Parthenolide Overcomes Vemurafenib Resistance in Melanoma

Nearly half of melanoma patients harbor an on-mutation of valine 600 to glutamine (V600E) in the BRAF gene. BRAF(V600E) inhibitors (BRAFi), including vemurafenib, have shown remarkable clinical efficacy in patients with BRAF(V600E)-mutant melanoma. However, most patients develop resistance to BRAFi within 6-8 months. Currently, no approved drug can overcome vemurafenib resistance. Safe and effective agents for treating vemurafenib-resistant melanoma are needed. Parthenolide, a sesquiterpene found in diverse Chinese medicinal herbs, exerts anti-melanoma effects, while whether it overcomes vemurafenib resistance in melanoma is still unknown. Studies showed that parthenolide downregulates protein levels of BRAF and c-Myc, and inhibits the activation of nuclear factor-kappa B (NF-κB), ERK, and STAT3 pathways. These parthenolide-affected molecules/pathways have been shown to promote vemurafenib resistance in melanoma. This study aimed to determine whether parthenolide overcomes vemurafenib resistance in melanoma.

The results showed that parthenolide dose- and time-dependently reduced viability of, and induced apoptosis and S phase arrest in, vemurafenib-resistant A375 (A375-VR) melanoma cells. Parthenolide inhibited A375-VR tumour growth without obvious toxicity in mice. To explore the mechanisms of action of parthenolide, we performed drug-targets-disease network analysis, and identified heat shock protein 90α (Hsp90α) as a key mediator of the effects of the compound. Hsp90α is a chaperone of diverse oncoproteins implicated in vemurafenib resistance in melanoma and, therefore, serves as a promising target for overcoming vemurafenib resistance. Molecular docking and dynamic simulation results showed that parthenolide potentially binds Hsp90α. Surface plasmon resonance assays confirmed the direct parthenolide-Hsp90α interaction. Piper assays showed that parthenolide dose-dependently inhibits the ATPase activity of Hsp90α. Immunoblotting showed that parthenolide decreases levels of Hsp90 client proteins, including RTKs (EGFR, PDGFR-β, Axl, and IGF1-R), B-Raf (V600E), C-Raf, and Src, and inhibited their downstream PI3K/Akt, Ras/Raf/MEK and Src/STAT3 pathways in A375-VR cells. The protein synthesis inhibitor cycloheximide diminished the effects of parthenolide on the stability of Hsp90 clients. Also, parthenolide inhibited Hsp90 signaling in A375-VR xenografts.

We also found that in A375-VR cells, parthenolide induced mitochondrial shrinkage, a morphological sign of ferroptosis. We further found that parthenolide decreased mitochondrial membrane potential (ΔΨm), inhibited GPX4 activity, and elevated levels of Fe2+, LPO, and ROS, further suggesting ferroptosis induction. Immunoblotting and RT-qPCR showed that parthenolide downregulated protein levels of the ferroptosis suppressors SLC7A11 and GPX4 but not the mRNA levels of them.

In summary, we have, for the first time, demonstrated that parthenolide overcomes vemurafenib resistance in melanoma, and Hsp90 inhibition and ferroptosis induction might be involved in its effects. This work facilitates the development of parthenolide as a therapeutic agent for treating vemurafenib-resistant melanomas.