Benjamin G. Bitler

Mutant p53 binds and controls estrogen receptor activity to drive endocrine resistance in ovarian cancer

High-grade serous ovarian cancer (HGSOC) is a highly lethal gynecologic malignancy in women. Women diagnosed with HGSOC initially respond to chemotherapy, but there is a >80% rate of relapse. There is thus a significant unmet need for new therapeutic targets for HGSOC. Estrogen receptor α (ERα) is a particularly attractive candidate, as ∼70% of HGSOC tumors stain positively for ERα and there are approved inhibitors that show limited toxicity. However, unlike the case for breast cancer, endocrine therapy for HGSOC has not shown consistently promising results. In this work, we show that missense mutant forms of p53, which occur in >60% of HGSOC, bind and inhibit ERα function and confer resistance to fulvestrant and elacestrant. Mechanistically, we show that mutant p53 predominantly inhibits one arm of the ERα pathway—the transactivation of jointly regulated ERα–SP1 target genes such as the mTOR regulator DEPTOR . We show that silencing mutant p53 restores the ability of ERα to transactivate ERα–SP1 target genes and renders HGSOC markedly more sensitive to endocrine therapy. Consistent with this premise, we show that the p53 mutant Y220C refolding compound rezatapopt enhances fulvestrant response in a Y220C mutant cell line.

Leveraging Multi-omics to Disentangle the Complexity of Ovarian Cancer

Tumor-Intrinsic Activity of Chromobox 2 Remodels the Tumor Microenvironment in High-grade Serous Carcinoma

Abstract Chromobox 2 (CBX2), an epigenetic reader and component of polycomb repressor complex 1, is highly expressed in >75% of high-grade serous carcinoma. Increased CBX2 expression is associated with poorer survival, whereas CBX2 knockdown leads to improved chemotherapy sensitivity. In a high-grade serous carcinoma immune-competent murine model, knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME), understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with specific immune cell types in the TIME. RNA sequencing and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found that modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2-overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53-null, Brca2-null ID8 syngeneic murine model (ID8 Trp53−/−Brca2−/−) led to decreased tumor progression compared with the control. NanoString immuno-oncology panel analysis suggested that knockdown in Cbx2 shifts immune cell composition, with an increase in macrophages. Multispectral immunohistochemistry (mIHC) further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of protumor macrophages, and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies. Significance: CBX2 expression correlates with the TIME. CBX2 modulation shifts the macrophage population, potentially leading to an immunosuppressive microenvironment, highlighting CBX2 as a target to improve efficacy of immunotherapy.

The Spatial Structure of the Tumor Immune Microenvironment Can Explain and Predict Patient Response in High-Grade Serous Carcinoma

Abstract Ovarian cancer is the deadliest gynecologic malignancy, and therapeutic options and mortality rates over the last three decades have largely not changed. Recent studies indicate that the composition of the tumor immune microenvironment (TIME) influences patient outcomes. To improve spatial understanding of the TIME, we performed multiplexed ion beam imaging on 83 human high-grade serous carcinoma tumor samples, identifying approximately 160,000 cells across 23 cell types. From the 77 of these samples that met inclusion criteria, we generated composition features based on cell type proportions, spatial features based on the distances between cell types, and spatial network features representing cell interactions and cell clustering patterns, which we linked to traditional clinical and IHC variables and patient overall survival (OS) and progression-free survival (PFS) outcomes. Among these features, we found several significant univariate correlations, including B-cell contact with M1 macrophages (OS HR = 0.696; P = 0.011; PFS HR = 0.734; P = 0.039). We then used high-dimensional random forest models to evaluate out-of-sample predictive performance for OS and PFS outcomes and to derive relative feature importance scores for each feature. The top model for predicting low or high PFS used TIME composition and spatial features and achieved an average AUC score of 0.71. The results demonstrate the importance of spatial structure in understanding how the TIME contributes to treatment outcomes. Furthermore, the present study provides a generalizable roadmap for spatial analyses of the TIME in ovarian cancer research.

Claudin-4 Modulates Autophagy via SLC1A5/LAT1 as a Mechanism to Regulate Micronuclei

Abstract Genome instability is a hallmark of cancer crucial for tumor heterogeneity and is often a result of defects in cell division and DNA damage repair. Tumors tolerate genomic instability, but the accumulation of genetic aberrations is regulated to avoid catastrophic chromosomal alterations and cell death. In ovarian cancer tumors, claudin-4 is frequently upregulated and closely associated with genome instability and worse patient outcomes. However, its biological association with regulating genomic instability is poorly understood. Here, we used CRISPR interference and a claudin mimic peptide to modulate the claudin-4 expression and its function in vitro and in vivo. We found that claudin-4 promotes a tolerance mechanism for genomic instability through micronuclei generation in tumor cells. Disruption of claudin-4 increased autophagy and was associated with the engulfment of cytoplasm-localized DNA. Mechanistically, we observed that claudin-4 establishes a biological axis with the amino acid transporters SLC1A5 and LAT1, which regulate autophagy upstream of mTOR. Furthermore, the claudin-4/SLC1A5/LAT1 axis was linked to the transport of amino acids across the plasma membrane as one of the potential cellular processes that significantly decreased survival in ovarian cancer patients. Together, our results show that the upregulation of claudin-4 contributes to increasing the threshold of tolerance for genomic instability in ovarian tumor cells by limiting its accumulation through autophagy. Significance: Autophagy regulation via claudin-4/SLC1A5/LAT1 has the potential to be a targetable mechanism to interfere with genomic instability in ovarian tumor cells.

WNT4 Regulates Cellular Metabolism via Intracellular Activity at the Mitochondria in Breast and Gynecologic Cancers

Abstract Wnt ligand WNT4 is critical in female reproductive tissue development, with WNT4 dysregulation linked to related pathologies including breast cancer (invasive lobular carcinoma, ILC) and gynecologic cancers. WNT4 signaling in these contexts is distinct from canonical Wnt signaling yet inadequately understood. We previously identified atypical intracellular activity of WNT4 (independent of Wnt secretion) regulating mitochondrial function, and herein examine intracellular functions of WNT4. We further examine how convergent mechanisms of WNT4 dysregulation impact cancer metabolism. In ILC, WNT4 is co-opted by estrogen receptor α (ER) via genomic binding in WNT4 intron 1, while in gynecologic cancers, a common genetic polymorphism (rs3820282) at this ER binding site alters WNT4 regulation. Using proximity biotinylation (BioID), we show canonical Wnt ligand WNT3A is trafficked for secretion, but WNT4 is localized to the cytosol and mitochondria. We identified DHRS2, mTOR, and STAT1 as putative WNT4 cytosolic/mitochondrial signaling partners. Whole metabolite profiling, and integrated transcriptomic data, support that WNT4 mediates metabolic reprogramming via fatty acid and amino acid metabolism. Furthermore, ovarian cancer cell lines with rs3820282 variant genotype are WNT4 dependent and have active WNT4 metabolic signaling. In protein array analyses of a cohort of 103 human gynecologic tumors enriched for patient diversity, germline rs3820282 genotype is associated with metabolic remodeling. Variant genotype tumors show increased AMPK activation and downstream signaling, with the highest AMPK signaling activity in variant genotype tumors from non-White patients. Taken together, atypical intracellular WNT4 signaling, in part via genetic dysregulation, regulates the distinct metabolic phenotypes of ILC and gynecologic cancers. Significance: WNT4 regulates breast and gynecologic cancer metabolism via a previously unappreciated intracellular signaling mechanism at the mitochondria, with WNT4 mediating metabolic remodeling. Understanding WNT4 dysregulation by estrogen and genetic polymorphism offers new opportunities for defining tumor biology, precision therapeutics, and personalized cancer risk assessment.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR

AbstractOvarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Mechanisms of Ovarian Cancer-Associated Cachexia

Abstract Cancer-associated cachexia occurs in 50% to 80% of cancer patients and is responsible for 20% to 30% of cancer-related deaths. Cachexia limits survival and treatment outcomes, and is a major contributor to morbidity and mortality during cancer. Ovarian cancer is one of the leading causes of cancer-related deaths in women, and recent studies have begun to highlight the prevalence and clinical impact of cachexia in this population. Here, we review the existing understanding of cachexia pathophysiology and summarize relevant studies assessing ovarian cancer–associated cachexia in clinical and preclinical studies. In clinical studies, there is increased evidence that reduced skeletal muscle mass and quality associate with worse outcomes in subjects with ovarian cancer. Mouse models of ovarian cancer display cachexia, often characterized by muscle and fat wasting alongside inflammation, although they remain underexplored relative to other cachexia-associated cancer types. Certain soluble factors have been identified and successfully targeted in these models, providing novel therapeutic targets for mitigating cachexia during ovarian cancer. However, given the relatively low number of studies, the translational relevance of these findings is yet to be determined and requires more research. Overall, our current understanding of ovarian cancer–associated cachexia is insufficient and this review highlights the need for future research specifically aimed at exploring mechanisms of ovarian cancer–associated cachexia by using unbiased approaches and animal models representative of the clinical landscape of ovarian cancer.

The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval

Abstract Purpose: Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment. Experimental Design: We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC. Results: The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence. Conclusions: Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

The SETDB1–TRIM28 Complex Suppresses Antitumor Immunity

Abstract The tumor immune microenvironment is influenced by the epigenetic landscape of the tumor. Here, we have identified the SETDB1–TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR–Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a suppressor of PD-L1 expression. We then revealed that expression of the SETDB1–TRIM28 complex negatively correlated with infiltration of effector CD8+ T cells. Inhibition of SETDB1–TRIM28 simultaneously upregulated PD-L1 and activated the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) innate immune response pathway to increase infiltration of CD8+ T cells. Mechanistically, SETDB1–TRIM28 inhibition led to micronuclei formation in the cytoplasm, which is known to activate the cGAS–STING pathway. Thus, SETDB1–TRIM28 inhibition bridges innate and adaptive immunity. Indeed, SETDB1 knockout enhanced the antitumor effects of immune checkpoint blockade with anti–PD-L1 in a mouse model of ovarian cancer in a cGAS-dependent manner. Our findings establish the SETDB1–TRIM28 complex as a regulator of antitumor immunity and demonstrate that its loss activates cGAS–STING innate immunity to boost the antitumor effects of immune checkpoint blockade.

Assessing Genetic Variants in Matched Biocompartments From Patients With Serous Ovarian Cancer

The clinical use of molecular tumor profiling (MTP) is expanding and there is an increasing use of MTP data to manage patient care. At the University of Colorado, 18 patients were diagnosed with primary serous ovarian cancer between 9/2015 and 6/2019 and consented for banking and analysis of tumor, ascites and plasma. All 18 patients had tumor and plasma samples that were sent for MTP, and 13 of 18 patients additionally had ascites collected and sent for MTP. 50-gene panel testing and BRCA testing were performed on primary tumor. BRCA genetic variants were more likely to be identified in plasma as compared to ascites or tumor, though not statistically significant ( P = 0.17). Co-occurring genetic variants between plasma and ascites were less common in comparison to co-occurring variants between tumor and plasma or tumor and ascites, though not statistically significant ( P = 0.68). Variants in KDR (VEGFR2) and TP53 were most likely to be conserved across all 3 biocompartments. Mutant allele frequencies (MAF) of individual genetic variants varied across biocompartments, though tended to be highest in the tumor, followed by ascites.

KDM5A Inhibits Antitumor Immune Responses Through Downregulation of the Antigen-Presentation Pathway in Ovarian Cancer

Abstract The extent to which effector CD8+ T cells infiltrate into tumors is one of the major predictors of clinical outcome for patients with epithelial ovarian cancer (EOC). Immune cell infiltration into EOC is a complex process that could be affected by the epigenetic makeup of the tumor. Here, we have demonstrated that a lysine 4 histone H3 (H3K4) demethylase, (lysine-specific demethylase 5A; KDM5A) impairs EOC infiltration by immune cells and inhibits antitumor immune responses. Mechanistically, we found that KDM5A silenced genes involved in the antigen processing and presentation pathway. KDM5A inhibition restored the expression of genes involved in the antigen-presentation pathway in vitro and promoted antitumor immune responses mediated by CD8+ T cells in vivo in a syngeneic EOC mouse model. A negative correlation between expression of KDM5A and genes involved in the antigen processing and presentation pathway such as HLA-A and HLA-B was observed in the majority of cancer types. In summary, our results establish KDM5A as a regulator of CD8+ T-cell infiltration of tumors and demonstrate that KDM5A inhibition may provide a novel therapeutic strategy to boost antitumor immune responses.

Loss of Claudin-4 Reduces DNA Damage Repair and Increases Sensitivity to PARP Inhibitors

Abstract High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.

Utilizing Serum-Derived Lipidomics with Protein Biomarkers and Machine Learning for Early Detection of Ovarian Cancer in the Symptomatic Population

Abstract Ovarian cancer is the fifth leading cause of cancer-related deaths among women. Most patients are diagnosed at late stage (III/IV), resulting in a 5-year survival rate below 30%. This is driven by the presentation of vague abdominal symptoms that confound diagnosis at early stages (I/II) and a shortage of robust biomarkers. We are taking a novel approach for earlier ovarian cancer detection, leveraging lipids as biomarkers. We utilized untargeted ultrahigh pressure liquid chromatography–mass spectrometry to analyze sera from two large, independent cohorts (N = 433 and N = 399) designed to reflect the symptomatic population, including individuals with benign adnexal masses, early- and late-stage ovarian cancer, gastrointestinal disorders, and otherwise healthy women seeking care for symptoms. We identified a significantly altered lipid profile in ovarian cancer and early-stage ovarian cancer specifically across both cohorts compared with controls. We also profiled select protein biomarkers (cancer antigen 125, human epididymis protein 4, β-2 folate receptor α, and mucin 1) and, utilizing machine learning–based modeling, identified a proof-of-concept multiomic model consisting of less than 20 top-performing lipid and protein features. This model was trained on cohort 1 and tested on cohort 2, achieving AUCs of 92% (95% confidence interval, 87%–95%) for distinguishing ovarian cancer from controls and 88% (95% confidence interval, 83%–93%) for distinguishing early-stage ovarian cancer from controls. These findings demonstrate the clinical utility and robustness of lipids as proof-of-concept diagnostic biomarkers for early ovarian cancer within the clinically complex symptomatic population, particularly when applied in a multiomic approach. Significance: Patients with ovarian cancer endure delayed diagnosis and poor outcomes. We profiled lipids in two cohorts and integrated them with proteins in machine learning. This enabled early-stage detection in a complex range of controls.

Claudin-4 Stabilizes the Genome via Nuclear and Cell-Cycle Remodeling to Support Ovarian Cancer Cell Survival

Abstract Alterations in the interplay between the nucleus and the cell cycle during cancer development lead to a state of genomic instability, often accompanied by observable morphologic aberrations. Tumor cells can regulate these aberrations to evade cell death, either by preventing or eliminating genomic instability. In epithelial ovarian cancer, overexpression of claudin-4 significantly contributes to therapy resistance through mechanisms associated with genomic instability regulation. However, the molecular mechanisms underlying claudin-4 overexpression in epithelial ovarian cancer remain poorly understood. In this study, we modified claudin-4 expression and employed a unique claudin mimic peptide to investigate claudin-4’s function. Our findings show that claudin-4 supports ovarian cancer cell survival by stabilizing the genome through nuclear and cell-cycle remodeling. Specifically, claudin-4 induced nuclear constriction by excluding lamin B1 and promoting perinuclear F-actin accumulation, thereby altering nuclear structure and dynamics. Similarly, cell-cycle modifications due to claudin-4 overexpression resulted in fewer cells entering the S-phase and reduced genomic instability in tumors. Importantly, disrupting claudin-4’s biological effects using claudin mimic peptide and forskolin increased the efficacy of PARP inhibitor treatment, correlating with alterations in the oxidative stress response. Our data indicate that claudin-4 protects tumor genome integrity by modulating the crosstalk between the nucleus and the cell cycle, leading to resistance to genomic instability formation and the effects of genomic instability–inducing agents. Significance: High-grade serous ovarian carcinoma is marked by chromosomal instability, which can serve to promote disease progression and allow cancer to evade therapeutic insults. The report highlights the role of claudin-4 in regulating genomic instability and proposes a novel therapeutic approach to exploit claudin-4–mediated regulation.

Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation

Abstract 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 double-stranded RNA 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 antitumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

Targeting Tryptophan Catabolism in Ovarian Cancer to Attenuate Macrophage Infiltration and PD-L1 Expression

Abstract High-grade serous carcinoma (HGSC) of the fallopian tube, ovary, and peritoneum is the most common type of ovarian cancer and is predicted to be immunogenic because the presence of tumor-infiltrating lymphocytes conveys a better prognosis. However, the efficacy of immunotherapies has been limited because of the immune-suppressed tumor microenvironment (TME). Tumor metabolism and immune-suppressive metabolites directly affect immune cell function through the depletion of nutrients and activation of immune-suppressive transcriptional programs. Tryptophan (TRP) catabolism is a contributor to HGSC disease progression. Two structurally distinct rate-limiting TRP catabolizing enzymes, indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), evolved separately to catabolize TRP. IDO1/TDO2 are aberrantly expressed in carcinomas and metabolize TRP into the immune-suppressive metabolite kynurenine (KYN), which can engage the aryl hydrocarbon receptor to drive immunosuppressive transcriptional programs. To date, IDO inhibitors tested in clinical trials have had limited efficacy, but those inhibitors did not target TDO2, and we find that HGSC cell lines and clinical outcomes are more dependent on TDO2 than IDO1. To identify inflammatory HGSC cancers with poor prognosis, we stratified patient ascites samples by IL6 status, which correlates with poor prognosis. Metabolomics revealed that IL6-high patient samples had enriched KYN. TDO2 knockdown significantly inhibited HGSC growth and TRP catabolism. The orally available dual IDO1/TDO2 inhibitor, AT-0174, significantly inhibited tumor progression, reduced tumor-associated macrophages, and reduced expression of immune-suppressive proteins on immune and tumor cells. These studies demonstrate the importance of TDO2 and the therapeutic potential of AT-0174 to overcome an immune-suppressed TME. Significance: Developing strategies to improve response to chemotherapy is essential to extending disease-free intervals for patients with HGSC of the fallopian tube, ovary, and peritoneum. In this article, we demonstrate that targeting TRP catabolism, particularly with dual inhibition of TDO2 and IDO1, attenuates the immune-suppressive microenvironment and, when combined with chemotherapy, extends survival compared with chemotherapy alone.