Persistent mismatch repair deficiency following targeted correction of hMLH1

Abstract

The use of gene therapy to correct mutated or lost gene function for the treatment of human cancers has been an active, yet problematic area of biomedical research. Many technical difficulties, including efficient tissue-specific delivery, integration site specificity and general toxicity, are being addressed. Little is known, however, about the genetic and phenotypic stability that accompanies a successful gene-specific targeting event in a cancer cell. This question was addressed following the creation of a colon cancer cell line in which a mutated hMLH1 gene was corrected via targeted homologous recombination. This correction resulted in the expression of wild-type hMLH1 protein, restoration of the hPMS2 protein and mismatch repair (MMR) proficiency. One of two hMLH1-corrected clones, however, was found to retain defects in MMR activity. These cells continued to express the corrected hMLH1 protein, but had lost expression of another MMR protein, hMSH6. DNA sequence analysis of the hMSH6 gene revealed biallelic expansions of a cytosine repeat region in exon 5 that result in frameshifts leading to premature stop codons. These findings suggest that, similar to acquired drug resistance, the presence of genetically heterogeneous cancer cell populations or acquisition of compensatory mutations can result in 'resistance' to gene replacement therapy.

Cytosine-based nucleoside analogs are selectively lethal to DNA mismatch repair-deficient tumour cells by enhancing levels of intracellular oxidative stress

“…To determine whether our observations were generally applicable to models of dMMR, we assessed the effect of cytarabine in other isogenic MLH1 and MSH2-deficient cell line pairs. We used the CRC cell lines, BVEC F7bro (MLH1-deficient) and BVEC E2 (MLH1-proficient) (Weiss et al , 2007), as well as LoVo (MSH2-deficient) and LoVo+Chr2 (MSH2 proficient) (Watanabe et al , 2000). We found that cytarabine was selective for MMR-deficient cell lines in both systems (Figures 2A and B), suggesting that this phenotype was applicable to multiple models.…”

Knock-in of Mutant K-ras in Nontumorigenic Human Epithelial Cells as a New Model for Studying K-ras–Mediated Transformation

“…Indeed, we have shown recently the discovery of genetic heterogeneity between single-cell clones after targeted knock-in of the hMLH1 gene in the MIN Table 2. Details of the 16 human cancer cell lines harboring K-ras mutations categorized by K-ras expression and allelic status cancer cell line HCT 116 (50). This property of MIN may also explain the differing degrees of tumorigenicity seen in studies using the same MIN DLD-1 colon cancer K-ras knockout clones (20,47).…”

Novel 2-Benzoyl-6-(2,3-Dimethoxybenzylidene)-Cyclohexenol Confers Selectivity toward Human MLH1 Defective Cancer Cells through Synthetic Lethality

“…We performed a high-throughput synthetic lethal screen (48 h) to identify compounds that could selectively kill HCT116 MLH1 (–/–)-deficient cells but not its proficient counterpart, HCT116 MLH1 (+/–). The parental HCT116 MLH1 (–) cells carry a hemizygous point mutation (C755A) in exon 9 of the MLH1 gene, 24 resulting in a premature stop codon and loss of protein function, whereas the isogenic counterpart has a heterozygous knock-in of wild-type MLH1 allele (+/–) 25 ( Fig. 1a ).…”

“…To further support our observation, we generated an MLH1 isogenic line from HT29 cells using the CRISPR-Cas9 technique. Compared with the HCT116 isogenic, which is a knock-in of MLH1 25 , the HT29 isogenic contains a knockout of MLH1 . As shown in Figure 2c , loss of MLH1 protein was found in five out of nine MLH1 -edited HT29 lines, encoded as HT29(MLH1 -)1, HT29( MLH1 -)2a, HT29( MLH1 -)2b, HT29( MLH1 -)2c, and HT29( MLH1 -)3, respectively.…”