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.

Chronic kidney disease and associated comorbidities (diabetes, cardiovascular diseases) manifest with an accelerated ageing phenotype, leading ultimately to organ failure and renal replacement therapy. This process can be modulated by epigenetic and environmental factors which promote loss of physiological function and resilience to stress earlier, linking biological age with adverse outcomes post‐transplantation including delayed graft function (DGF). The molecular features underpinning this have yet to be fully elucidated. We have determined a molecular signature for loss of resilience and impaired physiological function, via a synchronous genome, transcriptome and proteome snapshot, using human renal allografts as a source of healthy tissue as an in vivo model of ageing in humans. This comprises 42 specific transcripts, related through IFNγ signalling, which in allografts displaying clinically impaired physiological function (DGF) exhibited a greater magnitude of change in transcriptional amplitude and elevated expression of noncoding RNAs and pseudogenes, consistent with increased allostatic load. This was accompanied by increased DNA methylation within the promoter and intragenic regions of the DGF panel in preperfusion allografts with immediate graft function. Pathway analysis indicated that an inability to sufficiently resolve inflammatory responses was enabled by decreased resilience to stress and resulted in impaired physiological function in biologically older allografts. Cross‐comparison with publically available data sets for renal pathologies identified significant transcriptional commonality for over 20 DGF transcripts. Our data are clinically relevant and important, as they provide a clear molecular signature for the burden of “wear and tear” within the kidney and thus age‐related physiological capability and resilience.

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.

No abstract

Introduction: Transcatheter aortic valve implantation (TAVI) is the standard of care for the majority of patients with severe symptomatic aortic stenosis (AS) at excessive-, high-and intermediate-surgical risk. A proportion of patients referred for TAVI do not undergo the procedure and proceed with an alternate treatment strategy. There is scarce data describing the final treatment allocation of such patients. Hence, we sought to evaluate the final treatment allocation of patients referred for TAVI in contemporary practice. Methods: We performed a single center prospective observational study, including all patients referred to our institution for treatment of severe aortic stenosis between February 2014 and August 2017. Baseline demographic and clinical data were recorded. Patients were categorized according to treatment allocation: TAVI, surgical aortic valve replacement (SAVR) or optimal medical therapy (OMT). Clinical outcomes were adjudicated according to VARC-2 definitions. All patients were discussed at a dedicated Heart Team meeting. Results: Total of 245 patients were referred for assessment to a dedicated TAVI clinic during the study period. Patients with moderate (N = 32; 13.1%) and asymptomatic (N = 31; 13.1%) AS were excluded. Subsequently, 53.9% (N = 132) received TAVI, 12.7% (N = 31) were managed with OMT, and 7.3% (N = 18) had SAVR. Reasons for OMT included primarily: patient's preference (N = 12; 38.7%); excessive surgical risk (N = 4; 12.9%) and severe frailty (N = 5; 16.1%). Reasons for surgical referral included low surgical risk (N = 11; 61.1%), excessive annulus size (N = 5; 27.8%), and aortic root dilatation (N = 2; 11.1%). Patients proceeding to SAVR had lower surgical risk than those in either the OMT or TAVI cohorts (P < 0.001). Mean STS score in SAVR group was 2.2 ± 1.3 vs. 4.5 ± 2.4 in OMT cohort and 6.1 ± 4.9 in TAVI cohort. Six-month all-cause mortality was 16.7, 19.4, and 9.3% among those receiving SAVR, OMT, and TAVI, respectively. Conclusions: Almost half of all patients with severe AS referred to a dedicated TAVI clinic did not receive a TAVI. A considerable proportion of patients were reclassified as Gorecka et al. Patient Disposition and Clinical Outcome moderate AS (13%), were asymptomatic (13%), or intervention was determined to be futile (13%) due to advanced frailty. Early detection and increased awareness of valvular heart disease are required to increase the number of patients that can benefit from TAVI.

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.

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.