ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine

Nora E Rahmani,Juseong Lee,Orsi Giricz,Tianyu Sun,Nandini Ramachandra,Kith Pradhan,Shahina Maqbool ,Aditi Shastri,Dharamveer Tatwavedi,Ulrich Steidl,Jacqueline Boultwood,K Visweswara Rao ,Mohammad Kazemi,Shalini Singh,B Peña ,Vickram Tittrea,Raul Olea,Jinghang Zhang,Omar Abdel‐Wahab ,Tushar D Bhagat,Daichi Inoue,Andrea López ,Shanisha Gordon-Mitchell,Srabani Sahu,Hamid Dolatshad,Yongmei Zhao,Andrea Pellagatti,Evripidis Gavathiotis,Nadege Gitego,Amit Verma

doi:10.1038/s41408-021-00541-0

Abstract

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.

Highlights

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are generally incurable malignancies characterized by high rates of chemo-refractoriness and intrinsic resistance to apoptosis
As the selectivity of malignant cells to this combination therapy is not well elucidated, we used a cellular model of a common leukemia-associated mutation in the epigenetic regulator ASXL1 to determine the expression levels of BCL2 and investigate the mechanistic basis of BCL2 overexpression to determine whether this regulates increased sensitivity to VEN and AZA
CRISPR/Cas9-mediated correction of the ASXL1 mutation leads to increased myeloid differentiation CRISPR/Cas9 mediated correction of the ASXL1G710X mutation in the KBM5 cell line [12] was confirmed by transcriptomic analysis

Summary

Introduction

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are generally incurable malignancies characterized by high rates of chemo-refractoriness and intrinsic resistance to apoptosis. Studies have demonstrated overexpression of antiapoptotic proteins, such as BCL2, in MDS/AML [1,2,3], the mechanistic basis of this upregulation is not well understood This becomes important as Venetoclax (VEN), a small molecule inhibitor of BCL2, has shown efficacy in conjunction with DNMTinhibitors, such as Azacytidine (AZA) [1, 4,5,6], resulting in high rates of complete remission [1, 4]. As the selectivity of malignant cells to this combination therapy is not well elucidated, we used a cellular model of a common leukemia-associated mutation in the epigenetic regulator ASXL1 to determine the expression levels of BCL2 and investigate the mechanistic basis of BCL2 overexpression to determine whether this regulates increased sensitivity to VEN and AZA. As the PRC2 acts through epigenetic modification of chromatin to maintain genes in a repressed state, an inactivating ASXL1 mutation leads to reduced PRC2 activity resulting in aberrant activation of genes [11, 12]

Methods

Results

Conclusion