Identifying Novel Genes Involved in Dysregulation of Homologous Recombination and Genomic Integrity in Myeloma

Genomic instability leads to ongoing acquisition of genomic changes which underlie development and progression of cancer. We have previously demonstrated that homologous recombination (HR), the most precise DNA repair mechanism, is dysregulated in myeloma and contributes to genomic instability and development of drug resistance. Purpose of this study was to identify novel genes and pathways involved in the acquisition of new genomic changes in MM patients.

We first developed a gene signature correlating with the genomic instability and survival in myeloma patients. To identify genes whose expression correlates with genomic instability, we used a myeloma patient dataset (gse26863, n=246) which had both the gene expression and CGH array-based copy number information for each patient. Genomic instability in each patient was determined by counting the total number of amplification and/or deletion events; an event was defined as a change in ≥3 and/or 5 consecutive probes. Genes whose elevated expression correlated with increased genomic instability in gse26863 dataset were tested for correlation with survival in two other datasets (IFM70, n=170; gse24080; n=559). Elevated expression of 287 genes correlated with poor overall (OS; P < 0.0001) as well as event free survival (EFS; P < 0.0001) in both these datasets. A pathway analysis identified cell cycle, chromosome maintenance, repair and replication as major pathways (P values < 0.00001). We confirmed their vaidity with other published genomic instability signatures. Of all the genes in our signature, 70% were common with Mitotic Chromosomal Instability signature (Sc Transl Med, 5 (181), 181ra50.[doi:17, 2013], 49% were present with published Chromosomal Instability Signature (CIN70; Nature Genetics, 38: 9, 2006) and 90% common with Chromosomal Instability Signature (CIN20; a subset of CIN70; Nature Genetics, 38: 9, 2006). We investigated the relevance of these genes with genomic instability, using a functional (HR activity) high throughput siRNA screen. Interestingly, >40% of the genes in our signature belonged to homologous recombination as indicated by our functional screen. We, therefore, initiated a functional siRNA screen of 124 genes to evaluate their impact on HR and nuclease activities. Based on the results, we next selected three genes for detailed evaluation. These included TTK (a dual specificity protein kinase), TPX2 (a spindle assembly factor required for normal assembly of mitotic spindles and microtubules) and USP21 (a deubiquitinase of histone H2A involved in epigenetic transcriptional repression). We suppressed these genes in MM cells using shRNA and confirmed that their suppression significantly reduces HR activity in MM cell lines. Suppression of USP21 was also associated with loss of RAD51 recombinase expression, suggesting its role in RAD51 stability. To monitor the impact of elevated expression of these genes on genomic integrity, we investigated knockdown cells for gH2AX levels using Western blotting. Knockdown of all 3 genes reduced gH2AX levels, indicating inhibition of spontaneous DNA breaks.

In summary, our data has identified TTK, TPX2 and USP21 as novel genes involved in dysregulation of HR and genomic integrity in MM cells with potential impact on our understanding of the disease progression.