Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role.,In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors.,Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.,We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy.,We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples.,In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples.,No NRAS mutations were detected in pre-resistant samples.,By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells.,The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.,We conclude that BRAF and NRAS co-mutations are not mutually exclusive.,However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy.,In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary.
Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors.,However, a limitation to such treatment is the occurrence of resistance.,Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent.,In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results.,However, also in this case resistance inevitably occurs.,It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.,Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D).,Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array.,Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies.,The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting.,The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.,In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT.,Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth.,We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.,On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.