Critical Role of Split Hand/Foot Malformation Type 1 (SHFM1) in Homologous Recombination and Cell Survival in Multiple Myeloma (MM)

Genomic instability is associated with development and progression of cancer. Our previous studies have demonstrated that myeloma cells display a marked genomic instability and the number of mutations correlates with clinical outcome. Based on published observation that dysregulated homologous recombination (HR) contributes to genomic instability in multiple myeloma (MM) we have investigated mechanisms underlying dysregulated HR. We conducted a functional high-throughput shRNA screen using HR assay and identified Split hand/foot malformation type 1 (SHFM1) as a novel regulator of HR in MM. SHFM1 has been shown to directly interact with BRCA2 and function in DNA binding and recombination. Based on these we have here investigated the role/s of SHFM1 in genomic evolution and impact on other oncogenic pathways/activities in MM. Using 2 different datasets (gse26863, n=246 and IFM n=170) with both expression data and copy number/SNP information, we analyzed the association of SHFM1 with genomic instability in vivo, Genomic instability in each patient was determined by counting the total number of copy number change events (deletions and amplifications) defined as changes in ≥ 5 consecutive SNPs. We observed that the higher SHFM1 expression correlated with increase in both the amplifications (p=0.0015) and deletions (p=0.0188) in gse26863 dataset, and correlated with increase in amplifications in 170 dataset (p=0.0006). When events were defined as changes in ≥ 3 consecutive SNPs, the increased SHFM1 correlated with increase in amplifications (p ≤ 0.0004) in both datasets. The increased SHFM1 expression also correlated with hyperdiploidy (P=0.0008) in MM patients. These observations suggest that elevated SHFM1 is associated with evolution, including hyperdiploidy, in myeloma. In a loss of function study, we suppressed SHFM1 in three different myeloma cell lines (ARP, RPMI, U266) using shRNA. In all three cell lines the SHFM1-KD was associated with reduction in live cell number (~50% of control non-targeting shRNA) at day 1 after selection, to complete cell death over a period of 1 - 3 weeks, in 3 different experiments. Both cell cycle and annexin V-labeling showed that mechanism of cell death was apoptotic. These data suggest that elevated SHFM1 has a critical role in survival and prevention of apoptosis in myeloma cells. To further understand the roles of SHFM1 in myeloma, we identified the genes whose expression correlated with SHFM1. In two different datasets, the expression of SHFM1 positively correlated with genes involved in DNA synthesis, homologous recombination (ATR, TOPBP1, RAD1, RAD50, POLD2, NBN, SSBP1, MRE11A), cell cycle progression and telomere maintenance, whereas negatively correlated with several apoptosis related genes (including TNFRSF12A, TNFRSF9 and DAPK2) and SUV420H2 (involved in epigenetic transcriptional repression). Investigation by real time PCR confirmed that SHFM1-KD was associated with ~2.0-fold increase in the expression of TNFRSF9; these and other associations are being further investigated by mass spectrometry. In summary, our data show that SHFM1 has critical roles in genomic evolution as well as prevention of apoptosis in myeloma cells, and can be targeted to make myeloma cells static.