The KDM5 Family of Histone Lysine Demethylases Are Targets of R-2-Hydroxyglutarate in IDH Mutant Tumors

Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in clonal hematopoiesis of unknown significance (CHIP), lower- and higher-grade myeloproliferative (MPN) and myelodysplastic (MDS) disorders, and primary and secondary AML. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity. To ask if the in vitro transforming activity of R-2HG directly correlates with TET2 inhibition, we treated TF-1 cells, a cytokine-dependent human AML cell line, with a dose range of cell-permeable esterified R-2HG. We found that R-2HG induces cytokine independence at concentrations that have no effect on 5hmC levels. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence. Likewise, treatment of TF-1 cells with KDM5c70, a specific inhibitor of KDM5 enzymes, strongly induces TF-1 cytokine independence. Of note, KDM5 inhibition has no effect on TET2 expression or 5hmC levels. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls. TET enzymes are not recurrently mutated in glioma and although there is a strong correlation between mutant IDH status and the CpG island methylator phenotype (CIMP), direct inhibition of TET2 by R-2HG has not been reproducibly demonstrated in glioma. To ask if TET2 activity is suppressed in IDH mutant glioma, we quantified 5hmC levels in a panel of primary IDH wild-type and IDH mutant glioma and AML samples by mass spectrometry. We found that, unlike in AML, in glioma there is no correlation between IDH1 mutation status and loss of 5hmC. We likewise saw no correlation between 5hmC levels and either IDH mutation status or intracellular R-2HG levels in patient derived xenograft (PDX) models of glioma. Given the lack of evidence that TET enzymes are tumor suppressor targets of R-2HG in IDH mutant glioma, we asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma PDX lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1. Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. Disclosures No relevant conflicts of interest to declare.

Inhibition of histone lysine demethylases with TACH101, a first-in-class pan-inhibitor of KDM4.

3105 Background: The KDM4 family of histone lysine demethylases consists of four main isoforms (KDM4A, B, C, D), all of which have been identified as key oncogenic drivers. They function as epigenetic regulators and control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play an important role in the epigenetic dysregulation in various cancers and is linked to more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform appears to not be effective. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Here we present data that show TACH101 has promising pre-clinical and pharmacologic properties as a cancer therapeutic. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines, apoptotic and cell cycle analyses, and efficacy studies in various xenograft tumor models and patient-derived organoid models. Results: In vitro, TACH101 was broadly effective in killing 67% (200 out of 300) of cancer cell lines screened. TACH101 demonstrated potent increase of H3K36me3 levels (EC50 < 0.001 mM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and potent anti-proliferative activity in multiple cell lines in OncoPanel. TACH101 treatment increased cancer cell population in S-phase in multiple cancer cell lines indicating cell-cycle arrest. TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. In vivo, TACH101 triggered effective tumor control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Further evaluation using a panel of patient-derived colorectal models and patient-derived organoids showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). TACH101 also reduced tumorigenic potential by 4.4-fold as determined by FACS analysis using sorted CD44High EpCAM+ population in Limiting Dilution Assays in vivo, suggesting that reduction of cancer stem cells by TACH101 may be effective in therapy-resistant settings. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Extensive preclinical work on TACH101 KDM4 inhibitor shows compelling data and broad applicability as a potential anti-cancer agent. Further evaluation is ongoing to advance the molecule into clinical trials.

JmjC-KDMs KDM3A and KDM6B modulate radioresistance under hypoxic conditions in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients’ survival.