The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAFV600E metastatic melanoma.,However, resistance to such compounds represents a formidable problem.,Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases.,Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway.,In addition to three independent amplifications and an aberrant form of BRAFV600E, we identified a new activating insertion in MEK1.,This MEK1T55delinsRT mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas.,Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis.,This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient.,In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting.,Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient’s melanoma.
Immune checkpoint inhibitors1 result in impressive clinical responses2-5 but optimal results will require combination with each other6 and other therapies.,This raises fundamental questions about mechanisms of non-redundancy and resistance.,Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) and reproduced this effect in mouse models.,Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common.,Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T cell exhaustion.,Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1.,Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio.,RT enhances the diversity of the T cell receptor (TCR) repertoire of intratumoral T cells.,Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones.,Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion.,Similar to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed.,Thus, PD-L1 on melanoma cells allows tumors to escape anti-CTLA4-based therapy, and the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms.