DiFi and Lim1215 were exposed to different doses of cetuximab as described in figure S2 to obtain the resistant variants. Cell viability was assessed by ATP content. Cells were seeded in 100μl medium in 96-well plastic culture plates. The experimental procedures for knock in of cancer mutations, the vectors, AAV production, cell infection and screening for recombinants have already been described elsewhere12 (link). Tumor specimens were obtained through protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (protocol 10-029) and Ospedale Niguarda Ca' Granda, Milano, Italy (protocols 1014/09 and 194/2010). Details about the clinical characteristics of patients are provided in Supplementary Table 2. Identification of cancer mutations in the KRAS, HRAS, NRAS, BRAF, PIK3CA and EGFR genes was performed with different sequencing platforms (Sanger, 454 pyrosequencing and Mass Spectrometry) as described in details in the supplementary methods. For immunoblot analysis, total cellular proteins were extracted by solubilizing the cells in boiling SDS buffer. Western blot detection was done by enhanced chemiluminescence. The analysis of KRAS activation was performed by immunoprecipitation assay with GST-Raf1-RBD. Real time PCR was performed using an ABI PRISM® 7900HT apparatus (Applied Biosytems). KRAS protein expression was evaluated by immunohistochemistry performed on 3μm thick tissue sections using a specific KRAS (F234) antibody (SC-30, mouse monoclonal IgG2a Santa Cruz Biotechnology). BEAMing was performed essentially as described previously10 (link), deviation from the original protocol are outlined in the supplementary methods. FISH experiments were conducted according with the histology FISH accessory kit (Dako, Glostrup, Danmark). Data are presented as the mean ± SD and n = 3. Statistical significance was determined by paired Student's t test. P < 0.05 was considered statistically significant.