Unravelling molecular mechanisms underlying therapy resistance in cutaneous melanoma

Sammanfattning: For many years the standard treatment of advanced metastatic melanoma with chemotherapeutic agents, including temozolomide (TMZ) and dacarbazine (DTIC), has been unsuccessful. The paradigm shift in melanoma treatment occurred with the identification of mutations in the BRAF gene that leads to a constitutively active BRAF V600E protein. This resulted in the development of BRAF mutant targeted therapies with small molecule inhibitors and showed favorable response in patients harboring BRAF mutations. However, most patients relapse due to acquired resistance to the inhibitors and biomarkers that can predict the therapy response is still lacking. In the first study we assessed the protein expression of melanosome related proteins in tumor biopsies from melanoma patients with different response to DTIC or TMZ. We found that expression of MITF and GPR143 was significantly higher in tumor samples from patients that did not respond to chemotherapy. In the second study we performed whole proteome profiling utilizing mass spectrometry based proteomics on pretreatment biopsies from melanoma patients receiving DTIC/TMZ. Our data showed a significant association between high expression of S100A13 protein and resistance to chemotherapy. Third study focused on identification of mediators of resistance to BRAF inhibitors. We therefore, established mutant BRAF inhibitor resistant sublines of BRAF V600E mutated melanoma cell line A375. By performing mass spectrometry based proteomics we identified several overexpressed proteins in the resistant sublines. We found two novel resistance mediators, aminopeptidase N (ANPEP/CD13) and FLI1 as well as the previously known receptor tyrosine kinase EPHA2 to be overexpressed and demonstrated to mediate vemurafenib resistance in our resistant sublines. Finally, we suggest that combination of vemurafenib with the multi kinase inhibitor dasatinib can overcome resistance in the melanoma cell lines. In the fourth study the efficiency of combining BRAF inhibitor PLX4720 with TMZ was evaluated in melanoma cell lines with variable sensitivity to BRAF inhibitors. We observed a schedule dependency in the response to the combination of PLX4720 with TMZ and further investigations indicated involvement of DNA damage response activation after PLX4720 treatment. Depletion of DNA repair protein MGMT by lomeguatrib abrogated the schedule dependency effect. Moreover, inhibition of ATR or disruption of the MDM2-p53 interaction by ATR inhibitor or nutlin-3, respectively, synergized with PLX4720 in induction of apoptosis. This thesis highlights some potential key molecular markers mediating resistance to chemo and targeted therapies in cutaneous malignant melanoma and emphasizes on the importance of using drug combination modalities as a way to overcome or bypass innate or acquired resistance