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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 MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment. To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). 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 library 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 were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells. 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 72 hours 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. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming. In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. Figure 1 Figure 1. Disclosures Konopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

In previous studies of recurrently amplified 1q21 genes in multiple myeloma (MM), we identified ILF2 (Interleukin Enhancer Binding Factor 2) as a key modulator of the DNA repair pathway, which promotes adaptive responses to genotoxic stress in a dose-dependent manner, explaining why 1q21 patients benefit less from high-dose chemotherapy than non-1q21 patients do (Marchesini, Cancer Cell 2017). These findings prompted us to develop strategies for blocking ILF2 signaling to enhance the effectiveness of available DNA-damaging agent-based treatments. Given that ILF2 is selectively overexpressed in 1q21 MM cells and is not easily amenable to small-molecule or antibody depletion, we collaborated with IONIS Pharmaceuticals to develop antisense nucleotides targeting ILF2 (ILF2 ASOs). To exclude on-target toxicities that could arise from ILF2 inhibition, we injected 14 different ASOs targeting mouse ILF2 into male Balb/c mice. Of the 14 ILF2 ASOs we tested, 6 did not induce either notable histopathological findings or hematological and biochemical alterations, which suggests that ILF2 inhibition is well-tolerated in normal tissues. Thus, ILF2 ASOs were used for functional validation studies in myeloma. Consistent with our previous work using ILF2-targeting shRNAs, we observed that ILF2 ASO-induced ILF2 depletion resulted in significantly inhibited cell proliferation, increased ATM/Chk2 pathway activation, γH2AX accumulation, and caspase 3-mediated apoptosis in KMS11 and JJN3 cells and sensitized these cells to melphalan, bortezomib and olaparib treatment (Fig 1). However, whereas KMS11 cells had a high level of DNA damage activation and a significantly higher apoptosis rate after more than 2 weeks of ILF2 ASO treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and apoptosis and became resistant to ILF2 ASO treatment. To gain insights into the molecular mechanisms by which MM cells can overcome ILF2 ASO-induced DNA damage activation, we subjected ILF2 ASO-treated KMS11 and JJN3 cells to RNA sequencing analysis at early and late treatment times. We found that the genes that were significantly downregulated in JJN3 but not KMS11 cells treated with ILF2 ASOs for more than 2 weeks as compared with those treated for 1 week were mostly involved in the regulation of the DNA damage response (Fig 2). These findings suggest that MM cells can activate compensatory mechanisms to overcome the deleterious effects of DNA damage and survive. To identify DNA repair effectors whose loss of function suppresses 1q21 MM cells' capability to overcome ILF2 ASO-induced DNA damage, we performed a CRISPR/Cas9 screening using a pool of single-guide RNAs (sgRNAs) targeting 196 genes involved in the DNA damage response. Using the drugZ algorithm to assess differences in the representation of all sgRNAs between cells treated with NT or ILF2 ASOs for 3 weeks (Fig 3), we found that sgRNAs targeting the DNA replication helicase/nuclease 2 (DNA2) were among the most significantly depleted sgRNAs in ILF2 ASO-treated JJN3 cells, whereas sgRNAs targeting the Fanconi anemia core complex-associated protein 24 (FAAP24) were significantly depleted in ILF2 ASO-treated KSM11 cells. Using the DNA2 inhibitor C5, we further validated that targeting DNA2 significantly enhances ILF2 ASO-induced apoptosis in JJN3 cells (Fig 4). Functional validation experiments using inducible sgRNAs are ongoing to evaluate whether the inhibition of DNA2 or FAAP24 is a synthetic lethal approach to targeting 1q21 MM cells in the setting of therapies with DNA-damaging agents. Collectively, our study demonstrates that ILF2 ASO therapy may be exploited to optimize the use of DNA-damaging agents in patients with 1q21 MM. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:Abbvie: Research Funding; Amgen: Research Funding; IONIS: Other: Intellectual property and research material IONIS).