Does dihydroartemisinin overcome vemurafenib resistance in melanoma by suppressing FLI1 expression?

Advanced melanoma is one of the deadliest forms of cancer. Around half of melanoma patients carry a BRAF mutation of valine 600 to glutamine (V600E). Although targeted therapies using selective BRAF(V600E) inhibitors (BRAFi), such as vemurafenib, have shown remarkable efficacy in patients with BRAF(V600E)-mutant melanomas, most patients develop resistance within 8 months. MEKi (mitogen-activated protein kinase kinase inhibitor) alone or in combination with ICB (immune checkpoint blockade antibody) has been shown to delay the emergence of BRAFi resistance, but it, still, typically develops within 15 months. Some smallmolecule agents have been tested for treating BRAFi-resistant patients; however, unfortunately, owing to serious adverse effects, clinical outcomes were not improved. Chinese medicinal herbderived multi-target and low-toxicity compounds have advantages in fighting drug resistance. Through screening, we found that dihydroartemisinin (DHA, an approved antimalarial drug and the active metabolite of artemisinin that occurs in Artemisia annua) reduces viability of, and induces apoptosis in, acquired vemurafenib-resistant A375 (A375-VR) and Colo829 (Colo829- VR) melanoma cells. Importantly, prophylactic dosing of DHA significantly suppresses A375- VR tumor growth in mice without affecting mouse weight; and DHA prolongs survival time of A375-VR melanoma-bearing mice. RNA-seq analyses revealed that mRNA level of FLI1 (friend leukemia integration site 1), an oncogene, is higher in A375-VR cells than in parental A375 cells, and DHA abolishes the resistance-associated upregulation. We also found that mRNA levels of several BRAFi resistance-related genes downstream of FLI1 are altered in A375-VR cells compared to those in A375 cells, and DHA reverses the alterations of most of these genes. Furthermore, DHA was found to be able to lower the protein level of FLI1 in A375-VR cultures and in mouse A375-VR xenografts. It has been reported that FLI1 is upregulated in BRAFiresistant melanoma cells; and silencing FLI1 is able to slow the growth of vemurafenib-resistant melanoma cells. We, therefore, hypothesize that DHA overcomes vemurafenib resistance in melanoma by suppressing FLI1 expression. The objectives of this study are as follows.

1) To investigate the effects, including toxicity, of DHA in overcoming vemurafenib resistance in melanoma mouse models;
2) To establish the role of FLI1 in the effects of DHA in overcoming vemurafenib resistance in melanoma cells; and
3) To determine whether DHA overcomes resistance to other BRAF(V600E)-targeted therapies in melanoma cells.

We expect that this study will, for the first time, demonstrate the ability of DHA to overcome vemurafenib resistance via, at least in part, suppressing FLI1 expression in BRAF(V600E)- mutant melanomas. This contribution will be significant because it is one step in a continuum of research that is expected to lead to the development of DHA and possibly other agents that can suppress FLI1 expression into adjuvant drugs for treating BRAFi-resistant melanomas.