BRAF inhibitor (BRAFi) combined with MEK inhibitor (MEKi) are used to treat melanomas harboring (V600E) mutation. While response is high, the majority of them relapsed. We found novel mechanisms to treat BRAFi resistant (BR) patients.
Five BR cells were established from known BRAF mutant cell lines A375, MEL-1220, A2058, UACC-62, and SKMEL28 by long-term exposure to BRAFi. These BR cells are hypersensitive to ADI-PEG20, an enzyme which degrades arginine to citrulline. ADI-PEG20 treatment resulted in 10-30% increase in apoptosis (Annexin V/PI) in BR cells compared to their paternal cells. The mechanisms involved are as follows: All BR cells express very low levels or do not express argininosuccinate synthetase (ASS, a key enzyme to generate arginine from citrulline) and also have attenuated glucose uptake. Thus, these BR cells rely primarily on exogenous arginine. Importantly, their ability to undergo autophagy upon nutritional stress is also impaired. The underlying mechanism for low levels of ASS is due to diminished c-Myc expression,a positive regulator for ASS. Additionally, AMPK-α 1, which governed autophagy and glucose uptake, was attenuated in BR cells. Overexpression of AMPK-α 1 using plasmid transfection in A2058BR and MEL-1220BR cells rescued 8-28% ADI-PEG20-induced apoptosis and enhanced autophagosome formation. Conversely, knockdown of AMPK-α 1 significantly enhanced ADI-PEG20-reduced cell viability in A2058 and MEL-1220 cells through attenuation of autophagy and glucose uptake. This is evidenced by decreased GLUT1 and LC3-II expression, and low activity of 2-NBDG uptake seen in BR cells. BMR (BRAF and MEK inhibitor) resistant cells also shared similar biochemical changes. Immunohistochemical staining further confirmed lower levels of ASS and AMPK-α 1 in A2058BR, BMR and in tumor tissues from 10 BR patients relative to their parental counterparts (average H-scores of ASS and AMPK in parental tissues vs. BR tissues are 58.2 vs. 7.8, and 146 vs. 78.3, respectively, P < 0.03). Importantly, these findings also apply to cell lines and tumor samples from patients who failed both BRAF and MEK inhibitor.
In summary, our data suggest that attenuated AMPK-α 1 mediated autophagy and glucose uptake and decreased c-Myc mediated ASS re-expression sensitize BR cells to arginine deprivation, and can be used as salvage therapy for BR patients.
Supported by 1RO1CA152197 and VA Merit Review BLR&D11860649.
