Peposertib suppresses generation of FLT3-internal tandem duplication formed by contralateral double nicks.

Abstract

FLT3-internal tandem duplication (ITD) is the most frequent gene mutation in acute myeloid leukemia. The consequences of FLT3-ITD have been analyzed in detail; however, the molecular mechanisms underlying FLT3-ITD generation have remained to be elucidated. We analyzed FLT3-ITDs in clinical samples using deep sequencing and identified not only oligoclonal ITDs but also rare deletion clones clustered at the palindrome-like sequence at FLT3 exon 14. We hypothesized that FLT3 exon 14 is genetically unstable due to the palindrome-like sequence at the region and that genomic damage at the site initiates FLT3-ITD formation. We used CRISPR/Cas9 to induce DNA damage for creating artificial FLT3-ITDs in human and mouse cell lines. We found that double nicks on the adjacent contralateral strand most efficiently generate ITDs. The artificial ITDs resembled clinical ITDs in the length distribution and characteristics at the joint. We further compared the inhibitory effects of olaparib and peposertib, specific inhibitors of single-strand break (SSB) and DSB repair, respectively. Peposertib remarkably reduced ITD formation, but olaparib did not affect the mutation pattern. The findings indicated that non-homologous end joining has a crucial role in the generation of ITDs. Our data shed light to the new role of peposertib, which potentially suppress generation of de-novo FLT3-ITDs caused by mis-repair event of the DNA damages in clinical course. Teaser Abstract: FLT3-ITD is the most common mutation in AML, yet its origins remain unclear. Using deep sequencing, we identified rare deletion clones clustering at a palindrome-like sequence in FLT3 exon 14, suggesting genetic instability. CRISPR/Cas9-induced DNA damage revealed that double nicks efficiently generate ITDs, resembling clinical cases. Peposertib, a DNA-PKcs inhibitor, significantly reduced ITD formation, highlighting the role of non-homologous end joining. Our findings suggest a potential therapeutic approach to prevent de novo FLT3-ITDs in AML.