Instructor Dana-Farber Cancer Institute, United States
Introduction: The dysregulation of cell cycle checkpoints and DNA repair pathways is a common feature of cancer cells and contributes to their continued growth and genomic evolution. Previously, we identified a six-gene kinase signature that correlated with genomic instability in nine human cancers including multiple myeloma (MM). PBK, a top kinase in the signature, was investigated further. The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4-p130. Upon cell cycle entry, MuvB dissociates from E2F4 and recruits FOXM1 for up-regulating mitotic gene expression.
Methods: RNA sequencing was done following PBK inhibition to investigate the genes/pathways regulated by PBK. Co-immunoprecipitation followed by western blotting and/or mass-spectrometry was done to find interacting partners of PBK, LIN54 and FOXM1. To investigate FOXM1 regulated genes following PBK inhibition, we performed Chromatin Immunoprecipitation (ChIP), followed by qPCR. Subcutaneous xenograft study using MM cells was done to evaluate the in vivo efficacy of the PBK inhibitor.
Results: RNA seq. analysis showed that E2F and FOXM1 were among the top six pathways down-regulated by the PBK inhibitor and that there was a notable overlap between the affected genes and target genes of the DREAM complex. Using Co-IP and ChIP experiments, we found that PBK interacts with and phosphorylates FOXM1, thus upregulating FOXM1 target genes such as RAD51, EXO1 and CDC25a. To get further insight into PBK-mediated regulation of the DREAM complex, we immunoprecipitated LIN54 (a major component of MuvB) in PBK-overexpression and PBK-knockdown MM cells. We found that PBK-overexpression increases the phosphorylated FOXM1-LIN54 interaction and disrupts the repressive DREAM complex, as evident from reduced LIN54-E2F4 interaction. On the contrary, PBK-knockdown or PBK inhibitor promoted the DREAM repressive complex (LIN54-E2F4 interaction) and reduced the phosphorylated FOXM1-LIN54 interaction. These data demonstrate that elevated PBK disrupts the repressive DREAM complex by dissociating MUVB from E2F4 and recruiting it to FOXM1, thus promoting mitosis and contributing to genomic instability. Consistently, the comparative gene expression profiling of paired myeloma cell samples collected at diagnosis and again after chemotherapy (melphalan) relapse (GSE19554), showed significant upregulation of PBK at relapse. In MM cells, treatment with PBK inhibitor inhibited spontaneous and chemotherapy-induced genomic instability (P < 0.05), as assessed by micronucleus assay. Moreover, in a subcutaneous mouse model of MM, treatment with PBK inhibitor also impaired tumor growth and significantly increased the efficacy of a chemotherapeutic agent (P = 0.0011).
Conclusions: Inhibitors of PBK have the potential to inhibit growth and increase the cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic instability in MM
