RKO exhibits all key traits of a distinct subpopulation of colorectal cancer patients, namely V600E mutant B Raf, microsatellite in stability, and the CpG island methylator pheno type. In addition, since RKO is wild type for KRAS, APC, and TP53, and lacks chromosomal nevertheless in stability, all relevant Inhibitors,Modulators,Libraries molecular features of other CRC subtypes are missing in these cells. We used this model system to study cancer cells traits de pending on B RafV600E and to identify agents selectively targeting BRAF mutant cells. Results and discussion BRAF targeting in RKO It has been shown that B RafV600E is sufficient to pro mote proliferation via Erk 1 2 signaling independently of exogenous growth factors and confers mechanisms to evade apoptosis.

However, these results are pri marily based on non quantitative Inhibitors,Modulators,Libraries RNA interference methods which are prone to artifacts in mamma lian cells due to nonspecific defense mechanisms. In contrast, somatic cell gene targeting enables quantitative knockouts of single alleles and the gener ation of endogenous models featuring well defined gen etic backgrounds. Utilizing this method, we have disrupted BRAF alleles in the colorectal cancer cell line RKO and established syngeneic clones which harbor a single BRAF allele of either wild type or mutant geno type. Despite its near diploid karyotype and MSI pheno type, the colorectal cancer cell line RKO carries a stable triplication of the BRAF gene locus with one wild type and two mutant alleles present in parental cells.

This genotype was verified by DNA sequencing Inhibitors,Modulators,Libraries in RKO E1, a subclone obtained from RKO that was found to be comparable to the parental cell line in terms of morphology and proliferation. In the first targeting round, an oncogenic allele of BRAF exon Inhibitors,Modulators,Libraries 15 was Inhibitors,Modulators,Libraries recombined and deleted by somatic cell gene targeting to generate the cell clone RBOW. Subsequently, either wild type or V600E mutant B Raf was disrupted by targeting a second allele in RBOW, yielding six BRAF mutant and one wild type clone from approximately 104 screened colonies. Out of these double positive clones, BRAF knockout cell lines RBO 1 and RBO 2 as well as RBW 1 were established. The apparent counterselection against inactivation of B RafV600E might indicate the presence of an oncogene addiction for B RafV600E as a cancer cell trait in RKO. For structural confirmation of the deleted alleles, DNA sequencing was performed and all genotypes were veri fied. Furthermore, all cells expressed BRAF protein at comparable levels. While the ex pression of Mek 1 2 and Erk 1 2 was independent of serum concentration and BRAF status, the phosphoryl ation of these effector kinases was constantly active in the BRAF mutant clones but low in BRAF wild selleck catalog type cells.