Identification of Epigenetic Modifiers Essential for Growth and Survival of AML1/ETO-Positive Leukemia

Abstract

Introduction Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with direct or indirect dysregulation of epigenetic modifiers. Previously, we and others have shown that the AML1/ETO (RUNX1/MTG8) fusion protein, encoded by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes (Rejeski et al., Oncogene 2021) Aim We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Materials and methods In the customized shRNA library screen, 2009 shRNAs developed to target 671 genes were pool-cloned into a retroviral vector allowing doxycycline-inducible expression to generate a plasmid shRNA library (Antonova et al., Cell Reports 2019). We transduced the AML1/ETO-positive Kasumi-1 and SKNO-1 cell lines, selected with blasticidine, and the expression vectors were induced with doxycycline. After 12 days of doxycycline treatment, double-positive dsRed/GFP cells were FACS-sorted, sequenced and compared with FACS-sorted single GFP-positive cells from day 0. Single shRNAs against two selected genes of interest (DNMT1, ATR), were cloned and validated following the same protocol used for shRNA library screen. The 9/10/17-LacZ and 9/14/18-AML1/ETO clones were described previously (Duque-Afonso et al., 2011). Briefly, individual U937 clones were stably transduced with an ecdysone-inducible system for the expression of AML1/ETO (9/14/18-AML1/ETO) or LacZ gene (9/10/7-LacZ as control cells). Gene expression was induced by 5 uM ponasterone A (PonA). RNA was isolated, RNA libraries were prepared and sequenced. Bioinformatic analysis were performed on the European Galaxy instance platform. Results Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. Among them, several epigenetic regulators as DNMT1, ATR, DMAP1, SMYD1, SMYD2, BRD4, SETD8, SETD1A and HDAC8 were depleted. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive (SKNO-1, Kasumi-1) and AML1/ETO-negative cell lines (OCI-AML3, HL60) with median IC50 for decitabine 74 nM (range 44-219), azacytidine 146 nM (range 14-267), berzosertib 219 nM (range 16-460) and ceralasertib 85 nM (range 42-328). We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1-inhibitor decitabine in a patient with an AML1/ETO-positive AML. To address the regulation of epigenetic modifiyer genes by AML1/ETO in an unbiased fashion, bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 1319 differentially expressed genes (DEGs) (822 up- and 497 downregulated). Four genes (DNMT1, PARP2, SMARCA4, and PRKCD) were both downregulated after AML1/ETO induction, and also detected in the survival shRNA screens of both cell lines as decreasing survival and proliferation. Conclusions To our knowledge we show here the first shRNA knock-down library screen for epigenetic modifiyer genes in AML1/ETO-positive cells. Using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. Some of these epigenetic regulators, such as DNMT1 and ATR, are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy, and confirmation studies (Baeten et al., ASH meeting 2022) appear warranted.