Exploring Signaling Mechanisms regulating genetic and non-genetic Drug Resistance in Melanoma

Melanoma is among the most heterogeneous and aggressive type of cancers. The BRAFV600E mutation, found in over 50% of metastatic melanomas, drives aberrant MAPK-ERK pathway activation and tumor progression. Selective BRAFV600E inhibitors lead to drug resistance and patient relapse in the clinic. A combination of BRAFV600E and MEK inhibition only delays such drug resistance. Additional pro-survival signaling pathways, such as the PI3K-Akt cascade also contribute to tumorigenic progression. Here we use biosensors to study ERK/AKT activity dynamics at the single cell level in primary, patient-derived BRAFV600E, as well as BRAFV600E/NRASQ61K cells in which the secondary NRASQ61K mutation emerged upon treatment with a BRAFV600E inhibitor. We find that BRAFV600E cells display sustained ERK activity and Akt activity that fluctuates on timescales of hours. Dabrafenib-mediated BRAFV600E inhibition leads to sustained Akt activity over timescales of hours. BRAFV600E/NRASQ61K cells displayed both sustained ERK and Akt activity. Elevated Akt activity contributes to survival. We then performed siRNA kinome screens targeting each three oncogenic signaling paradigms. We find that BRAFV600E cells are strongly resistant to kinome perturbations, with only a small number of kinases perturbations leading to decreased ERK activity. Surprisingly, BRAFV600E/NRASQ61K cells were much more sensitive to kinome perturbations. We speculate that the NRASQ61K mutation restores RTK inputs and pathway crosstalks to the MAPK network, rendering it sensitive to these additional inputs. We found that the PI3K/Akt network is much more sensitive to kinome perturbations, with perturbation of RTKs, or kinases involved in nutrient sensing or cytoskeletal dynamics being able to inhibit aberrant Akt activity in the different cell systems. Our screens revealed signaling nodes for which drugs were available. Finally, we show that drug-mediated PLK4 or PAK3 inhibition, that were identified in our screens, leads to immediate decrease of aberrant Akt signaling in BRAFV600E inhibited, BRAFV600E cells, or in BRAFV600E/NRASQ61K cells, implying a signaling crosstalk that involves direct protein interactions. Inhibition of these proteins leads to decreased survival. Our results provide new insights into the mechanisms of non-genetic and genetic origins of drug resistance, and identify new network vulnerabilities to intervene in drug resistance during targeted therapy