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Rational targeting of cooperating layers of the epigenome yields enhanced therapeutic efficacy against AML

ABSTRACT: Disruption of epigenetic regulation is a hallmark of Acute Myeloid Leukemia (AML), but therapeutic interventions are difficult by the interplay of epigenetic mechanisms controlling genomic elements. We hypothesized that concurrent targeting of aberrant promoter and enhancer epigenetic silencing improves efficacy against AML. To test this, we developed an ex vivo culturing system and treated 52 patient-derived AML with low-dose 5-Azacytidine and specific LSD1 inhibitors. Genetic analysis revealed that combination of 5-Azacytidine and LSD1 inhibition was most effective in TET2mut AML. Combination therapy further up-regulated expression of genes associated with both LSD1-bound enhancers and cytosine methylated promoters. Functional validation studies suggest that expression of these genes (e.g. GATA2, GFI1 and DLX2) contribute to drug efficacy. Mechanistically, combination therapy increased enhancer-promoter looping and chromatin-activating marks at the GATA2 locus. This gene induction effect was dampened by CRISPRi of the LSD1-bound enhancer in TET2mut AML. Concordant with TET2mut AML, knockdown of TET2 in human hematopoietic stem and progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Collectively, our platform revealed increased LSD1-mediated enhancer inactivation after TET2 deficiency that provided a basis for combinatorial epigenetic therapy to activate promoter-enhancer interactions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE89521 | GEO | 2019/10/24

REPOSITORIES: GEO

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Project description:Purpose: The tet oncogene family member 2 (TET2) gene was recently identified mutated in myeloid disorders including acute myeloid leukemia (AML). To date, there is increasing evidence for a functional role of TET2 mutations (TET2mut) in AML. Thus, we explored frequency, gene expression pattern, and clinical impact of TET2mut in a large cohort of AML patients, in the context of other AML-associated aberrations. Patients and Methods: Samples from 783 younger adult AML patients were analyzed for the presence of TET2mut (coding exons 3-11), and results were correlated with data from molecular genetic analyses, gene expression profiling, and clinical outcome. Results: In total, 66 TET2mut were found in 60 (60/783; 7.6%) patients, including missense (n=37), frameshift (n=16), and nonsense (n=13) mutations, which with one exception were all heterozygous. TET2mut were not correlated with distinct clinical features or genetic alterations, except for isocitrate dehydrogenase mutations (IDHmut) that were almost mutually exclusive with TET2mut (p<0.001). TET2mut were characterized by only a weak gene expression pattern, which nevertheless reflected TET2mut-associated biology. TET2mut did not impact response to induction therapy and clinical outcome; combining patients exhibiting TET2mut and/or IDHmut revealed shorter overall survival (p=0.03), although this association was not independent from known risk factors. Conclusion: TET2mut were identified in 7.6% of younger adult AML patients and did not impact response to therapy and survival. Mutations were mutually exclusive with IDHmut, thereby supporting recent data on a common mechanism of action, which might obscure the impact of TET2mut if compared against all other AML cases. We performed a supervised class comparison analysis comparing 31 TET-mutated AML cases (TET2 mut) vs. 302 TET2-wildtype AML cases (TET wt) to see if a specific gene expression pattern associated with the presence of the identified TET2 mutations could be determined. No technical replicates were performed.

Project description:Clinical responses to kinase inhibitor therapy in acute myeloid leukemia (AML) are limited by the inevitable development of resistance. A major contributor to resistance is early epigenetic adaptation, leading to persistence of a small number of leukemia cells. Here we show that inhibition of the epigenetic regulator lysine-specific deme-thylase 1 (LSD1) augments the response to inhibitors of the FLT3 kinase in AML. We demonstrate that combined FLT3 and LSD1 inhibition results in synergistic cell death of FLT3-mutant AML cells via proliferative arrest and apoptosis. The drug combination synergistically activates a pro-differentiative epigenetic and transcriptional program while simultaneously suppressing the activity of MYC target genes. High resolution multi-modal epigenetic analyses revealed that combined FLT3 and LSD1 resulted in the suppression of MYC-bound promoters and the activation of PU.1-bound enhanc-ers. Forced expression of MYC partially abrogated the drug effect, and regulon en-richment analysis in primary AML samples nominated STAT5 as a putative regulator of MYC gene expression. STAT5 is highly bound to the MYC blood super-enhancer and inhibition of FLT3 results in a loss of STAT5 binding and a loss of super enhancer acti-vation. Furthermore, knockdown of STAT5 augments LSD1-inhibitor induced cell death. LSD1 inhibition also directly represses MYC target genes which show specific accumulation of the repressive LSD1 substrate H3K9me1. We validated these findings in 67 primary AML samples including 19 FLT3-ITD positive AML samples, with the vast majority demonstrating improved responses with the drug combination. High MYC regulon activity was a predictor of response to the drug combination and RNA-seq on drug treated AML samples revealed suppression of MYC target genes. Finally, single cell ATAC seq on primary AML blasts treated ex-vivo with combined FLT3 and LSD1 inhibition results in a shift from MYC super enhancer-high to a MYC super enhancer-low cell state. Collectively, these studies provide preclinical rationale for the investiga-tion of dual FLT3 and LSD1 inhibition in clinical trial.

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Rational targeting of cooperating layers of the epigenome yields enhanced therapeutic efficacy against AML

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