Carol McCormick

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3] [4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins - in addition to their role in DNA-damage recognition - decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.

Interactions of the DNA mismatch repair proteins MLH1 and MSH2 with c-MYC and MAX

MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER™ fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2–3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.

Identification of a Selective G1-Phase Benzimidazolone Inhibitor by a Senescence-Targeted Virtual Screen Using Artificial Neural Networks

Cellular senescence is a barrier to tumorigenesis in normal cells, and tumor cells undergo senescence responses to genotoxic stimuli, which is a potential target phenotype for cancer therapy. However, in this setting, mixed-mode responses are common with apoptosis the dominant effect. Hence, more selective senescence inducers are required. Here we report a machine learning–based in silico screen to identify potential senescence agonists. We built profiles of differentially affected biological process networks from expression data obtained under induced telomere dysfunction conditions in colorectal cancer cells and matched these to a panel of 17 protein targets with confirmatory screening data in PubChem. We trained a neural network using 3517 compounds identified as active or inactive against these targets. The resulting classification model was used to screen a virtual library of ~ 2M lead-like compounds. One hundred and forty-seven virtual hits were acquired for validation in growth inhibition and senescence-associated β-galactosidase assays. Among the found hits, a benzimidazolone compound, CB-20903630, had low micromolar IC50 for growth inhibition of HCT116 cells and selectively induced senescence-associated β-galactosidase activity in the entire treated cell population without cytotoxicity or apoptosis induction. Growth suppression was mediated by G1 blockade involving increased p21 expression and suppressed cyclin B1, CDK1, and CDC25C. In addition, the compound inhibited growth of multicellular spheroids and caused severe retardation of population kinetics in long-term treatments. Preliminary structure-activity and structure clustering analyses are reported, and expression analysis of CB-20903630 against other cell cycle suppressor compounds suggested a PI3K/AKT-inhibitor–like profile in normal cells, with different pathways affected in cancer cells.