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 ...