HSP70 Inhibition Blocks Adaptive Resistance and Synergizes with MEK Inhibition for the Treatment of NRAS-Mutant Melanoma

Abstract

NRAS-mutant melanoma is currently a challenge to treat. This is due to an absence of inhibitors directed against mutant NRAS, along with adaptive and acquired resistance of this tumor type to inhibitors in the MAPK pathway. Inhibitors to MEK have shown some promise for NRAS-mutant melanoma. In this work, we explored the use of MEK inhibitors for NRAS-mutant melanoma. At the same time, we investigated the impact of the brain microenvironment, specifically astrocytes, on the response of a melanoma brain metastatic cell line to MEK inhibition. These parallel avenues led to the surprising finding that astrocytes enhance the sensitivity of melanoma tumors to MEK inhibitors (MEKi). We show that MEKi cause an upregulation of the transcriptional regulator ID3, which confers resistance. This upregulation of ID3 is blocked by conditioned media from astrocytes. We show that silencing ID3 enhances the sensitivity of melanoma to MEKi, thus mimicking the effect of the brain microenvironment. Moreover, we report that ID3 is a client protein of the chaperone HSP70, and that HSP70 inhibition causes ID3 to misfold and accumulate in a detergent-insoluble fraction in cells. We show that HSP70 inhibitors synergize with MEKi against NRAS-mutant melanoma, and that this combination significantly enhances the survival of mice in two different models of NRAS-mutant melanoma. These studies highlight ID3 as a mediator of adaptive resistance, and support the combined use of MEK and HSP70 inhibitors for the therapy of NRAS-mutant melanoma. Significance: MEKi are currently used for NRAS-mutant melanoma, but have shown modest efficacy as single agents. This research shows a synergistic effect of combining HSP70 inhibitors with MEKi for the treatment of NRAS mutant melanoma.

Targeting ErbB3 and Cellular NADPH/NADP+ Abundance Sensitizes Cutaneous Melanomas to Ferroptosis Inducers

“…Western blot analysis revealed that ferroptotic cell death is characterized by a marked loss of many proteins, including several with roles in ferroptosis, such as GPX4 and ACSL4 (acyl-CoA synthetase long-chain family member 4; Figure B, Figure S1B and C). These changes closely resemble those caused by an inhibitor of the stress-inducible molecular chaperone HSP70 − (Figure C). As shown (Figure S1D), treating cells with the HSP70 inhibitor AP-4-139B (hereafter 139B) , results in a similar dose-dependent cytotoxicity in all melanoma cell lines examined, regardless of genotype or differential ferroptosis sensitivity.…”

“…These changes closely resemble those caused by an inhibitor of the stress-inducible molecular chaperone HSP70 − (Figure C). As shown (Figure S1D), treating cells with the HSP70 inhibitor AP-4-139B (hereafter 139B) , results in a similar dose-dependent cytotoxicity in all melanoma cell lines examined, regardless of genotype or differential ferroptosis sensitivity. RSL3-mediated ferroptosis leads to an increase in ferrous iron and MDA (Figure S1E), which is a toxic product of lipid peroxidation; 139B-mediated HSP70 inhibition had little effect on these factors (Figure S1E).…”