Preclinical efficacy of a RAF inhibitor that evades paradoxical MAPK pathway activation in protein kinase BRAF -mutant lung cancer

Abstract

Oncogenic activation of protein kinase BRAF drives tumor growth by promoting mitogen-activated protein kinase (MAPK) pathway signaling. Because oncogenic mutations in BRAF occur in ∼2-7% of lung adenocarcinoma (LA), BRAF-mutant LA is the most frequent cause of BRAF-mutant cancer mortality worldwide. Whereas most tumor types harbor predominantly the BRAF V600E -mutant allele, the spectrum of BRAF mutations in LA includes BRAF V600E (∼60% of cases) and non-V600E mutant alleles (∼40% of cases) such as BRAF G469A and BRAF G466V. The presence of BRAF V600E in LA has prompted clinical trials testing selective BRAF inhibitors such as vemurafenib in BRAF V600E -mutant patients. Despite promising clinical efficacy, both innate and acquired resistance often result from reactivation of MAPK pathway signaling, thus limiting durable responses to the current BRAF inhibitors. Further, the optimal therapeutic strategy to block non-V600E BRAF-mutant LA remains unclear. Here, we report the efficacy of the Raf proto-oncogene serine/ threonine protein kinase (RAF) inhibitor, PLX8394, that evades MAPK pathway reactivation in BRAF-mutant LA models. We show that PLX8394 treatment is effective in both BRAF V600E and certain non-V600 LA models, in vitro and in vivo. PLX8394 was effective against treatment-naive BRAF-mutant LAs and those with acquired vemurafenib resistance caused by an alternatively spliced, truncated BRAF V600E that promotes vemurafenib-insensitive MAPK pathway signaling. We further show that acquired PLX8394 resistance occurs via EGFR-mediated RAS-mTOR signaling and is prevented by upfront combination therapy with PLX8394 and either an EGFR or mTOR inhibitor. Our study provides a biological rationale and potential polytherapy strategy to aid the deployment of PLX8394 in lung cancer patients.ncogenic mutations in the BRAF serine/threonine protein kinase occur in a wide spectrum of solid tumor malignancies, most notably melanoma, colorectal cancer, and lung adenocarcinoma (1). Mutant BRAF promotes tumor growth by hyperactivating the RAF-MEK-ERK signaling cascade (2-5). BRAF V600E is the most common oncogenic form of BRAF in most tumor types, and selective RAF inhibitors such as vemurafenib and dabrafenib have demonstrated clinical efficacy in melanoma and LA harboring BRAF V600E (6-9). Despite these findings, ∼15% of BRAF mutant cancers do not respond to BRAF inhibitors, and the majority of patients who achieve a response will inevitably acquire resistance to these targeted agents, predominantly through reactivation of MAPK pathway signaling (4, 10-16).The clinical efficacy of RAF inhibitors depends on the degree of suppression of MAPK pathway output (8). Vemurafenib and dabrafenib paradoxically activate the MAPK pathway in cells with oncogenic RAS or increased upstream receptor signaling, thereby enhancing cellular proliferation that can promote cutaneous squamous cell carcinomas and keratoacanthomas that often harbor RAS mutations, and potentially other RAS-driven malignancies (17-25). Combination therapy with a MEK i...

Targeting BRAF-Mutant Non-Small Cell Lung Cancer: From Molecular Profiling to Rationally Designed Therapy

“…In an effort to overcome this resistance, triple combination of BRAFi plus MEKi plus EGFR tyrosine kinase inhibitors is currently under evaluation in patients with BRAF ‐mutant colorectal cancer (NCT01750918). Preclinical data suggest that this strategy may have utility in patients with NSCLC, as acquired resistance to PLX8394 could be prevented by upfront combination with EGFR or mTOR inhibitors .…”

“…Although clinical data are limited among patients with non‐V600E mutations in NSCLC, combination of dabrafenib plus trametinib has demonstrated antiproliferative and proapoptotic effects in cell lines harboring both activating and inactivating BRAF non‐V600 mutations . Additionally, PLX8394 has demonstrated preclinical activity in both V600E and non‐V600E lung adenocarcinoma models .…”

The spectrum of cutaneous adverse events during encorafenib and binimetinib treatment in B‐rapidly accelerated fibrosarcoma‐mutated advanced melanoma

“…This might reduce the formation of (cutaneous) adverse events and hence improve drug tolerability. 37,38 In the present study, cutaneous adverse reactions that occurred in patients with encorafenib monotherapy included palmoplantar erythrodysesthesia, palmoplantar hyperkeratosis, alopecia, macular and maculopapular exanthemas, xerosis cutis and keratosis pilaris. In patients with binimetinib therapy, druginduced papulopustular eruptions predominated.…”

Novel BRAF fusions in pediatric histiocytic neoplasms define distinct therapeutic responsiveness to RAF paradox breakers

“…PLX8394 blocks BRAF kinase activity via disrupting BRAF dimerization 14 but we observed no disruption of MTAP-and MS4A6A-BRAF fusion dimerization with PLX8394 ( Figure 2C-D, PLX8394 lanes), thereby providing a plausible explanation for PLX8394 unresponsiveness in soft agar assays though MAPK/PI3K signaling remains discordantly suppressed by some unknown mechanism. This distinct unresponsiveness to pan-RAFi represents a significant departure from the current view that BRAF-fusions and other BRAF mutations should respond to second-generation RAFi such as PLX8394 9,15 . We found that this difference arises due to the contribution of N-terminal partners, MTAP (exons 1-7) and MS4A6A (exons 1-6), to respective fusion dimerization that is unaffected by PLX8394 ( Figure 2C-D, lanes 3,7).…”

“…Despite the lack of cytologic atypia or increased mitotic rate by H&E stain, a Ki-67 proliferation index stain revealed increased staining in lesional cells up to 40%. Targeted RNA-seq identified a fusion of MS4A6A (NM_022349.3) exons 1-6 and BRAF (NM_004333.4) exons [11][12][13][14][15][16][17][18]. During staging, the patient was found to have PET-avid dissemination to lymph nodes and lung ( Figure 1N-P).…”