Abstract Background and Aims: Genomic instability, a prominent feature of cancer and precancerous conditions, leads to constant acquisition of genomic changes some of which enable these cells to progress through worsening stages of disease, overcome immune surveillance and survive through therapeutic interventions. The purpose of this study was to identify and validate genes and pathways driving genomic evolution in human cancer, with special focus on myeloma (MM). Methods: Gene expression and copy number data of ten human cancers (MM, hepatocellular carcinoma, hormone positive breast cancer, triple negative breast cancer and adenocarcinomas of esophagus, stomach, pancreas, lung, prostate and ovary) were used in a stepwise manner in each cancer. First, the extent of genomic instability in each patient was assessed by calculating total copy number events and then integrated with expression data to identify genes whose expression correlated with genomic instability. Subsequently the genes in this signature were investigated for correlation with survival in different MM datasets. Polo-like kinase 4, a member of this signature, was investigated in loss and gain of function studies for its impact on growth and chromosomal integrity. Results: Expression of 32 genes correlated with genomic instability in ten human cancers including MM. Functional enrichment analysis demonstrated that microtubule motor, RNA/DNA hybrid ribonuclease, flap endonuclease and 5‘-3‘ exonuclease activities were prominent molecular functions whereas spindle organization, chromosome segregation and nuclear division were major biological processes associated with these genes. Expression of 11 out of 32 genes associated with poor OS and/or EFS in three different MM datasets MMRF, IFM70 (n=170) and GSE24080 (n=559). Of these 11 genes, polo-like kinase 4 (PLK4), with the highest relative expression in MMRF dataset was investigated further. Evaluation by Western blotting confirmed that expression of PLK4 was low in PBMC (n=2) whereas markedly elevated in all 10 MM cell lines tested. PLK4 knockdown significantly inhibited whereas its overexpression increased DNA breaks and genomic instability (as assessed from micronucleus assay) as well as growth rate of MM cells. Evaluation of cell cycle in PLK4-overexpressing MM as well as non-cancerous (HS5) cells identified a smaller third peak after G2 indicating a sub population of cells with DNA content 4N. Consistently, the karyotypic evaluation of these MM cells demonstrated that after 8 weeks in culture, control cells had only 1 out of 70 (1.4%) abnormal metaphases, whereas 8 out of 70 (11.4%) metaphases in PLK4-overexpressing cells were abnormal. Abnormalities in PLK4-overexpressing cells included fused, broken and polyploid chromosomes. In non-cancerous immortal (HS5) cells, 5 out of 70 (7%) abnormal metaphases were observed in control cells whereas 15 out of 70 (21.4%) were abnormal in PLK4-overexpressing cells. Abnormalities included fused, broken, triradial and polyploid chromosomes. Evaluation by fluorescence in situ hybridization (FISH) demonstrated c-myc amplification (with 3 to 5 copies) in 30/500 (6%) control cells whereas 56/500 (11.2%) PLK4-overexpressing cells. Similar observations were made when a centromere probe was used to investigate these samples by FISH, indicating an increase in intrachromosomal instability by PLK4. A specific PLK4 inhibitor caused inhibition of cell viability in all 4 MM cell lines tested (with IC50 ranging from 11 nM – 71 nM) with a minimal impact observed in 2 normal PBMC samples (IC50 50 M) indicating a high therapeutic index. Furthermore, we observed that PLK4 regulates expression and/or phosphorylation of PBK which in turn phosphorylates FOXM1 leading to increased genomic instability (through expression of DNA repair genes including RAD51) as well as contributes to ongoing proliferation of MM cells(through inhibition of DREAM complex). Conclusions: Using an integrated genomic strategy, we identified a global gene signature (comprised of 32 genes) associated with genomic instability and poor survival in human cancers including MM. One of the most prominent amongst these, PLK4, is identified as an important component of this signature impacting development of chromosomal changes including hyperdiploidy, a major defining feature in half of MM . With its role in cell cycle and and chromosome integrity, it is a promising target in MM to inhibit growth and progression.
Zhao et al. (Mon,) studied this question.