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Table S1 shows the cell viability inhibitory rate (%) in SCCOHT cell lines vs other cell lines in the epigenetic drug screen. Table S2 shows the effects of Quisinostat on the proteome of BIN67 cells after treatment for 24 or 72 hours. Table S3 shows the significantly altered proteins by Quisinostat treatment for 24 hours in BIN67 cells. Table S4 shows the significantly altered proteins by Quisinostat treatment for 72 hours in BIN67 cells. Table S5 shows the biological functions that were significantly affected by Quisinostat treatment for 72 hours in BIN67 cells. Table S6 shows the proteins that were significantly regulated by SMARCA4 re-expression in BIN67 cells. Table S7 shows the biological functions that were significantly affected by SMARCA4 re-expression in BIN67 cells. Table S8 shows the proteins that were significantly regulated by both Quisinostat treatment (72 hr) and SMARCA4 re-expression in BIN67 cells. Table S9 shows the biological functions that were significantly affected by both Quisinostat and SMARCA4 re-expression in BIN67 cells.

Supplementary Table from Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

Fig.S1 shows that SCCOHT cells are sensitive to HDAC inhibition. Fig. S2 shows that SCCOHT cells are sensitive to HDAC inhibition or depletion. Fig.S3 shows that SMARCA4/A2 dual deficiency is not associated with hypersensitivity to HDAC inhibitors in lung and pancreatic cancer cell lines. Fig.S4 shows that pan-HDAC inhibitors induce cell morphology changes in SCCOHT cells. Fig.S5 shows that SMARCA2 activation is partially required for the hypersensitivity of SCCOHT cells to HDAC inhibition. Fig. S6 shows the effect of quisinostat on mouse body weights in BIN67 mouse xenograft models. Fig.S7 shows the synergistic effects between HDAC inhibitors and EZH2 inhibitors in SWI/SNF deficiency-driven tumor cells. Fig. S8 shows that depletion of HDAC2 increased sensitivity of BIN67 cells to EPZ-6438 treatment. Fig.S9 shows the synergistic effects between HDAC inhibitors and EZH2 inhibitors in SCCOHT cells. Fig.S10 shows the effect of combined treatment of EPZ-6438 and quisinostat on mouse body weight and survival.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation in vitro and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.

Supplementary Data from Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a Promising Surface Target in Ewing Sarcoma

Cancer cells must overcome anoikis (detachment-induced death) to successfully metastasize. Using proteomic screens, we found that distinct oncoproteins upregulate IL1 receptor accessory protein (IL1RAP) to suppress anoikis. IL1RAP is directly induced by oncogenic fusions of Ewing sarcoma, a highly metastatic childhood sarcoma. IL1RAP inactivation triggers anoikis and impedes metastatic dissemination of Ewing sarcoma cells. Mechanistically, IL1RAP binds the cell-surface system Xc− transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and the glutathione antioxidant. Under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Therefore, IL1RAP maintains cyst(e)ine and glutathione pools, which are vital for redox homeostasis and anoikis resistance. IL1RAP is minimally expressed in pediatric and adult normal tissues, and human anti-IL1RAP antibodies induce potent antibody-dependent cellular cytotoxicity of Ewing sarcoma cells. Therefore, we define IL1RAP as a new cell-surface target in Ewing sarcoma, which is potentially exploitable for immunotherapy.Significance:Here, we identify cell-surface protein IL1RAP as a key driver of metastasis in Ewing sarcoma, a highly aggressive childhood sarcoma. Minimal expression in pediatric and adult normal tissues nominates IL1RAP as a promising target for immunotherapy.See related commentary by Yoon and DeNicola, p. 2679.This article is highlighted in the In This Issue feature, p. 2659

Myeloid ecotropic virus insertion site 1 (MEIS1) is essential for normal hematopoiesis and is a critical factor in the pathogenesis of a large subset of acute myeloid leukemia (AML). Despite the clinical relevance of MEIS1, its regulation is largely unknown. To understand the transcriptional regulatory mechanisms contributing to human MEIS1 expression, we created a knock-in green florescent protein (GFP) reporter system at the endogenous MEIS1 locus in a human AML cell line. Using this model, we have delineated and dissected a critical enhancer region of the MEIS1 locus for transcription factor (TF) binding through in silico prediction in combination with oligo pull-down, mass-spectrometry and knockout analysis leading to the identification of FLI1, an E-twenty-six (ETS) transcription factor, as an important regulator of MEIS1 transcription. We further show direct binding of FLI1 to the MEIS1 locus in human AML cell lines as well as enrichment of histone acetylation in MEIS1-high healthy and leukemic cells. We also observe a positive correlation between high FLI1 transcript levels and worse overall survival in AML patients. Our study expands the role of ETS factors in AML and our model constitutes a feasible tool for a more detailed understanding of transcriptional regulatory elements and their interactome.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry–based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies.Significance:These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

Detailed supplemental methods of cell lines and cell culture, cell cycle synchronization and inducible expression of FOXL2, quantitative mass spectrometry analysis of induced FOXL2 expression, FOXL2 ChIP-seq analysis, RNA-seq analysis, and electrophoretic mobility shift assay.

Known transcription factor motifs enriched in shared and FOXL2 mutant-specific peaks predicted by Homer motif analysis.

IC50 curves for ADI-PEG20 in ovarian cancer cell lines. A, ASS1-deficient ovarian cancer cell lines and B, ASS1-proficient ovarian cancer cell lines.

Table containing proteins differentially expressed in at least two rarer ovarian cancer subtypes when compared to HGSC.

Purpose:Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes.Experimental Design:We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT.Results:Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo.Conclusions:Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

File containing supplemental figures and legends

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W–inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program.Significance:A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

Supplemental material and methods

Supplementary Data from Multiomics Characterization of Low-Grade Serous Ovarian Carcinoma Identifies Potential Biomarkers of MEK Inhibitor Sensitivity and Therapeutic Vulnerability

Clear cell ovarian carcinoma (CCOC) is the second most common subtype of epithelial ovarian carcinoma. Late-stage CCOC is not responsive to gold-standard chemotherapy and results in suboptimal outcomes for patients. In-depth molecular insight is urgently needed to stratify the disease and drive therapeutic development. We conducted global proteomics for 192 cases of CCOC and compared these with other epithelial ovarian carcinoma subtypes. Our results showed distinct proteomic differences in CCOC compared with other epithelial ovarian cancer subtypes including alterations in lipid and purine metabolism pathways. Furthermore, we report potential clinically significant proteomic subgroups within CCOC, suggesting the biologic plausibility of stratified treatment for this cancer. Taken together, our results provide a comprehensive understanding of the CCOC proteomic landscape to facilitate future understanding and research of this disease. © 2022 The Pathological Society of Great Britain and Ireland.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. Analysis of sequencing data from 1,727 unique AML patients revealed frequent mutations in ubiquitin ligase family genes. Loss of function of the Skp1/Cul1/Fbox (SCF) E3 ubiquitin ligase complex genes are found in 8 - 9% of adult AML patients including recurrent mutations in FBXO11. FBXO11 is the most significantly downregulated gene of the SCF complex in AML samples. Depletion of Fbxo11 promotes myeloid-biased stem cell maintenance and cooperates with AML1-ETO and mutant KRAS to generate serially transplantable mouse and human AML in in vivo models. FBXO11 mediates K63-linked polyubiquitination of the LONP1 mitochondrial protease, and loss of FBXO11 impairs LONP1 activity thereby reducing mitochondrial membrane potential, imparting stem cell properties and driving leukemogenesis. Our findings suggest that loss of FBXO11 function primes HSPC for myeloid-biased self-renewal through attenuation of LONP1-mediated regulation of mitochondrial function.

CIC encodes a transcriptional repressor and MAPK signalling effector that is inactivated by loss-of-function mutations in several cancer types, consistent with a role as a tumour suppressor. Here, we used bioinformatic, genomic, and proteomic approaches to investigate CIC’s interaction networks. We observed both previously identified and novel candidate interactions between CIC and SWI/SNF complex members, as well as novel interactions between CIC and cell cycle regulators and RNA processing factors. We found that CIC loss is associated with an increased frequency of mitotic defects in human cell lines and an in vivo mouse model and with dysregulated expression of mitotic regulators. We also observed aberrant splicing in CIC-deficient cell lines, predominantly at 3′ and 5′ untranslated regions of genes, including genes involved in MAPK signalling, DNA repair, and cell cycle regulation. Our study thus characterises the complexity of CIC’s functional network and describes the effect of its loss on cell cycle regulation, mitotic integrity, and transcriptional splicing, thereby expanding our understanding of CIC’s potential roles in cancer. In addition, our work exemplifies how multi-omic, network-based analyses can be used to uncover novel insights into the interconnected functions of pleiotropic genes/proteins across cellular contexts.

There are two serous ovarian cancer histotypes, low and high grade (LGSOC and HGSOC), which are distinct clinical and biological entities. LGSOC is a rare histotype with a relatively stable genome, while HGSC is more common and genomically unstable. Somewhat surprisingly LGSOC expresses high levels of the stimulator of the interferon genes (STING). The STING pathway recognizes cytoplasmic double-stranded DNA and mounts innate cellular immunity through interferon-beta type I production. Our objective is to investigate the aberrant STING signaling in LGSOC and test the effectiveness of oncolytic viruses against LGSOC. We used immunohistochemistry on tissue microarrays (TMAs) to assess STING protein expression in different ovarian cancer histotypes. Whole proteome analysis was applied to identify differentially expressed proteins in LGSOC and HGSOC patient samples (both n=9). Further, a semi-targeted proteomics approach was used to evaluate the expression levels of the STING pathway-related proteins in LGSOC, HGSOC, and LGSOC precursor tumors (each subtype, n=20). To evaluate the key transcription, phosphorylation, and translocation events in STING signaling, we treated LGSOC cell lines with an agonist (dsDNA90) and performed qPCR, immunoblotting, and immunofluorescence experiments, respectively. We tested the viability of the LGSOC cell lines in response to Vaccinia Virus (VV), and Vesicular Stomatitis Virus (VSV) based oncolytic vectors with or without immunostimulatory transgenes. Our results show that STING protein levels were consistently higher in LGSOC TMAs relative to other histotypes. Proteomics analysis showed that the half of the 16 most differentially expressed proteins were the effectors of STING signaling with unexpectedly lower expression in LGSOC, suggesting that despite the robust levels of STING in LGSOC tumors, the pathway is not fully active. Attenuated STING translocation and expression of IFNB1 and other cytokines in LGSOC cell lines confirm the aberrancy in the STING pathway. Semi-targeted proteomics revealed the considerable overexpression of STIM1 in LGSOC patient tumors, which has previously been shown to sequester STING in the endoplasmic reticulum. The treatment with VV and VSV oncolytic viruses significantly reduced the proliferation of LGSOC cell lines. In summary, we find attenuated STING signaling in LGSOC, possibly due to overexpression of STIM1 preventing STING translocation. Although oncolytic viruses show promising results in LGSOC cell lines, more research is needed to determine the optimal treatment strategy, testing oncolytic viruses expressing various transgenes and combination therapies. Citation Format: Almira Zhantuyakova, Dawn Cochrane, Gian Negri, Sandra E. Spencer Miko, Taha Azad, Jutta Huvila, Marta Llaurado Fernandez, Mark Carey, Gregg B. Morin, John Bell, David Huntsman. Deciphering aberrant STING pathway and exploring oncolytic viruses therapy in low grade serous ovarian carcinoma [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 6417.

Introduction: Immunotherapeutic strategies have produced remarkable results in some malignancies. However, optimal cell surface targets in many childhood cancers remain elusive and tools for novel target discovery are limited. We developed a proteogenomic approach to identify high confidence cell surface proteins for immunotherapy development and applied it to neuroblastoma, an often fatal childhood cancer. Through the Pediatric Immunotherapy Discovery and Development Network (PI-DDN), we have extended this approach to 14 high-risk childhood cancers. This effort includes MS-based surfaceome data generation for 175 patient-derived xenograft (PDX) models, 30 primary patient tumors, and 10 human derived cell line models. Methods/Results: To optimize the utility of our approach and data, we are developing a web-based application called PIONEER (Pediatric Integrative Omics Network Enhancing Early Research). The goal of PIONEER is to disseminate data to the broader scientific community and to provide the necessary analysis, query and visualization tools to make these data accessible to everyone, regardless of computational expertise. A pilot version of PIONEER was developed using R Shiny Dashboard and is derived from our neuroblastoma efforts. The application is comprised of two main categories: (1) target discovery data and prioritization (2) target validation and preclinical development. Modules include proteomics, transcriptomics, epigenomics, multi-omics, validation and pre-clinical drug development. Cancer ‘omics data currently housed in PIONEER include tumor and cancer cell line mass-spectrometry based proteomics, RNA-sequencing, and chromatin immunoprecipitation (ChIP) sequencing. Extensive normal tissue expression data from GTEx and mass spectrometry will be integrated. Surface proteins are prioritized through an integrative multi-omic analysis of tumor and normal tissue data. Users can perform queries and cancer subtype and cross-histotype studies, apply custom cutoffs, and generate plots for visualization. We are currently adding functionality to support automatic data analysis and integration for surface proteins (SPACE: Surface Protein Analysis for Collaborative Efforts). Through the target validation and preclinical development modules, users can view an antibody repository, immunofluorescence, immunohistochemistry, drugs in development for each protein, and efficacy in patient derived xenograft models. PIONEER will be deployed using R Connect; data for additional histotypes will be incorporated as available. Conclusion: PIONEER will provide a comprehensive characterization of the surfaceome of high-risk pediatric cancers and a web-based application for data integration, visualization and sharing. This interface facilitates the discovery of optimal immunotherapeutic drug targets in high-risk childhood cancers. Citation Format: Amber K. Weiner, Hemma Murali, Rawan Shraim, Karina L. Conkrite, Alexander B. Radaoui, Daniel Martinez, Brian Mooney, Sandra E. Spencer Miko, Gian Negri, Alberto Delaidelli, Caitlyn de Jong, Yuankun Zhu, Allison P. Heath, Jennifer Pogoriler, Yael P. Mosse, Deanne M. Taylor, Poul H. Sorensen, Gregg B. Morin, Benjamin A. Garcia, John M. Maris, Sharon J. Diskin. PIONEER: harnessing multi-omics data to enhance immunotherapeutic target discovery and development. [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 4515.

Low-grade serous ovarian carcinoma (LGSOC) is a rare tumor subtype with high case fatality rates in patients with metastatic disease. There is a pressing need to develop effective treatments using newly available preclinical models for therapeutic discovery and drug evaluation. Here, we use multiomics integration of whole-exome sequencing, RNA sequencing, and mass spectrometry–based proteomics on 14 LGSOC cell lines to elucidate novel biomarkers and therapeutic vulnerabilities. Comparison of LGSOC cell line data with LGSOC tumor data enabled predictive biomarker identification of MEK inhibitor (MEKi) efficacy, with KRAS mutations found exclusively in MEKi-sensitive cell lines and NRAS mutations found mostly in MEKi-resistant cell lines. Distinct patterns of Catalogue of Somatic Mutations in Cancer mutational signatures were identified in MEKi-sensitive and MEKi-resistant cell lines. Deletions of CDKN2A/B and MTAP genes were more frequent in cell lines than tumor samples and possibly represent key driver events in the absence of KRAS/NRAS/BRAF mutations. These LGSOC cell lines were representative models of the molecular aberrations found in LGSOC tumors. For prediction of in vitro MEKi efficacy, proteomic data provided better discrimination than gene expression data. Condensin, minichromosome maintenance, and replication factor C protein complexes were identified as potential treatment targets in MEKi-resistant cell lines. This study suggests that CDKN2A/B or MTAP deficiency may be exploited using synthetically lethal treatment strategies, highlighting the importance of using proteomic data as a tool for molecular drug prediction. Multiomics approaches are crucial to improving our understanding of the molecular underpinnings of LGSOC and applying this information to develop new therapies. Significance: These findings highlight the utility of global multiomics to characterize LGSOC cell lines as research models, to determine biomarkers of MEKi resistance, and to identify potential novel therapeutic targets.

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W–inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. Significance: A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

Purpose: Many rare ovarian cancer subtypes, such as small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT), have poor prognosis due to their aggressive nature and resistance to standard platinum- and taxane-based chemotherapy. The development of effective therapeutics has been hindered by the rarity of such tumors. We sought to identify targetable vulnerabilities in rare ovarian cancer subtypes. Experimental Design: We compared the global proteomic landscape of six cases each of endometrioid ovarian cancer (ENOC), clear cell ovarian cancer (CCOC), and SCCOHT to the most common subtype, high-grade serous ovarian cancer (HGSC), to identify potential therapeutic targets. IHC of tissue microarrays was used as validation of arginosuccinate synthase (ASS1) deficiency. The efficacy of arginine-depriving therapeutic ADI-PEG20 was assessed in vitro using cell lines and patient-derived xenograft mouse models representing SCCOHT. Results: Global proteomic analysis identified low ASS1 expression in ENOC, CCOC, and SCCOHT compared with HGSC. Low ASS1 levels were validated through IHC in large patient cohorts. The lowest levels of ASS1 were observed in SCCOHT, where ASS1 was absent in 12 of 31 cases, and expressed in less than 5% of the tumor cells in 9 of 31 cases. ASS1-deficient ovarian cancer cells were sensitive to ADI-PEG20 treatment regardless of subtype in vitro. Furthermore, in two cell line mouse xenograft models and one patient-derived mouse xenograft model of SCCOHT, once-a-week treatment with ADI-PEG20 (30 mg/kg and 15 mg/kg) inhibited tumor growth in vivo. Conclusions: Preclinical in vitro and in vivo studies identified ADI-PEG20 as a potential therapy for patients with rare ovarian cancers, including SCCOHT.

The current World Health Organization classification does not distinguish transitional cell carcinoma of the ovary (TCC) from conventional tubo-ovarian high-grade serous carcinoma (HGSC), despite evidence suggesting improved prognosis for patients with TCC; instead, it is considered a morphologic variant of HGSC. The immunohistochemical (IHC) markers applied to date do not distinguish between TCC and HGSC. Therefore, we sought to compare the proteomic profiles of TCC and conventional HGSC to identify proteins enriched in TCC. Prognostic biomarkers in HGSC have proven to be elusive, and our aim was to identify biomarkers of TCC as a way of reliably and reproducibly identifying patients with a favorable prognosis and better response to chemotherapy compared with those with conventional HGSC. Quantitative global proteome analysis was performed on archival material of 12 cases of TCC and 16 cases of HGSC using SP3 (single-pot, solid phase—enhanced, sample preparation)-Clinical Tissue Proteomics, a recently described protocol for full-proteome analysis from formalin-fixed paraffin-embedded tissues. We identified 430 proteins that were significantly enriched in TCC over HGSC. Unsupervised co-clustering perfectly distinguished TCC from HGSC based on protein expression. Pathway analysis showed that proteins associated with cell death, necrosis, and apoptosis were highly expressed in TCCs, whereas proteins associated with DNA homologous recombination, cell mitosis, proliferation and survival, and cell cycle progression pathways had reduced expression. From the proteomic analysis, three potential biomarkers for TCC were identified, claudin-4 (CLDN4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), and minichromosome maintenance protein 7 (MCM7), and tested by IHC analysis on tissue microarrays. In agreement with the proteomic analysis, IHC expression of those proteins was stronger in TCC than in HGSC (p < 0.0001). Using global proteomic analysis, we are able to distinguish TCC from conventional HGSC. Follow-up studies will be necessary to confirm that these molecular and morphologic differences are clinically significant.

A high organic content CE-MS/MS (HOCE-MS/MS) method was developed for the proteomic analysis of envelope proteins extracted from spinach leaves. Separation was performed in a 1-m long hydroxypropyl cellulose coated capillary, using 8% (v/v) formic acid in 70% (v/v) methanol and 22% water as the BGE. A flow-through microvial interface was used to couple the CE system with an Orbitrap Fusion Lumos mass spectrometer, and field-amplified sample stacking was used to improve the concentration sensitivity. Using this optimized method, 3579 peptides and 1141 proteins were identified using the Proteome Discoverer software with a 1% false discovery rate at the protein level. Relative to conventional aqueous CE, HOCE-MS did a better job of discovering hydrophobic peptides and provided more peptide and protein identifications. Relative to nano-LC-MS, it achieved comparable peptide and protein identification performance and detected peptides not identified by LC-MS: of the full set of peptides identified using the two techniques, 19% were identified only using HOCE-MS. It also outperformed nano-LC-MS with respect to the detection of low molecular weight peptides.