Abstract 1103: Linkage between endogenous Chk2 activation and p14ARF expression in p53-mutated cells revealed by proteomic and genomic analyses of the NCI60

Chk2 is a multi-substrate kinase involved in the DNA damage response and genomic stability. Chk2 inhibitors are currently being evaluated in cancer models, with the rationale of enhancing the activity of DNA damaging agents in p53-deficient tumors while protecting normal tissues. Part of the ATM-Chk2-p53 axis, Chk2 is overexpressed or activated in various malignant tumors. Here we determined Chk2 copy number, total and single-exon transcriptional levels, and total and activated (pT68) protein across the 60 cell lines of the NCI DTP screen. We correlated these parameters with p53 mutational status, miRNA expression (Agilent miRNA Microarray V2), and performed supervised transcript (Agilent Whole Human Genome Oligo Microarray) class comparison analysis between high and low pT68 Chk2 cell lines using BRB Array Tools 3.8. All the lines used for this purpose were known to be p53-mutated. Our phosphoproteomic analyses demonstrate that a number of cell lines have high endogenous Chk2 activation (ovarian OVCAR3 and OVCAR4, renal RFX-393, lung NCI-H322M, and EKVX, melanoma UACC-62 and −257, leukemia RPMI-8226 and breast BT-549) whereas the wild-type p53 tumors almost invariably show undetectable Chk2 activation. Expression of total Chk2 protein and mRNA were generally positively correlated to each other. Exon array analyses across the NCI-60 suggest exon 3 splicing in almost all Chk2 high expresser cell lines and premature truncation between exons 8 and 10 in the ovarian SKOV3 cells, which could be related to the low level of Chk2 expression in that cell line. One microRNA, hsa-miR-455-3p, showed a highly significant negative correlation with both Chk2 transcript levels and with total protein levels (Spearman's r =-0.48, p < 10-3 and r=-0.35, p < 0.01 respectively) and was predicted to hybridize to Chk2 mRNA by miRBase (www.mirbase.org). Among the 181 probes differentially expressed in cells with highly activated Chk2, 123 mapped to known gene IDs, and 58 were eligible for network analysis with Ingenuity Pathways Analysis 8.0 (Ingenuity Systems Inc.). Functions associated with the networks enriched in genes differentially expressed by high pT68 cells included: cell cycle, cell-to-cell signaling, cell growth and proliferation, cancer. Remarkably, the tumor suppressor CDKN2A (p14ARF) showed a 132-fold difference between the two groups considered (unpaired t test, two-tailed p < 10-7; false discovery rate < 10-7), being highly overexpressed in high pT68 Chk2 cell line