Mitochondrial biogenesis meets chemoresistance in BRAF-mutant melanoma

ABSTRACT

The acquisition of resistance to current mitogen activated protein kinase (MAPK) inhibitors in B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutant melanoma is almost inevitable. Our recent findings identify therapy-induced mitochondrial biogenesis (MitoBiogenesis) and aberrant tumor bioenergetics as therapeutic escape mechanisms and offer a rational combinatorial strategy to further improve the efficacy of MAPK inhibitors.

KEYWORDS:

• BRAF-mutant melanoma
• mitochondrial biogenesis
• targeted therapies

Stage IV melanomas have poor clinical prognosis, although this has improved with new therapies.^1,2 Approximately 50% of melanomas harbor a point mutation in the B-Raf proto-oncogene, serine/threonine kinase (BRAF), either V600E or V600K, which results in constitutively active mitogen activated protein kinase (MAPK) signaling.³ Advancements in the development of small-molecule inhibitors targeting mutant BRAF and immune checkpoint blockade therapies targeting immune inhibitory molecules such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed death-ligand 1 (PD-L1) have provided unprecedented improvements in patient outcomes. These proven therapeutic advances began in 2011, when the BRAF inhibitor vemurafenib was shown to improve both overall and progression-free survival rates for patients with BRAF-mutant melanomas in a phase III clinical trial.⁴ Subsequently, combined BRAF and mitogen-activated protein kinase kinase (MEK) inhibition, which now is standard of care for treating BRAF-mutant melanomas, has further improved the rate of progression-free survival compared to BRAF inhibition alone.⁵ Despite the initial clinical efficacy of BRAF-targeted therapies, tumor relapse is almost inevitable due to survival of a small tumor cell population that is dependent on reactivation of the MAPK and phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B (PI3K/AKT) signaling pathways to invade therapy.⁶

A comprehensive understanding of the molecular mechanisms underlying both intrinsic and acquired drug resistance to MAPK inhibitors (MAPKi) is still being elucidated and will be essential for the development of combinatorial strategies to overcome therapy resistance. Mitochondrial biogenesis (MitoBiogenesis) and tumor metabolism are aberrantly altered in cancer cells under a variety of cellular stress types. The Warburg effect is a shift toward glycolysis that is frequently utilized by many types of tumors, but some melanomas balance their metabolic needs depending on the interplay between microphthalmia-associated transcription factor (MITF), which is a key regulator of MitoBiogenesis, and peroxisome proliferator-activated receptor gamma coactivator 1-α (PPARGC1α).⁷ Melanoma cells with high expression of MITF and PPARGC1α exhibit increased oxidative phosphorylation (OxPhos) and resistance to oxidative stress. Interestingly, the treatment of BRAF-mutant melanomas with BRAF inhibitors leads to addiction of tumor cells to OxPhos.⁸

In our study, we built upon these findings for MitoBiogenesis and mechanistically dissected intrinsic therapy resistance to develop a rational approach for overcoming both intrinsic and acquired drug resistance.¹ We analyzed expression of the MitoBiogenesis transcriptional signature in a panel of BRAF-mutant melanomas using both cell lines and patients' tumors. We found that MAPKi downregulate MitoBiogenesis in a subset of MAPKi-sensitive cells with high basal levels of MitoBiogenesis and upregulate it in MAPKi-resistant cells with low basal levels (Fig. 1). These intrinsically resistant cells depend on upregulated MitoBiogenesis for survival and have increased mitochondrial DNA (mtDNA) copy number, mitochondrial mass, maximal oxygen consumption rate, and reactive oxygen species production. We were also able to validate upregulation of the MitoBiogenesis gene signature, mtDNA content, and representative OxPhos subunits in a subset of early on- and post-treatment tumor biopsies derived from BRAF-mutant melanoma patients treated with BRAF-targeting therapies.

Figure 1. Mitochondrial biogenesis (MitoBiogenesis) is a drug resistance mechanism and therapeutic target in melanoma. BRAF mutant melanoma cells with an intrinsic low basal level of MitoBiogenesis upregulate MitoBiogenesis, increase mitochondrial DNA (mtDNA) content, and alter tumor bioenergetics to survive mitogen activated protein kinase (MAPK) inhibition. Gamitrinib treatment or the knockdown of tumor necrosis factor receptor-associated protein 1 (TFAM) or mitochondrial transcription factor A (TRAP1) synergizes with MAPK inhibitors to kill resistant melanoma cells.

Previously, PPARGC1α was implicated as the key mediator of MitoBiogenesis in cancer cells.⁷ We found that depletion of tumor necrosis factor receptor-associated protein 1 (TRAP1) or mitochondrial transcription factor A (TFAM), but not of PPARGC1α, substantially enhanced the efficacy of MAPK inhibition. Both TRAP1 and TFAM are important regulators of MitoBiogenesis and play roles in mitochondrial protein folding and genome replication, respectively. Our siRNA-based screening data lead us to hypothesize that gamitrinib, an inhibitor of TRAP1, would be effective in inhibiting MitoBiogenesis. Indeed, the combination of gamitrinib and MAPKi impaired MitoBiogenesis and inhibited aberrant tumor bioenergetics in vitro. The synergistic effect of the 2 inhibitors significantly suppressed tumor growth in vivo. Analyses of The Cancer Genome Atlas (TCGA) melanoma cases revealed that melanoma patients with higher expression of mitochondrial biogenesis transcriptional signature or co-expression of glycolysis and OxPhos have markedly worse overall survival rates. Thus, there is a clinical relevance to this phenotype and further investigations are warranted.

For future studies, we envision that gamitrinib will synergize with MAPKi in the treatment of BRAF-mutant melanomas with acquired drug resistance. An increase in expression of MITF and PPARGC1α in neuroblastoma RAS viral oncogene homolog (NRAS) mutant melanoma cells upon MEK inhibition has already been shown.⁹ Thus, gamitrinib may be effective in combination with a MEK1/2 inhibitor to treat NRAS-mutant melanomas.¹⁰ It is likely that the MitoBiogenesis transcriptional signature will also be modulated in tumors derived from patients who progressed on anti-CTLA4, anti-PD-1, and/or anti-PD-L1 therapies and it will be interesting to investigate whether and how MitoBiogenesis affects response and resistance to immune checkpoint blockade therapies.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.