Keeping Tumors Out of the MAPK Fitness Zone

The majority of human tumors are driven by dysregulation of RAS/RAF/MEK/ERK (MAPK pathway) signaling. The introduction of ATP-competitive BRAF inhibitors (BRAFi), including vemurafenib and dabrafenib, was a major therapeutic advance for treatment of the 50% of cutaneous melanomas with BRAF^V600E mutations. Most patients responded, often with dramatic reductions in tumor volume, but progressive disease occurred within a period of months through diverse mechanisms that reactivate MAPK signaling, including upregulation of BRAF or CRAF expression, alternative splicing leading to BRAF deregulation, and activation of RAS/MAPK pathway signaling by mutations in RAS, MEK1, or MEK2 genes (1, 2). Moreover, metastases in an individual patient can acquire resistance through different genetic mechanisms (2). These issues are even more challenging in the approximately 7% of nonmelanoma solid tumors with BRAF mutations, which are not as responsive as melanoma to MAPK inhibitors or to immune checkpoint therapies.

Wild-type (WT) RAF proteins are normally regulated by receptor tyrosine kinase activation of RAS proteins. GTP-loaded RAS binds to RAF, promoting formation of active RAF dimers. Type I BRAFi preferentially inhibit the common driver BRAF^V600E, which is active in monomeric form, but these agents paradoxically promote activity of BRAF/CRAF heterodimers, driving downstream signaling through MEK and ERK and yielding side effects, including squamous cell carcinoma/keratoacanthoma (3).

Adaptive mechanisms attenuate responses to inhibition of BRAF^V600E, enhancing fitness of tumor cells that can be further selected for therapeutic resistance. Activation of MAPK and dependent transcription factors by BRAF^V600E results in homeostatic feedback inhibition of the pathway upstream of RAS. For example, MAPK directly phosphorylates and inhibits receptor kinases, and the MAPK pathway enhances expression of pathway antagonists SPRY and DUSP. Inhibition of BRAF^V600E melanomas with vemurafenib relieves this negative regulation, thereby enhancing upstream activation of RAS and consequent dimerization of (WT) RAF proteins with limited vemurafenib sensitivity (1). Over time, alternative splicing to produce truncated constitutively dimeric BRAF, and/or overexpression of BRAF or CRAF, can be selected, and will also promote RAF dimerization and vemurafenib resistance.

These findings prompted efforts for vertical control of the RAF/MEK/ERK kinase cascade through drug combinations. The combination of BRAFi dabrafenib or vemurafenib with MEKi trametinib or cobimetinib, respectively, both reduces paradoxical activation of WT RAF proteins in nontumor tissue and attenuates feedback-dependent RAF dimerization. Although this combination is superior to BRAFi therapy alone, progression occurs within 2 years (4). Two publications now describe complementary approaches for attacking MAPK tumors by preventing outgrowth of resistant tumor subclones.

In this issue of Cancer Discovery, Hong and colleagues describe an approach to improved MAPK pathway targeting by exploiting the phenomenon of inhibitor addiction of BRAF- and NRAS-driven tumors (Fig. 1, top; ref. 5). Single-agent and double-drug resistant (DDR) cells selected with BRAFi/MEKi combinations often become addicted to the inhibitors such that they proliferate more slowly and/or lose viability when the inhibitors are withdrawn (6, 7). Reduced fitness with drug withdrawal is linked to MAPK pathway activity. Drug washout in MEKi-resistant BRAF melanoma or NRAS melanoma induces rebound pERK, indicative of MAPK activity that is quantitatively associated with the degree of drug dependence (5, 7). This rebound ERK signaling is important for drug withdrawal cellular toxicity: Deleterious effects of drug washout are mitigated with ERK inhibition, and overexpression of BRAF^V600E intensifies the withdrawal phenotypes.

Hong and colleagues were able to categorize DDR cell lines with a range of resistance mechanisms into two phenotypic categories (5). After drug removal, slow-cycling lines reduce proliferation, with little cell death, and eventually regrow after drug removal. Cell death–predominant lines respond to drug withdrawal with substantial cell death and senescence of the persisters. This extreme addiction phenotype is associated with greatest rebound reactivation of MAPK signaling. The slow-cycling phenotype is mediated by p38-dependent upregulation of FRA1/JUNB and induction of p21^CDKN1A. Pharmacologic induction of DNA damage with DNA checkpoint inhibitors and PARPi enhances death of these cells by apoptosis. In contrast, parthanatos is the foremost mechanism of death in cell death–predominant lines and is induced through a pathway involving mitochondrial dysfunction, leading to DNA damage and cleavage of apoptosis-inducing factor. Combining MAPKi withdrawal with agents that interfere with DNA repair substantially increases cell death for both slow-cycling and cell death–predominant lines.

On the basis of these findings, the authors hypothesized that enhancement of MAPK signaling by drug withdrawal can be exploited to control treatment-refractory tumors. MEKi therapy has short-term impact on the 20% to 25% of patients whose melanoma is NRAS-driven, but they become resistant (8). In experimental models that signal through RAF dimerization (BRAFV^S365L or NRAS mutation) and that were selected for MEKi resistance, MAPK signaling is enhanced by withdrawal of MEKi. As predicted, addition of the BRAFi vemurafenib at the time of MEKi withdrawal augments rebound MAPK activation and substantially increases the delay in tumor growth associated with MEKi withdrawal. In a trametinib-resistant NRAS xenograft model, the combination of BRAFi to superactivate ERK signaling with PARPi to exacerbate DNA damage further sustained tumor control.

Thus far, MAPK pathway vertical targeting does not translate to durable responses for most patients. As ERKi are now advancing into clinical trials, Xue and colleagues have assessed strategies for combining BRAFi, MEKi, and ERKi (Fig. 1, bottom; ref. 9). BRAF overexpression is a common route to single-agent MAPKi resistance of BRAF^V600 tumors. Single-cell copy-number analysis of a RAFi-resistant patient-derived xenograft (PDX) from a BRAF^V600 tumor selected for ERKi resistance revealed that multiple independent BRAF-amplified cell clones expand in parallel, counter to the expectation that a single dominant clone with greater fitness would overtake the tumor population. Importantly, BRAF copy-number gains were found to be common in BRAF^V600E tumors before treatment, so that sequential treatment with RAF/MEK/ERK pathway inhibitors may be readily defeated by selection of BRAF-amplified clones (9).

The authors proposed a fitness threshold to be overcome for MAPK signaling and proliferation in the face of drug exposure. With BRAF amplification as a common resistance mechanism for RAFi, MEKi, and ERKi, the impact of different MAPK pathway–targeted drugs and drug combinations can be graded according to the level of experimentally induced BRAF^V600E expression required for cells to prevail. Thus, BRAF^V600E cells were less sensitive to single-agent ERKi than to BRAFi or MEKi. The three-drug combination had the greatest antitumor effects on PDX models selected for inhibitor resistance. Moreover, although tumors treated and released from the two-drug combination had increased BRAF copy number, this copy-number gain was not selected with the triple combination, suggesting that the fitness threshold for this cocktail was too high to surpass with RAF amplification. The treatment schedule was further optimized (in mice) with intermittent scheduling of drug combinations in PDX to maximize therapeutic index. Intermittent combination treatments were effective and led to sustained remission posttherapy. Importantly, the triple-agent combination inhibited tumor growth across 11 of 11 lung and melanoma PDX models tested with BRAF^V600 mutations and diverse secondary alterations.

Together, these two reports describe new approaches validated in xenograft models that may significantly improve control of melanoma and other RAS/MAPK–driven tumors by targeted agents. As DNA damage exacerbates both slow-cycling and cell death–predominant inhibitor-addicted BRAF^V600 melanoma, the addition of PARPi or other agents to interfere with DNA repair at the time of inhibitor withdrawal would be a new tactic for patients developing resistance to BRAFi/MEKi combination therapy. The possibility of bringing in BRAFi plus PARPi and other agents to promote DNA damage for therapy of MAPKi-resistant NRAS and BRAF^variant melanoma with drug withdrawal is an innovative idea for therapy of these genotypes that may also incite interest in approaches for enhancing parthanatotic tumor death.

The sustained control of PDX with triple BRAFi, MEKi, ERKi combination is very encouraging. Although the triple combination fitness threshold was insurmountable under the conditions of tumor evolution in PDX models, differences in parameters including tumor cell number and genomic plasticity of subclones at the tolerated drug doses may lead to different outcomes in patients. Indeed, if treatment-selected resistance becomes an overriding factor at tolerable drug concentrations in humans, augmenting cell death with PARPi and BRAFi (for BRAF^variant and NRAS tumors) may improve outcomes.

These and other studies underscore opportunities for significant improvement in use of signaling targeted therapies. Tumor fitness can be negatively affected by high BRAF because copy number is reduced with removal of ERKi (9), there is an acute impact of excessive MAPK signaling with drug withdrawal (5), and clinical studies suggest that drug holidays are advantageous (6). Transient treatment cessation during intermittent therapy will slow growth of addicted tumors and also reduce selective pressure for gene amplification and other compensatory changes. The relative activity of BRAFi for inhibition of monomeric and dimeric RAF signaling, which has different treatment implications depending on RAS and RAF driver genotypes, varies among different agents and can be fine-tuned (3), and in the triple BRAFi/MEKi/ERKi combination, BRAFi reduces toxicity of the less tumor-selective MEKi and ERKi, possibly through relief by paradoxical activation of RAF dimer signaling (9). Nonetheless, heterogeneity in MAPK signaling in different metastatic microenvironments and with differential epigenetic and genetic changes (including BRAF copy number) will make it challenging to find a sweet spot with normalized MAPK signaling in tumors without overwhelming impact on nontumor tissue.

The difficulties encountered in obtaining durable responses by targeting cancer signaling pathways reflect the fact that there is still much to learn about cancer signaling networks and evolution of tumor cell subpopulations. Progress in use of MAPK-directed therapies in melanoma and other solid tumors has been accelerated by investigation at multiple scales beginning with structure-based mechanisms of drug activity on- and off-target, and including exhaustive investigation of pathway-level, epigenetic, and genetic mechanisms for treatment resistance. Newer approaches, including single-cell analyses of tumor evolution and modeling drug responses at the cellular level (10), are bootstrapping a systems-level understanding that will surely continue to improve therapies.

No potential conflicts of interest were disclosed.