Dysregulation of SHFM1, a Novel Target for Prevention of Genomic Instability in Myeloma, Is Associated with Epigenetic Changes at Specific CpG Sites

We have previously demonstrated that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. We, therefore, hypothesized that mechanisms underlying dysregulation of HR, drive genomic instability and have potential to serve as novel markers of progression and target to make cancer cells static. In an attempt to identify molecular intermediates underlying increased HR, we conducted a high-throughput shRNA screen using HR activity in MM cells as an endpoint, and identified split hand/split foot malformation locus 1 - SHFM1 - as a novel regulator of HR in myeloma. We demonstrate that suppression of SHFM1 using specific lentiviral shRNA knockdown inhibits HR activity in myeloma as well as other (esophageal) cancer cells. Suppression of HR activity is also associated with reduction in spontaneous DNA breaks thus having a positive impact on genomic integrity. We have also observed that suppression of SHFM1 gene lead to reduction in acquisition of new genomic changes by both copy number alteration as well as SNPs. Evaluation of expression profile in 330 MM patients and 18 normal plasma cells indicated that the gene is overexpressed in myeloma, relative to normal plasma cells (P = 0.0059). Expression of this gene in myeloma also significantly correlates with its copy number (R=0.43; P<0.00000004). To understand the molecular mechanism of its overexpression in myeloma, we evaluated DNA methylation changes associated with altered expression of this gene. We investigated CpG sites in the promoter region of SHFM1 gene using primers with sodium bisulfite converted DNA-specific design. The primers were designed such that they avoid annealing to CpG sites at the region of interest to the maximum extent possible. Samples were bisulfite converted and amplification of all samples using specific primer pairs was performed, and then massively parallel sequenced and analyzed. We observed methylated CpG at two SHFM1 gene locations in PBMC but unmethylated in all 12 out of 12 myeloma cell lines at first location and in 9 out of 12 myeloma cell lines at second cite (Fisher’s exact test for comparisons of PBMC vs. average methylation ratios in caner samples for two locations was P=1.38E-13 and 0.05, respectively).

In summary our results highlight SHFM1 as an important gene modulating genomic instability and its control by methylation provides an insight into epigenomic control of DNA stability.