(PA-328) Inhibition of DNA Damage Response Factor DNA-PKcs-mediated H2AX Phosphorylation Enhances Selinexor-induced Anti-multiple Myeloma Effects

Abnormal DNA damage response (DDR) in multiple myeloma (MM) promotes genomic instability and drug resistance. DNA-PKcs is a key DDR factor in non-homologous end joining (NHEJ) repair for DNA double-strand breaks. Under certain conditions, it can compensate for ATM to initiate H2AX phosphorylation (γH2AX), which coordinates DDR signaling and promotes DNA repair. Selinexor, an exportin 1 (XPO1) inhibitor, targets the nucleocytoplasmic transport of macromolecules. It suppresses DDR gene expression and homologous recombination (HR) repair. However, it remains unclear whether XPO1 regulates the activation and transport of DDR proteins like DNA-PKcs, and if DNA-PKcs modulates MM cells’ sensitivity to Selinexor by influencing DNA damage repair. This study explores the role of DNA-PKcs in Selinexor-induced DNA damage and apoptosis in MM.

Methods: n/a

Results:

We first discovered XPO1 inhibition by Selinexor or knockdown promoted DNA-PKcs phosphorylation at S2056 and increased γH2AX expression in MM cells, without affecting ATM or ATR activation. Subcellular fractionation showed inhibiting XPO1 reduced cytoplasmic DNA-PKcs but recruited more DNA-PKcs to chromatin and promoted DNA-PKcs phosphorylation on chromatin. We then found Selinexor-induced stimulation of chromatin-bound DNA-PKcs primarily regulated H2AX phosphorylation. Inhibiting DNA-PKcs, via knockdown or its inhibitor Nedisertib, reduced Selinexor-induced γH2AX expression. This reduction likely impairs the recruitment of various DDR factors, disrupting DNA damage repair. Indeed, Using CRISPR/Cas9 gene-editing system and NHEJ/HR reporters in U2OS cells, we observed Selinexor inhibited HR repair without affecting NHEJ repair, and the addition of DNA-PKcs inhibitor Nedisertib enhanced Selinexor’s suppression of HR repair and even induced its inhibition of NHEJ repair in U2OS cells. Consistent with these findings, Comet and TUNEL assays confirmed DNA-PKcs inhibition enhanced Selinexor-induced DNA damage in MM cells. Since excessive DNA damage can lead to cell death, we next investigated the role of DNA-PKcs inhibition in MM sensitivity to Selinexor. Results showed DNA-PKcs knockdown promoted Selinexor-induced apoptosis and increased MM sensitivity to Selinexor. The combination of Nedisertib and Selinexor exhibited a significantly synergistic killing effects on MM cells in vitro; in vivo, this combination treatment slowed MM tumor growth. Notably, Nedisertib did not enhance Selinexor-induced apoptosis in PBMCs from healthy donors.

Conclusions:

DNA-PKcs-mediated H2AX phosphorylation promotes DNA damage repair and survival of myeloma cells in response to XPO1 inhibitor Selinexor. DNA-PKcs inhibition increases Selinexor-induced DNA damage and enhances MM sensitivity to Selinexor. The combination of DNA-PKcs inhibitor Nedisertib with Selinexor demonstrates strong synergistic anti-MM effects both in vitro and in vivo. These findings suggest that dual targeting of DNA-PKcs and XPO1 could serve as a promising new strategy for treating MM.