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1048 Cooperative Gene Repression By DNA Methylation and LSD1-Mediated Enhancer Inactivation in Acute Myeloid Leukemia

Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation
Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: DNA-Methylation
Monday, December 5, 2016: 5:15 PM
Room 31 (San Diego Convention Center)

Cihangir Duy, PhD1*, Matt Teater, M.S.2*, Francine E. Garrett-Bakelman, MD, PhD1, Tak C Lee, M.S.1*, Cem Meydan, PhD3*, Jacob Lowell Glass, MD, PhD4, Meng Li, PhD1*, Helai Mohammad, PhD5*, Kimberly Smitheman5*, Alan H. Shih, MD, PhD4, Omar Abdel-Wahab, MD4, Elisabeth Paietta, Ph.D.6, Martin S. Tallman, MD4*, Monica L. Guzman, PhD7, Ryan G. Kruger, PhD5*, Caretha L. Creasy, PhD5*, Ross L. Levine, MD8, Martin Carroll, MD9 and Ari Melnick, MD10

1Department of Medicine, Division of Hematology & Medical Oncology, Weill Cornell Medicine, New York, NY
2Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY
3Institute for Computational Biomedicine and Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY
4Memorial Sloan Kettering Cancer Center, New York, NY
5GlaxoSmithKline, Collegeville, PA
6Oncology, Montefiore Medical Center, Bronx, NY
7Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY
8Memorial Sloan-Kettering Cancer Center, New York, NY
9Division of Hematology and Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA
10Division of Hem./Onc., Dept. of Medicine, Weill Cornell Medical College, New York, NY

Epigenetic silencing of tumor suppressor genes, mediated by aberrant DNA hypermethylation and repressive histone modifications, is a hallmark of acute myeloid leukemia (AML). Novel epigenetic therapies are emerging, but single-agent approaches targeting only one category of epigenetic marks have limited therapeutic activity. We show that combinatorial treatment strategy targeting epigenetic modifications of both promoter and enhancer elements can improve therapeutic intervention.

As current cell lines do not accurately represent the heterogeneous nature of AML, we established an ex vivo culturing system enabling us to propagate primary AML specimens long enough to test epigenetic therapeutics (48 specimens expanded 4-6 weeks; 6 specimens expanded >12 months; 39 specimen fail to expand). We tested 52 expandable primary AMLs with a combination therapy utilizing the DNA demethylating agent 5-Azacytidine (5-Aza) and a novel small molecule inhibitor against LSD1, a histone demethylase that removes enhancer-associated marks (mono-/di-methylated histone H3 lysine 4; H3K4me1/2). In order to avoid DNA damage-induced cytotoxicity, we applied a non-DNA damaging dose of 5-Aza (≤ 200 nM). 5-Aza treatment had modest inhibition of cell growth for the majority of cases, but did not appreciably alter cell viability. Treatment with the LSD1 inhibitor (LSD1i) substantially impaired cell growth and survival of ~80% (p<0.001) of cultured primary AMLs. Notably, we found that dual combination therapy using LSD1i and 5-Aza demonstrated significantly improved inhibition of cell viability and growth in most LSD1i-resistant AML cases. Targeted resequencing analysis of 200 genes recurrently mutated in myeloid malignancies revealed an association of mutations with therapeutic activity; TET2mut AML cases without concurrent DNMT3A mutations were most substantially impaired by combinatorial treatment (p<0.001). Also, leukemia burden was significantly reduced after combination therapy in NSG mice transplanted with luciferase-labelled TET2mut patient-derived AML cells (p<0.0002).

To gain insight into mechanism, we performed a genome-wide integrative analysis of the DNA methylome (Enhanced Reduced Representation Bisulfite Sequencing), LSD1 occupancy sites (ChIP-sequencing) and gene expression profiling (RNA-sequencing) in a TET2mut patient-derived AML sample. Single-agent LSD1i treatment led to induction of genes associated with myeloid differentiation. Even though 5-Aza induced substantial hypomethylation, there was only a minor effect on gene expression. However, combination therapy did induce greater de-repression of genes, in particular those where 5-Aza induced hypomethylation at promoters and at LSD1-occupied enhancers. Combination treatment especially potentiated induction of differentiation genes, suggesting potential anti-leukemia stem cell effects. Accordingly, combination therapy resulted in impaired colony formation potential (p=0.007). Moreover, limiting dilution analysis in NSG mice demonstrated significant impairment of leukemia-initiating cells (p=0.0025) after 5-Aza+LSD1i combination therapy when compared to either drug alone.

To determine what genes mediate the effect of combination therapy, we studied genes preferentially induced by 5-Aza+LSD1i. GATA2, a transcription factor involved in hematopoietic differentiation, was more potently induced by combination therapy in primary AML samples vs. either drug alone. We found LSD1 occupied at the GATA2 distal hematopoietic enhancer and verified by using quantitative ChIP analysis that inhibition of LSD1 induced an increase of H3K4me2 and H3K27ac, a mark that reflects enhancer activation. Overexpression of GATA2 using viral transduction in two patient-derived AML cases caused partial differentiation and attenuated leukemia growth. These results indicated that cellular responses to combination therapy are in part mediated by induction of GATA2.

In summary, our results provide a novel link for therapeutic approaches targeting redundant epigenetic silencing of tumor suppressors via cooperative enhancer and enhancer-promoter activation.

Disclosures: Duy: GlaxoSmithKline: Research Funding. Mohammad: GlaxoSmithKline: Employment. Smitheman: GlaxoSmithKline: Employment. Guzman: Cellectis: Research Funding. Kruger: GlaxoSmithKline: Employment. Creasy: GlaxoSmithKline: Employment. Levine: Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees; CTI: Membership on an entity's Board of Directors or advisory committees. Carroll: Incyte: Consultancy, Research Funding. Melnick: Roche: Consultancy, Research Funding; GSK: Research Funding; Eli Lilly: Consultancy, Research Funding; Epizyme: Consultancy; Boehringer-Ingelheim: Consultancy; Janssen: Research Funding.

*signifies non-member of ASH