Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Abstract

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.cancer mutation ͉ oncogene addiction ͉ pharmacogenomic ͉ targeted therapies ͉ tumor progression model T he construction of model systems that accurately recapitulate the genetic alterations present in human cancer is a prerequisite to understand the cellular properties imparted by the mutated alleles and to identify genotype and tumor-specific pharmacological responses. In this regard, mammalian cell lines have been widely used as model systems to functionally characterize cancer alleles carrying point mutations and to develop and validate anticancer drugs. These models typically involve the ectopic expression (by means of plasmid transfection or viral infection) of mutated cDNAs in human or mouse cells (1). Although these approaches have yielded remarkable results, they are typically hampered by at least two caveats. First, the expression is achieved by transient or stable transfection of cDNAs, often resulting in over-expression of the target allele at levels that do not recapitulate what occurs in human cancers. Second, the expression of the mutated cDNA is achieved under the control of nonendogenous viral promoters. As a result, the mutated alleles cannot be appropriately (endogenously) modulated in the target cells. While such systems in which mutated oncogenes are ectopically expressed under exogenous promoters have been instrumental in dissecting their oncogenic properties, they have also led to controversial results. For example, studies focused on oncogene-mediated transformation and senescence have generated conflicting data depending on whether the cancer alleles were ectopically expressed or permanently introduced in the genome of mouse or human cells (2-5). To address the limitation of current models, we have used targeted homologous recombination to introduce (knock-in, KI) a panel of cancer alleles in human somatic cells. Specifically, we focused on EGFR, KRAS, BRAF, and PIK3CA mutated alleles that are found in multiple cancer types. Mutant cells have then been used to study the biochemical and transforming potential of common cancer alleles and to identify genotype-specific ...

BRAF V600E Is a Determinant of Sensitivity to Proteasome Inhibitors

“…Isogenic cell models able to effectively recapitulate single genetic aberrations have been employed extensively to establish binary drug-genotype associations (20,30,31). Nevertheless, limited efforts have been dedicated to dissect the role of combinations of mutations in determining drug response.…”

“…All experimental procedures for BRAF V600E targeting vector construction, adeno-associated virus (AAV) production, cell infection, and screening for recombinants have been described previously (20,21).…”

“…In Supplementary Table S1, we have reported a list of tested compounds, their chemical formula, their molecular weight (MW), the solvent used for suspension, the concentration of stock solutions, the concentrations tested in the experiments, and the storage conditions used for the stock. Each compound was preliminarily tested on HME-1 parental cells infected with scramble shRNA to determine the concentration referred to as the highest no-observed effect level (NOEL), the IC 50 , and the IC 90 values, as previously reported (20). The three concentrations of each compound tested on the entire isogenic cell panel were selected on the basis of these premises.…”

“…Normalized data was then clustered and plotted on an array using the GEDAS software (22). This approach was previously developed and described for the analysis of differential drug activity in KI isogenic models and is defined as a "Pharmarray" (20). Cell lines and drugs were clustered on the basis of their response profile.…”

Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells

“…SW48 cells are wt for KRAS, whereas SW48 KRAS G13D cells were created by knock-in of an activating mutation into one KRAS allele (Di Nicolantonio et al, 2008). We found that Snail2 knockdown using two small interfering RNA (siRNA) oligos differentially killed SW48 KRAS G13D cells, but had a minimal effect on SW48 wt cells (Figure 1d, Po0.01), even though Snail2 knockdown effects were stronger in SW48 wt cells (Supplementary Figure S1b).…”