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

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.

Targeting the IRE1α/XBP1s pathway suppresses CARM1-expressing ovarian cancer.

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers.

Targeting glutamine dependence through GLS1 inhibition suppresses ARID1A-inactivated clear cell ovarian carcinoma.

Alterations in components of the SWI/SNF chromatin-remodeling complex occur in ~20% of all human cancers. For example, ARID1A is mutated in up to 62% of clear cell ovarian carcinoma (OCCC), a disease currently lacking effective therapies. Here we show that ARID1A mutation creates a dependence on glutamine metabolism. SWI/SNF represses glutaminase (GLS1) and ARID1A inactivation upregulates GLS1. ARID1A inactivation increases glutamine utilization and metabolism through the tricarboxylic acid cycle to support aspartate synthesis. Indeed, glutaminase inhibitor CB-839 suppresses the growth of ARID1A mutant, but not wildtype, OCCCs in both orthotopic and patient-derived xenografts. In addition, glutaminase inhibitor CB-839 synergizes with immune checkpoint blockade anti-PDL1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation. Our data indicate that pharmacological inhibition of glutaminase alone or in combination with immune checkpoint blockade represents an effective therapeutic strategy for cancers involving alterations in the SWI/SNF complex such as ARID1A mutations.

Mouse models of epithelial ovarian cancer for preclinical studies.

Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer-related mortality in the developed world. EOC is a heterogeneous disease represented by several histological and molecular subtypes. Therefore, exploration of relevant preclinical animal models that consider the heterogenic nature of EOC is of great importance for the development of novel therapeutic strategies that can be translated clinically to combat this devastating disease. In this review, we discuss recent progress in the development of preclinical mouse models for EOC study as well as their advantages and limitations.

Targeting CARM1 in ovarian cancer with EZH2 and PARP inhibitors.

Coactivator-associated arginine methyltransferase 1 (CARM1)-expressing high-grade serous ovarian cancers are characterized by poor prognosis and limited therapeutic options. Here we discuss a novel therapeutic strategy to target CARM1-expressing ovarian cancer based on a combination of poly (ADP-ribose) polymerase (PARP) and enhancer of zeste homology 2 (EZH2) inhibitors.

EZH2 Inhibition Sensitizes CARM1-High, Homologous Recombination Proficient Ovarian Cancers to PARP Inhibition.

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts.

HDAC6 Inhibition Synergizes with Anti-PD-L1 Therapy in ARID1A-Inactivated Ovarian Cancer.

ARID1A, encoding a subunit of the SWI/SNF complex, is the most frequently mutated epigenetic regulator in human cancers and is mutated in more than 50% of ovarian clear cell carcinomas (OCCC), a disease that currently has no effective therapy. Inhibition of histone deacetylase 6 (HDAC6) suppresses the growth of ARID1A-mutated tumors and modulates tumor immune microenvironment. Here, we show that inhibition of HDAC6 synergizes with anti-PD-L1 immune checkpoint blockade in ARID1A-inactivated ovarian cancer. ARID1A directly repressed transcription of CD274, the gene encoding PD-L1. Reduced tumor burden and improved survival were observed in ARID1Aflox/flox/PIK3CAH1047R OCCC mice treated with the HDAC6 inhibitor ACY1215 and anti-PD-L1 immune checkpoint blockade as a result of activation and increased presence of IFNγ-positive CD8 T cells. We confirmed that the combined treatment limited tumor progression in a cytotoxic T-cell-dependent manner, as depletion of CD8+ T cells abrogated these antitumor effects. Together, these findings indicate that combined HDAC6 inhibition and immune checkpoint blockade represents a potential treatment strategy for ARID1A-mutated cancers. SIGNIFICANCE: These findings offer a mechanistic rationale for combining epigenetic modulators and existing immunotherapeutic interventions against a disease that has been so far resistant to checkpoint blockade as a monotherapy.See related commentary by Prokunina-Olsson, p. 5476.

N6-Methylation of Adenosine of FZD10 mRNA Contributes to PARP Inhibitor Resistance.

Despite the high initial response rates to PARP inhibitors (PARPi) in BRCA-mutated epithelial ovarian cancers (EOC), PARPi resistance remains a major challenge. Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of mRNA, yet the role of m6A modification in PARPi resistance has not previously been explored. Here, we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/ß-catenin pathway in BRCA-mutated EOC cells. Global m6A profile revealed a significant increase in m6A modification in FZD10 mRNA, which correlated with increased FZD10 mRNA stability and an upregulation of the Wnt/ß-catenin pathway. Depletion of FZD10 or inhibition of the Wnt/ß-catenin sensitizes resistant cells to PARPi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity. Moreover, combined inhibition of PARP and Wnt/ß-catenin showed synergistic suppression of PARPi-resistant cells in vitro and in vivo in a xenograft EOC mouse model. Overall, our results show that m6A contributes to PARPi resistance in BRCA-deficient EOC cells by upregulating the Wnt/ß-catenin pathway via stabilization of FZD10. They also suggest that inhibition of the Wnt/ß-catenin pathway represents a potential strategy to overcome PARPi resistance. SIGNIFICANCE: These findings elucidate a novel regulatory mechanism of PARPi resistance in EOC by showing that m6A modification of FZD10 mRNA contributes to PARPi resistance in BRCA-deficient EOC cells via upregulation of Wnt/ß-catenin pathway.

Repurposing Pan-HDAC Inhibitors for ARID1A-Mutated Ovarian Cancer.

ARID1A, a subunit of the SWI/SNF complex, is among the most frequently mutated genes across cancer types. ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCCs), diseases that have no effective therapy. Here, we show that ARID1A mutation confers sensitivity to pan-HDAC inhibitors such as SAHA in ovarian cancers. This correlated with enhanced growth suppression induced by the inhibition of HDAC2 activity in ARID1A-mutated cells. HDAC2 interacts with EZH2 in an ARID1A status-dependent manner. HDAC2 functions as a co-repressor of EZH2 to suppress the expression of EZH2/ARID1A target tumor suppressor genes such as PIK3IP1 to inhibit proliferation and promote apoptosis. SAHA reduced the growth and ascites of the ARID1A-inactivated OCCCs in both orthotopic and genetic mouse models. This correlated with a significant improvement of survival of mice bearing ARID1A-mutated OCCCs. These findings provided preclinical rationales for repurposing FDA-approved pan-HDAC inhibitors for treating ARID1A-mutated cancers.

CARM1-expressing ovarian cancer depends on the histone methyltransferase EZH2 activity.

CARM1 is an arginine methyltransferase that asymmetrically dimethylates protein substrates on arginine residues. CARM1 is often overexpressed in human cancers. However, clinically applicable cancer therapeutic strategies based on CARM1 expression remain to be explored. Here, we report that EZH2 inhibition is effective in CARM1-expressing epithelial ovarian cancer. Inhibition of EZH2 activity using a clinically applicable small molecule inhibitor significantly suppresses the growth of CARM1-expressing, but not CARM1-deficient, ovarian tumors in two xenograft models and improves the survival of mice bearing CARM1-expressing ovarian tumors. The observed selectivity correlates with reactivation of EZH2 target tumor suppressor genes in a CARM1-dependent manner. Mechanistically, CARM1 promotes EZH2-mediated silencing of EZH2/BAF155 target tumor suppressor genes by methylating BAF155, which leads to the displacement of BAF155 by EZH2. Together, these results indicate that pharmacological inhibition of EZH2 represents a novel therapeutic strategy for CARM1-expressing cancers.

BET Bromodomain Inhibition Synergizes with PARP Inhibitor in Epithelial Ovarian Cancer.

PARP inhibition is known to be an effective clinical strategy in BRCA mutant cancers, but PARP inhibition has not been applied to BRCA-proficient tumors. Here, we show the synergy of BET bromodomain inhibition with PARP inhibition in BRCA-proficient ovarian cancers due to mitotic catastrophe. Treatment of BRCA-proficient ovarian cancer cells with the BET inhibitor JQ1 downregulated the G2-M cell-cycle checkpoint regulator WEE1 and the DNA-damage response factor TOPBP1. Combining PARP inhibitor Olaparib with the BET inhibitor, we observed a synergistic increase in DNA damage and checkpoint defects, which allowed cells to enter mitosis despite the accumulation of DNA damage, ultimately causing mitotic catastrophe. Moreover, JQ1 and Olaparib showed synergistic suppression of growth of BRCA-proficient cancer in vivo in a xenograft ovarian cancer mouse model. Our findings indicate that a combination of BET inhibitor and PARP inhibitor represents a potential therapeutic strategy for BRCA-proficient cancers.

Progress toward overcoming hypoxia-induced resistance to solid tumor therapy.

Hypoxic tumors are associated with poor clinical outcome for multiple types of human cancer. This may be due, in part, to hypoxic cancer cells being resistant to anticancer therapy, including radiation therapy, chemotherapy, and targeted therapy. Hypoxia inducible factor 1, a major regulator of cellular response to hypoxia, regulates the expression of genes that are involved in multiple aspects of cancer biology, including cell survival, proliferation, metabolism, invasion, and angiogenesis. Here, we review multiple pathways regulated by hypoxia/hypoxia inducible factor 1 in cancer cells and discuss the latest advancements in overcoming hypoxia-mediated tumor resistance.

Hypoxia/HIF1α induces lapatinib resistance in ERBB2-positive breast cancer cells via regulation of DUSP2.

ERBB2/HER2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can promote tumor progression. Breast cancer patients with ERBB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that specifically blocks EGFR/ERBB2 signaling. Here, we show that hypoxia, via HIF-1, induces resistance to lapatinib-mediated effects in ERBB2-expressing mammary epithelial and ERBB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three-dimensional (3D) cultures are decreased under hypoxic conditions. Hypoxia can maintain activation of signaling pathways downstream from ERBB2 including AKT and ERK in the presence of lapatinib. HIF-1 is both required and sufficient to induce lapatinib resistance as overexpression of stable HIF-1 in ERBB2-expressing cells blocks lapatinib-mediated effects and maintains ERBB2-downstream signaling under normoxic conditions. Under hypoxia, activation of ERK signaling is required for lapatinib resistance as treatment with MEK inhibitor trametinib reverses hypoxia-mediated lapatinib resistance. HIF-1 can bypass the lapatinib-treated inhibition of the ERK pathway via inhibition of the dual-specificity phosphatase 2 (DUSP2). Indeed, overexpression of DUSP2 in ErbB2-positve breast cancer cells reverses hypoxia-mediated lapatinib resistance. Thus, our results provide rationale for therapeutic evaluation of the treatment of hypoxic ERBB2 expressing breast tumors with a combination of lapatinib and MEK inhibitors.

The oncogene HER2/neu (ERBB2) requires the hypoxia-inducible factor HIF-1 for mammary tumor growth and anoikis resistance.

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.