Two Worlds Collide: Unraveling the Role of the Immune System in BRAF–MEK Inhibitor Responses

Melanoma is the deadliest form of skin cancer. Over the past 9 years, significant progress has been made in the development of therapies that can deliver long-term responses to patients with advanced disease. Among these, targeted therapies such as the BRAF–MEK inhibitor combination are associated with a 5-year overall survival of approximately 33%, and the immune checkpoint inhibitors (ICI; such as anti–PD-1 therapy) have been shown to be effective in >30% of patients (1, 2). One major question for the melanoma field is how best to combine targeted therapy and immunotherapy so that response durations can be maximized and off-target effects are minimized.

BRAF inhibitors and the BRAF–MEK inhibitor combination were initially developed as tumor-intrinsic therapies that target the uncontrolled cell growth of melanoma cells driven by mutant BRAF. It is now becoming clear that oncogenic BRAF can also modulate the ability of the immune system to recognize melanoma cells. Acquisition of a BRAF mutation leads to constitutive signaling through the MAPK pathway, which in turn contributes to immune escape through the recruitment of regulatory T cells (Treg), decreased antigen presentation (via downregulation of MHC class I), and the inhibition of IFNγ, IL2, and TNFα release (3). As would be expected, inhibition of BRAF in BRAF-mutant melanoma cells reverses these processes and can restore tumor–immune recognition (4). In preclinical studies, it was found that BRAF inhibition led to increased CD40L expression and IFNγ release from CD4⁺ T cells, reduced accumulation of myeloid-derived suppressor cells (MDSC) and Tregs, and decreased levels of multiple cytokines including IL1, IL6, and IL10 (3). In coculture studies of melanoma cells and dendritic cells (DC), BRAF inhibition restored IL12 and TNFα expression and increased levels of the T-cell stimulatory molecules CD80, CD83, and CD86. In transgenic mouse melanoma models of BRAF-mutant melanoma, inhibition of BRAF improved the ratio of CD8⁺ T cells to MDSCs in the tumor. There were initial concerns that the BRAF–MEK inhibitor combination might lead to immunosuppression, particularly as MEK is required for T-cell activation. Interestingly, this did not seem to be the case, and although MEK inhibition was found to impair naïve T-cell priming, it paradoxically increased the numbers of tumor-associated T cells, in part by suppressing the apoptosis that followed chronic T-cell receptor (TCR) stimulation (5). The potential for the MEK–BRAF inhibitor combination to synergize with immune checkpoint blockade was demonstrated in mouse melanoma models, with the antitumor effects observed being associated with decreased tissue-associated macrophage (TAM) and Treg accumulation, improved IFNγ release, and enhanced antigen presentation (6).

Acquisition of BRAF inhibitor resistance is associated with a reduced immune response. In an analysis of pre- and post-BRAF inhibitor–treated melanoma patient samples, numbers of tumor-infiltrating CD8⁺ T cells declined, and the proportion of suppressive immune cells including MDSCs, Tregs, and TAMs increased, as the individuals failed therapy. These observations paved the way for immunotherapy/targeted therapy combinations to be evaluated clinically. Initial attempts to develop targeted therapy/immunotherapy combinations (particularly with ipilimumab) were not successful due to severe toxicity. More success has been experienced by combining BRAF–MEK inhibitors with anti–PD-1, with some improvements in response being noted (7, 8). Mechanistically this combination was associated with enhanced CD8⁺ T-cell accumulation and the increased expression of MHC I and II. As with ipilimumab, the targeted therapy/anti–PD-1 combination was associated with serious off-target effects (58% grade 3–5 toxicity). At this time, a rational mechanistic basis for combining immunotherapy and targeted therapy is still lacking.

In this issue of Cancer Discovery, Erkes and colleagues report on a new mechanism by which the immune system contributes to the BRAF–MEK inhibitor response in vivo (9). For their studies, the authors chose to forego the usual BRAF-mutant human melanoma cell lines and instead used murine BRAF-mutant melanoma cell lines that had been subjected to multiple rounds of UV irradiation to increase their immunogenicity. These cell lines have the capacity to respond to both immunotherapy and targeted therapy in vivo, and can be grown efficiently in immunocompetent mice. The authors began by making the striking observation that BRAF–MEK inhibitor responses in vivo were significantly longer in duration when the melanoma cell lines were grown in immunocompetent mice compared with the immunocompromised mice (9). In the immunocompetent mice, BRAF–MEK inhibition was associated with a vigorous immune response characterized by increased infiltration of CD4⁺ and CD8⁺ T cells and reduced levels of MDSCs and TAMs (Fig. 1). This immune response was critical to the efficacy of the BRAF–MEK inhibitor combination, with the depletion of CD4⁺ and CD8⁺ T cells significantly blunting the BRAF–MEK inhibitor response. As recognition of the tumor by T cells requires antigen presentation (typically by DCs), the authors next asked whether the BRAF–MEK inhibitor–treated melanoma cells released factors that could activate and prime the DCs. It was found that drug treatment increased the expression of multiple immunostimulatory molecules including HMGB1, calreticulin, and IL1α from the melanoma cells and that this was associated with increased expression of MHC class II expression on the tumor-resident DCs. It therefore seemed likely that BRAF–MEK inhibition was leading to a specialized form of cell death in the melanoma cells, and stimulated an immune response.

Multiple forms of programmed cell death are known to exist, including apoptosis, necroptosis, ferroptosis, NETosis, parthanosis, and pyroptosis. Among these, pyroptosis (which is derived from the Greek word for fire, “pyro”) is linked to pathogen infection and involves the release of proinflammatory cytokines. Classic pyroptosis is dependent upon the caspase-1–mediated cleavage of gasdermin D (GSDMD), whose N-terminal portion assembles into cell membrane–spanning pores that mediate cytokine release and lytic cell death. Previous work by the authors demonstrated a role for caspase-3–mediated cleavage of gasdermin E (GSDME) in pyroptotic cell death, providing a potential link between BRAF inhibitor–induced apoptosis and pyroptosis-associated immune stimulation (10). Silencing of Gsdme in the melanoma cells through CRISPR knockdown was shown to reduce both pyroptotic cell death and immune cell accumulation in BRAF–MEK inhibitor–treated tumors, but curiously did not alter the dynamics of the initial antitumor response. Although these data appeared conflicting, it was found that the Gsdme-silenced tumors were more likely to recur following cessation of therapy, demonstrating a role for pyroptosis in the immune regulation of residual disease. The apparent lack of effect of Gsdme silencing upon the initial antitumor responses seen by Erkes and colleagues suggested the involvement of additional immune-mediated effects of BRAF–MEK inhibition that were pyroptosis-independent (9).

The pyroptotic response was blunted following the acquisition of BRAF–MEK inhibitor resistance, an effect associated with reduced caspase-3 activity, GDSME cleavage, and HMGB1 release. This decrease in pyroptosis following acquisition of BRAF–MEK inhibitor resistance was also associated with reduced levels of T-cell accumulation in the tumor, further supporting the link between pyroptosis and a continued antitumor immune response. As the final part of their study, the authors asked whether there was any therapeutic benefit to restoring pyroptosis in BRAF–MEK inhibitor–resistant tumors. A number of clinically relevant drugs including CDK4/6 inhibitors, BET inhibitors, ERK inhibitors, and chemotherapeutic agents were evaluated for their ability to induce pyroptosis. Among these, the chemotherapy drug etoposide was found to induce pyroptosis, and some improvement in mouse survival was observed when this drug was used as a salvage therapy (9). The authors did not determine whether etoposide restored T-cell infiltration to the resistant tumors—a potential issue given that drugs such as etoposide can be myelosuppressive. Although the results with etoposide were intriguing, it is worth noting that previous clinical experience of etoposide in melanoma has not been positive (11). It therefore remains to be determined whether etoposide will prove beneficial as a salvage therapy in patients with melanoma who have failed BRAF–MEK inhibitor therapy.

The study by Erkes and colleagues provides important new insights into how BRAF–MEK inhibition generates immune responses in vivo, perhaps pointing toward a rationale for future targeted therapy/immunotherapy combination trials. The author's use of immunocompetent animal models is an important demonstration of why the microenvironmental/immune effects of targeted therapy drugs should always be considered in preclinical studies. The potential role of pyroptotic cell death and its role in shaping the immune responses in patients with melanoma treated with the BRAF–MEK inhibitor combination has profound clinical implications, particularly in the context of targeted therapy/ICI therapy combinations. New insights into how the immune system contributes to targeted therapy responses are likely to prove critical in designing better therapy combinations and sequences that can deliver durable responses to patients with melanoma.

No potential conflicts of interest were disclosed.