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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.
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