Cutaneous Malignant Melanoma (CMM) is the deadliest form of skin cancer responsible for approximately 500 deaths annually in Sweden, with an increasing incidence over last few years. Early CMM detection enables good prognosis by treatment with surgery, whereas disseminated disease is associated with poor prognosis due to intrinsic and acquired drug resistance. One of the most difficult therapeutic challenges in CMM is to overcome resistance to systemic therapies and thereby avoid disease progression. We have identified afatinib (EGFR/HER2 inhibitor) and crizotinib (MET/ALK inhibitor) as an efficacious and thereby a promising alternative targeted combination therapy option independent of BRAF/NRAS mutational background for CMM patients. We hypothesize that there are other drug targets beyond these canonical targets which may be more important in mediating anti-tumorigenic responses as observed both in vitro and in vivo. For elucidating the underlying molecular mechanisms, we are using three different approaches- RPPA, Mass-spectophotometry (MS) and pRTK arrays. For the different approaches, we have taken into consideration that we would want to capture early (3h) and thereby direct targets of afatinib and crizotinib and compare it to later time point (24h) to correlate the differential expression changes mediated by the cellular stress induced by these drugs. Moreover, we have extracted tumor protein lysates from mice (n=20) exposed to vehicle, afatinib or crizotinib or both in combination and analysed the differences in molecular signatures using RPPA to compare between combination and the different treatment arms. We have used t-test statistical analysis and an in-depth bioinformatics approach to further evaluate our MS and RPPA results respectively. Our results highlighted that genes involved in mTOR pathway, DNA repair and mitochondrial fusion were among the top significantly changed genes (fold change of >2 fold for in vitro data and fold change > 30% for in vivo data; p<0.05). Currently we are further selecting candidate genes of potential interest to evaluate their role in CMM. For this, we will be using both CMM patient samples and PDX models. Despite an increased understanding of the genetics and biochemistry of advanced CMM, the mechanisms underlying their complex interactions are still partly deciphered. Further understanding will give a potential for more successful therapeutic strategies and evidence-based management of patients with disseminated CMM. This study presents itself as a novel therapy regime with a potential application in the clinic, which could be used by CMM patients irrespective of BRAF/NRAS mutational status, and also for CMM patients with acquired BRAF resistance. Moreover, identifying key molecular targets could help us to better predict therapy efficacy as well as design novel and more efficacious treatment regimens for CMM patients.

Elucidate the underlying molecular mechanisms of the combination treatment effects of Afatinib (EGFR/HER2 inhibitor) and Crizotinib (MET inhibitor) in cutaneous malignant melanoma (CMM)