PF230 THE RNA HELICASE DDX17 PROTECTS ACUTE MYELOID LEUKEMIA CELLS AGAINST DNA DAMAGE

Abstract

Background:Alternative splicing (AS) is essentially regulated by RNA‐binding proteins (RBPs) and is one of many deregulated cellular processes in Acute Myeloid Leukemia (AML). The alternative usage of exons substantially increases the coding potential of mRNAs and frequently leads to the expression of tumor‐promoting mRNA variants in cancer modulating apoptosis, proliferation, invasion and migration of cancer cells. This underlines the great plasticity of AS in cancer and justifies the need to elucidate the function of nuclear RBPs in specific cancer types. We found that the RNA helicase DDX17 is significantly more highly expressed in primary human AML specimens with high versus low Leukemia Stem Cell (LSC) frequency.Aims:In this study, we aimed to understand the functional role of DDX17 in normal hematopoietic stem and progenitor cells (HSPCs) and in AML cells. In particular, we sought to determine whether DDX17 exerts its function in the hematopoietic system primarily via AS or AS‐unrelated mechanisms.Methods:We performed knockdown (KD) experiments using small hairpin (sh) RNAs to determine whether DDX17 was essential for primary human cord blood CD34+ HSPCs and leukemia cells. Furthermore, mRFP tagged DDX17 was overexpressed through lentiviral transduction to track protein localization by immunofluorescence (IF) imaging. Lentivirally transduced HSPCs and leukemia cells were analyzed in vitro for proliferative output, viability, immunophenotype, and colony formation potential. RNA sequencing (RNA‐Seq) was used to determine how DDX17 KD affected gene expression and alternative exon usage. Pull down experiments combined with mass spectrometry revealed binding partners in presence and absence of chemotherapeutic agents. Finally, we performed homologous recombination (HR) and drug sensitivity assays to determine the role of DDX17 in DNA damage.Results:Knockdown of DDX17 induced DNA damage and severely impaired the function of healthy HSPCs and TP53 wild type AML cells. TP53 null cells maintained their proliferative capacity during short term in vitro expansion in absence of DDX17, but were significantly sensitized towards DNA damaging agents. In line, overexpression of DDX17 protected the cells against double strand break (DSB) inducing drugs. IF imaging revealed direct recruitment of DDX17 to DNA damage sites, while RNA‐Seq showed suppression of multiple DNA repair genes.Summary/Conclusion:RNA‐Seq, IF imaging, proteomics, and HR assays suggest an essential role of DDX17 in DNA repair via different mechanisms including direct recruitment to DNA damage sites, alternative splicing, and regulation of DNA repair genes on the transcriptional level.