did not find cohort-specific focal SCNA that could explain biological behavior. However, ploidy was significant higher in late-stage (median 3.0) than early-stage (median 1.9) samples. CN signature exposures were significantly different between early and late stage cohorts. Relative exposure of signature 3 was greater in early-stage and greater signature 4 in late-stage. Unsupervised clustering based on CN signatures identified three clusters that were prognostic. Summary:This project identifed that early stage and late stage HGSC have highly similar patterns of mutation and focal SCNA. However, genome-wide analysis indicates that the abnormalities seen in advanced disease are not present in early stage disease, which may represent a discrete subset with reduced metastatic potential. By identifying and characterizing the copy number signature changes, these data suggest that diagnosis at early-stage might reflect biological differences and not fortuitous chance. These data improve understanding of HGSC biology and may reveal potential new treatment strategies. Citation Format: Zhao Cheng, Hasan B. Mirza, Darren P. Ennis, Philip Smith, Lena Morrill Gavarró, Chishimba Sokota, Gaia Giannone, Teodora Goranova, Thomas Bradley, Anna Piskorz, Michelle Lockley, Baljeet Kaur, Naveena Singh, Laura A. Tookman, Jonathan Krell, Jackie McDermott, Geoff Macintyre, Florian Markowetz, James D. Brenton, Iain A. McNeish. The copy number landscape of early stage ovarian high grade serous carcinoma [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 53.

Background It has been proposed that T helper (Th)2-driven immune deviation in early life can be countered by Th1 inducing childhood infections and that such counter-regulation can protect against allergic asthma. Objective To test whether Th1-inducing infection with Bordetella pertussis protects against allergic asthma using well-characterized murine models. Methods Groups of mice were sensitized to ovalbumin (OVA) in the presence or absence of B. pertussis, a well-characterized Th1 inducing respiratory infection. Immunological, pathological and physiological parameters were measured to assess the impact of infection on immune deviation and airway function. Results We demonstrate that OVA sensitization does not affect the development of B. pertussisspecific immune responses dominated by IgG2a and IFN-g and does not impair Th1-mediated clearance of airway infection. In contrast, B. pertussis infection at the time of sensitization modulated the response to OVA and significantly reduced total serum and OVA-specific IgE. The pattern of cytokine responses, in particular OVA-specific IL-5 responses in the spleen was also modulated. However, B. pertussis did not cause global suppression as IL-10 and IL-13 levels were enhanced in OVA-stimulated spleen cell cultures and in lavage fluid from infected co-sensitized mice. Histopathological examination revealed that B. pertussis infection prior to OVA sensitization resulted in increased inflammation of bronchiolar walls with accompanying hyperplasia and mucous metaplasia of lining epithelia. These pathological changes were accompanied by increased bronchial hyper-reactivity to methacholine exposure. Conclusion Contrary to the above premise, a Th1 response induced by a common childhood infection does not protect against bronchial hyper-reactivity, but rather exacerbates the allergic asthmatic response, despite modulation of immune mediators.

The prevalence of asthma and allergic disease has increased in many countries, and there has been speculation that immunization promotes allergic sensitization. Bordetella pertussis infection exacerbates allergic asthmatic responses. We investigated whether acellular pertussis vaccine (Pa) enhanced or prevented B. pertussis -induced exacerbation of allergic asthma. Groups of mice were immunized with Pa, infected with B. pertussis , and/or sensitized to ovalbumin. Immunological, pathological, and physiological changes were measured to assess the impact of immunization on immune deviation and airway function. We demonstrate that immunization did not enhance ovalbumin-specific serum immunoglobulin E production. Histopathological examination revealed that immunization reduced the severity of airway pathology associated with sensitization in the context of infection and decreased bronchial hyperreactivity upon methacholine exposure of infected and sensitized mice. These data demonstrate unequivocally the benefit of Pa immunization to health and justify selection of Pa in mass vaccination protocols. In the absence of infection, the Pa used in this study enhanced the interleukin-10 (IL-10) and IL-13 responses and influenced airway hyperresponsiveness to sensitizing antigen; however, these data do not suggest that Pa contributes to childhood asthma overall. On the contrary, wild-type virulent B. pertussis is still circulating in most countries, and our data suggest that the major influence of Pa is to protect against the powerful exacerbation of asthma-like pathology induced by B. pertussis .

The prevalence of asthma and allergic disease has increased in many countries and there has been speculation that immunization promotes allergic sensitization. Bordetella pertussis infection exacerbates allergic asthmatic responses. We investigated whether whole-cell pertussis vaccine (Pw) enhanced or prevented B. pertussis induced exacerbation of allergic asthma. Groups of mice were immunized with Pw, infected with B. pertussis and/or sensitized to ovalbumin. Immunological, pathological and physiological changes were measured to assess the impact of Pw immunization on immune deviation and airway function. Pw immunization modulated ovalbumin-specific serum IgE production, and reduced local and systemic IL-13 and other cytokine responses to sensitizing allergen. Histopathological examination revealed Pw immunization reduced the severity of airway pathology and decreased bronchial hyperreactivity to methacholine exposure. Pw does not enhance airway IL-13 and consequently does not enhance but protects against the exacerbation of allergic responses. We find no evidence of Pw contributing to allergic asthma, but rather provide evidence of a mechanism whereby whole-cell pertussis vaccination has a protective role.