Genetic and Pharmacological Inhibition of TUT4/TUT7 Sensitizes AML Cells to BCL-2 Inhibition

Abstract

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.