Abnormally formed FUS/EWS/TAF15 (FET) fusion oncoproteins are essential oncogenic drivers in many human cancers. Interestingly, at the molecular level, they also form biomolecular condensates at specific loci. However, how these condensates lead to gene transcription and how features encoded in the DNA element regulate condensate formation remain unclear. Here, we develop an in vitro single-molecule assay to visualize phase separation on DNA. Using this technique, we observe that FET fusion proteins undergo phase separation at target binding loci and the phase separated condensates recruit RNA polymerase II and enhance gene transcription. Furthermore, we determine a threshold number of fusion-binding DNA elements that can enhance the formation of FET fusion protein condensates. These findings suggest that FET fusion oncoprotein promotes aberrant gene transcription through loci-specific phase separation, which may contribute to their oncogenic transformation ability in relevant cancers, such as sarcomas and leukemia.

Growing evidence has demonstrated that epigenetic dysregulation is a common pathological feature in human cancer cells. Global alterations in the epigenetic landscape are prevalent in malignant cells across different solid tumors including, prostate cancer, non-small-cell lung cancer, renal cell carcinoma, and in haemopoietic malignancy. In particular, DNA hypomethylation and histone hypoacetylation have been observed in acute myeloid leukemia (AML) patient blasts, with histone methylation being an emerging area of study. Histone 3 lysine 9 trimethylation (H3K9me3) is a post-translational modification known to be involved in the regulation of a broad range of biological processes, including the formation of transcriptionally silent heterochromatin. Following the observation of its aberrant methylation status in hematological malignancy and several other cancer phenotypes, recent studies have associated H3K9me3 levels with patient outcome and highlighted key molecular mechanisms linking H3K9me3 profile with AML etiology in a number of large-scale meta-analysis. Consequently, the development and application of small molecule inhibitors which target the histone methyltransferases or demethylase enzymes known to participate in the oncogenic regulation of H3K9me3 in AML represents an advancing area of ongoing study. Here, we provide a comprehensive review on how this particular epigenetic mark is regulated within cells and its emerging role as a potential therapeutic target in AML, along with an update on the current research into advancing the generation of more potent and selective inhibitors against known H3K9 methyltransferases and demethylases.

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed-lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of the KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing downregulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing down-regulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

Acute Myeloid Leukemia (AML) is an aggressive heterogeneous hematological malignancy. Persistence of leukemia stem cells (LSC) drive AML leukemogenesis, responsible for drug resistance and disease relapse following conventional chemotherapy. Growing evidence demonstrates that epigenetic-based therapies pose a unique and rational avenue for eradication of LSCs, enabling long-term remission and cure. In this study, we identified the histone lysine specific demethylase 4A, KDM4A, as an essential regulator of AML oncogenic potential. KDM4A inhibition by shRNA knock-down (KD) or KDM4A inhibitors, selectively promoted myeloid differentiation of AML cells, resulting in significant AML cell death whilst sparing normal human CD34+ hemopoietic stem and progenitorcells (HSPC). Despite substantial evidence demonstrating that KDM4A is amplified and overexpressed in various malignancies including breast, lung, ovarian, prostate cancer and leukemia, there is little information to date as to its defined role in AML leukemogenesis nor whether it represents a viable therapeutic target. To investigate the molecular mechanisms underlying the leukemia-selective dependence on KDM4A, we performed ChIP-seq and RNA-seq to profile the epigenomic and transcriptional consequence of KDM4A KD in MLL-AF9-driven human AML THP-1 cells, followed by validation in primary AML patient blasts and normal human CD34+ HSPCs. KDM4A KD leads to a significant global enrichment of its substrate, H3K9me3 and surprisingly H3K27me3 (substrate of the Polycomb Repressive Complex 2, PRC2), and within the promoter regions of a number of KDM4A bound genomic loci. Both H3K9me3 and H3K27me3 are the repressive histone modifications, which correlate with the transcriptional down-regulation of PRC2 target genes upon KDM4A KD including Nuclear Factor of Activated T Cells 2 (NFATC2) and RNA Polymerase Associated Factor 1 (PAF1). KD of either NFATC2 or PAF1 leads to AML cell apoptosis, while over-expression of NFACT2 in THP-1 cells partially overcomes KDM4A inhibition. Together we have established NFATC2 and PAF1 as two key KDM4A direct downstream targets, both of which have well-established roles in AML leukemogenesis, promoting AML cell survival. Interestingly inhibition of de novo H3K27me3 using the pharmacological inhibitor of histone methyltransferase EZH2 EPZ6438, resulting a reduction of H3K27me3, reduces accumulation of H3K9me3 and partially rescues the detrimental phenotype of KDM4A KD in THP-1 cells, suggesting that KDM4A epigenetic regulation in MLL-AF9 AML is PRC2 activity dependent. Taken together, our data have uncovered a new insight of cross-talk between two repressive epigenetic modifications of H3K9me3 and H3K27me3 regulated by KDM4A and required for MLL-AF9 cell survival. To further address the clinical relevance of KDM4A and its downstream targets in AML, we employed meta-analysis on publicly available patient datasets. Our analysis of 461 AML patient samples, shows that KDM4Ahighpatients are enriched amongst poorly differentiated subtype of AML (p=0.01505; Fisher's exact test) and rare amongst more differentiated AML (p=0.07153; Fisher's exact test). High expression of NFATC2relates to worse clinical outcome in a large cohort of AML patients. Furthermore, using Lasso regression algorithm, we relate KDM4A KD induced global transcriptional changes to patient survival in a large AML dataset, yielding an optimal 21-gene signature (KDM4A-21). The KDM4A-21 score can be calculated for each patient as the weighted sum of expressions of the 21 genes. The scorehigh patients in a number of large de novo AML cohorts confer significantly poor overall survival compared with scorelow population (p<0.05; Mantel-Cox log-rank test). We are further evaluating these genes as potential cellular biomarkers of KDM4A inhibition for future preclinical trials. To our knowledge, these data for the first time delineate an essential and selective role for KDM4A in AML oncogenesis. Our results reveal a novel tractable regulatory network of KDM4A-PAF1/NFATC2 that is sustained by KDM4A mediated H3K9me3/H3K27me3 epigenetic crosstalk and is indispensable in MLL-AF9 AML cells. Our study provides strong evidence to establish that KDM4A and its downstream targets could represent propitious novel therapeutic targets, and the associated transcriptional network may be used to guide personalised epigenetic therapies in AML. Disclosures No relevant conflicts of interest to declare.