P-106: Targeting DNA2 overcomes myeloma cells’ metabolic reprogramming in response to DNA damage

Abstract

<h3>Background</h3> DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in 1q21 MM. <h3>Method</h3> We continuously treated MM cells with an ILF2-targeting antisense (ILF2-ASO) or control non-targeting antisense (NT-ASO) for 3 weeks to see if ILF2-ASO exposure leads to the selection of MM clones intrinsically resistant to DNA damage or activates compensatory mechanisms to overcome ILF2 depletion-induced DNA damage. <h3>Results</h3> Single-cell RNA sequencing (scRNA-seq) analysis revealed that DNA damage-resistant ILF2-ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Metabolomic analysis of MM cells after long-term exposure to ILF2-ASO showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates. Consistent with these results, ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs in ILF2-ASO-treated JJN3 cells were significantly depleted after 3 weeks of treatment, suggesting that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO- and ILF2-ASO-treated MM cells exposed to NSC for 3 days showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. <h3>Conclusion</h3> In conclusion, our study has revealed a novel mechanism through which MM cells counteract oxidative DNA damage and maintain mitochondrial respiration after metabolic reprogramming.