Summary Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM.

SUMMARY Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including srsf2. RNA-Seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling and histone modification, which are also enriched in mouse CMP haploinsufficient for srsf2 and in CD34+ CMML patient cells harboring srsf2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing and myeloid progenitor differentiation.

Myelodysplastic syndromes (MDS) are heterogeneous neoplastic disorders of hematopoietic stem cells (HSCs). The current standard of care for patients with MDS is hypomethylating agent (HMA)-based therapy; however, almost 50% of MDS patients fail HMA therapy and progress to acute myeloid leukemia, facing a dismal prognosis due to lack of approved second-line treatment options. As cancer stem cells are the seeds of disease progression, we investigated the biological properties of the MDS HSCs that drive disease evolution, seeking to uncover vulnerabilities that could be therapeutically exploited. Through integrative molecular profiling of HSCs and progenitor cells in large patient cohorts, we found that MDS HSCs in two distinct differentiation states are maintained throughout the clinical course of the disease, and expand at progression, depending on recurrent activation of the anti-apoptotic regulator BCL-2 or nuclear factor-kappa B-mediated survival pathways. Pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden in experimental systems. Further, patients with MDS who progressed after failure to frontline HMA therapy and whose HSCs upregulated BCL-2 achieved improved clinical responses to venetoclax-based therapy in the clinical setting. Overall, our study uncovers that HSC architectures in MDS are potential predictive biomarkers to guide second-line treatments after HMA failure. These findings warrant further investigation of HSC-specific survival pathways to identify new therapeutic targets of clinical potential in MDS.

Activation of blood coagulation and endothelial inflammation are hallmarks of respiratory infections with RNA viruses that contribute significantly to the morbidity and mortality of patients with severe disease. We investigated how signaling by coagulation proteases affects the quality and extent of the response to the TLR3-ligand poly(I:C) in human endothelial cells. Genome-wide RNA profiling documented additive and synergistic effects of thrombin and poly(I:C) on the expression level of many genes. The most significantly active genes exhibiting synergistic induction by costimulation with thrombin and poly(I:C) included the key mediators of 2 critical biological mechanisms known to promote endothelial thromboinflammatory functions: the initiation of blood coagulation by tissue factor and the control of leukocyte trafficking by the endothelial-leukocyte adhesion receptors E-selectin (gene symbol, SELE) and VCAM1, and the cytokines and chemokines CXCL8, IL-6, CXCL2, and CCL20. Mechanistic studies have indicated that synergistic costimulation with thrombin and poly(I:C) requires proteolytic activation of protease-activated receptor 1 (PAR1) by thrombin and transactivation of PAR2 by the PAR1-tethered ligand. Accordingly, a small-molecule PAR2 inhibitor suppressed poly(I:C)/thrombin–induced leukocyte-endothelial adhesion, cytokine production, and endothelial tissue factor expression. In summary, this study describes a positive feedback mechanism by which thrombin sustains and amplifies the prothrombotic and proinflammatory function of endothelial cells exposed to the viral RNA analogue, poly(I:C) via activation of PAR1/2.

In the last decade, significant effort has been directed toward the stratification of multiple myeloma (MM) patients for targeted therapy, and many studies have shown that some genetic alterations, especially t(4;14) translocation, loss of the short arm of chromosome 17, and amplification of chromosome 1q21, are associated with a poor outcome. The 1q21 amplicon spans a region of 10-15 Mb and contains a large number of possible candidate genes; it is among the most frequent chromosomal aberrations in patients with MM and is associated with poor prognosis, disease progression, and drug resistance. Therefore, the identification of critical 1q21 genes may yield potential therapeutic targets for this high-risk MM subgroup and provide a rationale for patient stratification. In an effort to accomplish this goal, we first identified a high-priority list of 78 copy number-driven 1q21 MM-relevant genes by integrating high-resolution array comparative genomic hybridization (aCGH) and matched expression profiling of the 254 MM samples deposited in the Multiple Myeloma Research Consortium (MMRC) database. Then, we performed a high-throughput systematic shRNA screen in vitroto identify 1q21 genes whose loss of function resulted in the selective death and/or growth inhibition of MM cells carrying the 1q21 amplification. We used shRNA targeting (excluding shRNAs that displayed cytotoxic activity regardless of 1q21 amplification) and a GFP competitive growth assay to identify 1q21-resident targets whose downregulation significantly decreased the percentage of GFP-positive MM cells with 1q21 amplification over a time of 7 days. These assays identified UBAPL2, INTS3, LASS2, KRTCAP2, and ILF2 as key targets for further analysis. Secondary validation experiments in the MM cell lines JJN3 and H929 confirmed that the downregulation of all of our top five candidate genes induced significant levels of apoptosis, inhibition of proliferation, and cell cycle arrest. Integration of copy number analysis, expression profiling, and clinical outcome indicated that only UBAPL2 and ILF2 were highly significant prognostic genes, and target validation in NOD-SCID mice showed that ILF2, but not UBAPL2, downregulation had a significant impact on in vivo survival. Therefore, we sought to further characterize ILF2’s role in 1q21-amplified MM. ILF2 encodes NF45, the regulatory subunit of NF90/NF110 complexes, which are involved in mitotic control, DNA break repair, and RNA splicing regulation. Downregulation of ILF2 in MM cells with 1q21 amplification resulted in multinucleated phenotypes and abnormal nuclear morphologies (nucleoplasmic bridges and buds and micronuclei) that were associated with a significant accumulation of phospho-H2AX foci and DNA damage response activation, increased sensitivity to the DNA damaging agent melphalan, and impaired activation of DNA repair pathways. Experiments of immunoprecipitation combined with mass spectometry showed that ILF2 interacts with numerous RNA binding proteins directly implicated in DNA repair or regulation of DNA damage response by modulating alternative splicing and stability of specific pre-mRNAs. Accordingly, RNA-seq analysis of ILF2-depleted MM cells, when compared to cells carrying scrambled shRNAs, identified specific changes in RNA splicing patterns both before and after treatment with melphalan. In conclusion, our studies have revealed an unanticipated link between 1q21 amplification, DNA damage response, and RNA splicing. We identified ILF2 as a key driver of this interaction, and our findings support the development of strategies designed to modulate ILF2 expression in patients with high-risk MM carrying 1q21 amplification. Disclosures No relevant conflicts of interest to declare.