Identification of EHMT2 As a New Target in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) constitutes a family of genetically heterogeneous lymphoid neoplasms and is the most common cancer in childhood. T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive subtype of ALL and accounts for approximately 15-20% of all cases. Despite remarkable improvement in the prognosis of childhood ALL over the past few decades, many patients will require additional therapy for relapsed/refractory disease, and ALL remains the second leading cause of cancer-related death in children. Genomic studies have identified recurrent somatic alterations in genes involved in DNA methylation and post-translational histone modifications in ALL, suggesting emerging opportunities for novel therapeutic interventions.

To identify new potential targets in T-ALL we tested a collection of inhibitors of epigenetic modifiers, including inhibitors of the histone acetyltransferase (HAT) p300 (CTPB and C646) and inhibitors of protein methyltransferases (PMTs), including targeting EZH2 (GSK126), DOT1L (SGC0946), and G9a (EHMT2)/G9a-like (GLP) (EHMT1) (UNC0642 and BIX01294). We demonstrated that the G9a/GLP inhibitors significantly impaired cell viability compared to other molecules as measured by the reduction of cellular ATP content.

G9a/EHMT2 and GLP/EHMT1 are conserved protein lysine methyltransferases that contain a Su(Var), Enhancer of Zeste, Trithorax (SET) domain. G9a localizes in euchromatin regions and regulates gene expression and chromosome structure through de novo mono- and di- methylation of histone H3 lysine 9 (H3K9me1/2). In particular, di- and trimethylation of H3K9 in gene promoters have been associated with transcriptional repression. Despite recent evidence that G9a is involved in different tumor types, the role of the G9a/GLP complex has not been systematically studied in T-ALL.

Because cancer driver genes and response to targeted therapy are frequently correlated with activating somatic mutations and/or expression levels we analyzed several transcriptional databases, including the Differentiation Map (DMAP), Cancer Cell Line Encyclopedia, and primary T-ALL datasets. Collectively, the results revealed that EHMT2 is a preferential marker of lymphoid differentiation and is highly expressed in T-ALL compared to other cancer subtypes or normal bone marrow cells. We next validated the effects of UNC0642 and extended testing to BRD4770, another selective G9a/GLP inhibitor. Competitive inhibition of G9a with both molecules impaired cell growth and triggered starvation-induced autophagic activity as shown by the detection of LC3-II by immunoblotting and formation of cytoplasmic autophagic vacuoles. Moreover, di-methylated histone H3 lysine 9 (H3K9me2) levels were decreased as measured by western blotting after histone extraction, supporting the notion that phenotypic changes upon small molecule treatment may be due to H3K9me2 loss and epigenetic reprogramming.

To genetically validate G9a as a potential new target in T-ALL we intersected our chemical screening results with a previously published short hairpin RNA (shRNA) screen targeting nearly 350 chromatin regulator genes (Knoechel B. et al., Cancer Cell, 2014). EHMT2 scored among the top 20 hits that impaired the viability of T-ALL cells, further supporting our initial observation of the dependency of T-ALL on EHMT2. Because shRNA-based screens are limited by the occurrence of off-target effects and potentially the incomplete repression of target genes, we took a CRISPR/Cas9 approach and designed sgRNA guides targeting the SET domain of EHMT2. Consistently, genetic knockout of EHMT2 in T-ALL cell lines impaired cell growth and induced apoptosis measured by Annexin V/ Propidium iodide staining and quantified by flow cytometry.

In conclusion, the intersection of multiple chemical, genetic, and genomic approaches led to the identification of G9a/EHMT2 as a druggable candidate in T-ALL. Our preliminary data strongly support the further investigation of EHMT2 in this aggressive subset of leukemia.