PDZ Binding Kinase (PBK) - a Novel Gene Driving Genomic Evolution in Multiple Myeloma

As in all cancers, genomic instability leads to ongoing acquisition of new genetic changes in multiple myeloma (MM). This adaptability underlies the development of drug resistance and progression in MM. This genomic instability is driven by cellular processes, mainly related with DNA repair and perturbed by functional changes in limited number of genes. Since kinases play a critical role in the regulation of biological processes, including DNA damage/repair signaling and are relatively easy to screen for inhibitors, we investigated for novel genes involved in the acquisition of new genomic changes in MM. Using a large genomic database which had both the gene expression and CGH array-based copy number information (gse26863, n=246), we first identified a total of 890 expressed kinases in MM and correlated their expression with genomic instability defined as a change in ≥3 and/or 5 consecutive amplification and/or deletion events. We identified 198 kinases whose elevated expression correlated with increased genomic instability (based on FDR ≤ 0.05). Amongst these kinases, using univariate Cox survival analysis, elevated expression of 15 kinases correlated with poor overall as well as event free survival (P ≤0.05) in two MM datasets (IFM70, n=170; gse24080; n=559). We further confirmed the correlation of these 15 genes in both EFS and OS in additional two MM datasets (MMRF CoMMpass Study, IFM-DFCI 2009) as well as in additional solid tumor datasets from TCGA from patients with lung and pancreatic adenocarcinoma (P values ranging from 0.01 to <0.000002). A pathway analysis identified phosphorylation and regulation of proteasome pathway, mitotic spindle assembly/checkpoint, chromosomal segregation and cell cycle checkpoints as among major pathways regulated by these genes.

To investigate the relevance of these genes with genomic instability, we performed a functional siRNA screen to evaluate impact of their suppression on homologous recombination (HR). PDZ Binding Kinase (PBK) was one of the top genes whose knockdown caused the maximal inhibition of HR activity in initial screen. To investigate it further in detail, we suppressed PBK in MM cells using shRNA and confirmed that its suppression significantly reduces HR activity. PBK-knockdown also reduced gH2AX levels (marker of DNA breaks) measured by Western blotting and decreased number of micronuclei (a marker of ongoing genomic rearrangements and instability) as assessed by flow cytometry . A small molecule inhibitor of PBK also confirmed a similar reduction in gH2AX levels as well as micronuclei, indicating inhibition of spontaneous DNA breaks and genomic instability. Using mass spectrometry and co-immunoprecipitation, we identified that PBK interacts with FEN1, a nuclease with roles in base excision repair and HR pathways. We confirmed that PBK induces phosphorylation of FEN1 and that inhibition of PBK, suppressed the phosphorylation of FEN1, RAD51 expression and gH2AX levels and it reversed FEN1-induced HR activity. These results confirm that phosphorylation of FEN1 nuclease by PBK contributes to its ability to impact DNA breaks, HR and genome stability in MM. PBK inhibition also significantly sensitized MM cells to melphalan and inhibited cell viability in a panel of MM cell lines (IC50 in MM cell lines ~20-30 nM vs ~100 nM in normal PBMCs) at the same time also reversed melphalan-induced genomic instability, as assessed by micronucleus assay. These data identify PBK as an important target affecting genomic instability, and its inhibitor as a potential drug, to inhibit genomic evolution and MM cell growth.