Transsulfuration Reprogramming: A Metabolic Driver of BRAF-V600E Resistance in Melanoma

Recently, a study by Péter Nagy's team [1] published in Cell Metabolism identified that the upregulation of cystathionine γ-lyase (CSE) and the inhibition of cystathionine β-synthase (CBS) are key factors contributing to the development of resistance to B-Raf proto-oncogene, serine/threonine kinase (BRAF) inhibitors (BRAFi) in treatment. Coadministration of the CSE inhibitor d,l-propargylglycine (PAG) with BRAFi significantly improved therapeutic efficacy and delayed the onset of resistance, offering a novel therapeutic strategy for patients with BRAF V600E-mutant (a valine-to-glutamic acid substitution at position 600 in the BRAF protein) melanoma.

Malignant melanoma is a highly aggressive tumor originating from melanocytes, with approximately 50% of patients harboring the BRAF V600E mutation [1]. This mutation leads to the activation of the downstream mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) signaling pathway, which in turn results in an increase in aerobic glycolysis, thereby supporting the proliferation of melanoma cells [2]. BRAF V600E inhibitors, such as vemurafenib (V) and dabrafenib (D), have been approved by the Food and Drug Administration (FDA) for the treatment of melanoma. However, resistance to these therapies often develops. Even when combining dabrafenib with the MEK inhibitor trametinib, resistance remains an inevitable challenge [3].

Existing research has indicated that treatment with dabrafenib-trametinib (DT) inhibits the BRAF/MEK/ERK pathway, leading to a shift in melanoma cell metabolism from aerobic glycolysis to mitochondrial respiration, which is unfavorable for melanoma cell proliferation [4]. Concurrently, the increased mitochondrial oxidative phosphorylation and electron transport chain (ETC) pathways result in enhanced reactive oxygen species (ROS) production. Excessive ROS production can disrupt the cellular redox balance and trigger oxidative stress responses. Building on this, the study discovered significant expression of cytochrome P (CYP)450 enzymes in dabrafenib- and trametinib-treated cells (DTC), with upregulation of CYP1B1 and CYP2F1 in dabrafenib- and trametinib-double resistant cells (DTR). These enzymes contribute to ROS production. To counteract the effects of ROS accumulation, antioxidant enzymes, such as superoxide dismutase 2 (SOD2), thioredoxin reductase 1 (TrxR1), catalase, 14-kDa human thioredoxin (Trx)-related protein (TRP14), glucose-6-phosphate dehydrogenase (G6PD), and glutathione (GSH) peroxidase 1 and 4 (GPX1, 4), are upregulated in DTC. However, this antioxidant response is limited, and the cells become more sensitive to exogenous oxidants, making them more susceptible to oxidative stress-induced damage. To support the function of antioxidant enzymes, DTC increasingly rely on the pentose phosphate pathway (PPP) to generate more nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate for these enzymes, which results in the loss of the aerobic glycolysis pathway, thus impeding melanoma cell proliferation. However, in DTR cells, glycolytic activity is restored, ROS accumulation decreases, and the cells resume proliferation. Although DT treatment initially inhibits cell proliferation effectively, with continued antioxidant and metabolic adaptation, the cancer cells eventually recover their growth capacity and develop resistance. This phenomenon suggests that other pathways may assist DTC in overcoming the oxidative stress induced by DT, ultimately leading to the emergence of DTR.

Péter Nagy's team has found that the transsulfuration pathway plays a key role in the development of drug resistance. Homocysteine (Hcy) is converted into cysteine (Cys) through the combined actions of CBS and CSE, which is the canonical function of the transsulfuration pathway. The demand for Cys is significantly increased in DTC. Cys can be used to synthesize GSH or reactive sulfur species (RSS), such as hydrogen sulfide (H₂S) and cysteine persulfides (Cys-SSH) [5]. GSH can reduce ROS accumulation via glutathione peroxidase 4 (GPX4), thereby mitigating ferroptosis caused by ROS. On the other hand, RSS can protect against ferroptosis and oxidative stress, safeguard proteins from oxidative damage, provide energy for ATP synthesis in the ETC, and regulate aerobic glycolysis. The increase in RSS is a key factor in tumor cell survival under oxidative stress conditions. However, further analysis revealed that in DTC, CBS levels decrease and CSE levels increase, whereas in DTR, CBS levels increase and CSE levels decrease. This suggests that DTC does not rely on the canonical activity of the transsulfuration pathway to support the high demand for Cys and thus generate more RSS. Instead, DTC increase the uptake of cystine disulfide (CySSCy) via the cystine-glutamate transporter (xCT) on the cell membrane. CySSCy can either be converted into Cys-SSH via the action of CSE or CBS, or be processed through the Trx pathway to generate Cys, subsequently producing H₂S and other RSS. The researchers further validated this pathway through intracellular amino acid metabolism analysis and isotope labeling, confirming that this pathway is crucial for DTC survival. Gene knockout experiments also demonstrated that CSE is the critical factor for the acquisition of drug resistance in DTC. Elevated expression of CSE in melanoma patients undergoing DT treatment further supports this hypothesis and suggests the potential for targeting CSE in combination with DT as a therapeutic strategy for melanoma.

The research team utilized PAG to inhibit CSE activity and combined it with DT treatment. Experiments showed that the combination of PAG and DT significantly delayed the development of resistance. In melanoma mouse models, the combination therapy significantly extended progression-free survival and inhibited tumor growth. This suggests that combining CSE inhibitors could be a crucial strategy to enhance the efficacy of BRAF V600E targeted therapy. However, it is noteworthy that this combination was not applied to DTR. CSE was downregulated in resistant cells, indicating that PAG treatment might be ineffective once resistance has emerged. Therefore, future investigations are warranted to evaluate the timing and context-specific application of CSE inhibition, and to explore whether early intervention could maximize therapeutic benefit while avoiding resistance.

In summary, the study demonstrates that melanoma cells undergo extensive metabolic reprogramming under DT combination therapy. DT treatment induces oxidative stress in DTC, prompting an increase in the PPP activity, which boosts the activity of the cellular redox system. Meanwhile, DTC increase the uptake of CySSCy, and through CSE, generate Cys-SSH, which play a critical role in protecting DTC from oxidative stress-induced damage and contribute to the development of drug resistance. This study reveals a novel mechanism underlying resistance to DT treatment and provides new insights for the development of more effective combination therapy strategies. Figure 1 summarizes the resistance mechanism of DTC.

However, the study has some limitations. xCT activity is crucial for DTC survival, suggesting that further inhibition of xCT alongside PAG and DT may enhance the therapeutic effect. Given these findings, it is worth considering whether DTR still retain targetable metabolic vulnerabilities. Although downregulation of CSE may render PAG treatment ineffective, future research could explore whether further modulation of the transsulfuration pathway might resensitize resistant cells to therapy. Such strategies may help expand the therapeutic window and improve the efficacy of targeted treatment for melanoma. Additionally, the new combination therapy remains in preclinical stages, and clinical trials need to be conducted to confirm the feasibility of this approach in humans. Overall, the study broadens the perspective for melanoma treatment strategies and warrants further investigation.

Juntong Chen: conceptualization, writing – original draft. Guoqing Ding: funding acquisition, supervision, resources. Jie Zhang: funding acquisition, writing – review and editing, supervision, resources. All authors have read and approved the final article.

The authors have nothing to report.

The authors declare no conflicts of interest.