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.

Organophosphorus-catalyzed Staudinger ligation between carboxylic acids and azides in the presence of phenylsilane reductant produces amides. NMR-based mechanistic investigations revealed that the catalytic Staudinger ligation does not proceed via reduction of phosphine oxide but rather via reduction of iminophosphorane, which can subsequently undergo several transformations to produce the amide product.

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.

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 & IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation > 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (>15% reduction) and increased apoptosis (>20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.