Edward J. Hartsough

Resistance to RAF Inhibitors Revisited

In early 2011, we reviewed the initial success of the RAF inhibitor, vemurafenib, in mutant V600 BRAF melanoma patients. It was soon evident that the response to RAF inhibitor is heterogeneous and that the short-term benefits are burdened by the development of resistance. The field has progressed rapidly with the FDA-approval of vemurafenib and the development of other RAF and MEK inhibitors. Despite these advances, the issue of RAF inhibitor resistance remains. Here, we review recent clinical advances in the field, the growing number of resistance mechanisms, preclinical evidence for combinatorial trials using RAF inhibitors as the building blocks, and the new challenges that are arising.

Inhibition of mutant BRAF splice variant signaling by next-generation, selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAFV600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAFV600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAFV600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAFV600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAFV600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.

In Vivo MAPK Reporting Reveals the Heterogeneity in Tumoral Selection of Resistance to RAF Inhibitors

Activation of the ERK1/2 mitogen-activated protein kinases (MAPK) confers resistance to the RAF inhibitors vemurafenib and dabrafenib in mutant BRAF-driven melanomas. Methods to understand how resistance develops are important to optimize the clinical use of RAF inhibitors in patients. Here, we report the development of a novel ERK1/2 reporter system that provides a noninvasive, quantitative, and temporal analysis of RAF inhibitor efficacy in vivo. Use of this system revealed heterogeneity in the level of ERK1/2 reactivation associated with acquired resistance to RAF inhibition. We identified several distinct novel and known molecular changes in resistant tumors emerging from treatment-naïve cell populations including BRAF V600E variants and HRAS mutation, both of which were required and sufficient for ERK1/2 reactivation and drug resistance. Our work offers an advance in understanding RAF inhibitor resistance and the heterogeneity in resistance mechanisms, which emerge from a malignant cell population.

Beneficial effects of RAF inhibitor in mutant BRAF splice variant-expressing melanoma.

Resistance to RAF inhibitors such as vemurafenib and dabrafenib is a major clinical problem in the treatment of melanoma. Patients with mutant BRAF melanoma that progress on RAF inhibitors have limited treatment options, and drug removal from resistant tumors may elicit multiple effects. A frequent mechanism of resistance to RAF inhibitors is caused by expression of mutant BRAF splice variants. RAF inhibitor–resistant cell lines, generated in vivo, were tested as to whether or not mutant BRAF splice variants confer a fitness advantage in the presence of RAF inhibitor. Critically, cells expressing distinct mutant BRAF splice variants grow more efficiently in vitro and in vivo in the presence of the vemurafenib analog, PLX4720, compared with in the absence of inhibitor. PLX4720-treated BRAF splice variant–expressing cells exhibited levels of phospho-extracellular signal–regulated kinase (ERK)1/2 comparable to untreated parental cells. In addition, a reduction in phospho-ERK1/2 levels following treatment with the MEK inhibitor, trametinib (GSK1120212) phenocopied the fitness benefit provided by PLX4720. These data indicate that mutant BRAF splice variant–expressing melanoma cells are benefited by defined concentrations of RAF inhibitors. Implications: This study provides evidence that RAF inhibitor–resistant melanoma cells benefit from continued therapy. Mol Cancer Res; 12(5); 795–802. ©2014 AACR.

Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

ERBB3/HER3 expression and signaling are upregulated in mutant BRAF melanoma as an adaptive, prosurvival response to FDA-approved RAF inhibitors. Because compensatory ERBB3 signaling counteracts the effects of RAF inhibitors, cotargeting ERBB3 may increase the efficacy of RAF inhibitors in mutant BRAF models of melanoma. Here, we corroborate this concept by showing that the ERBB3 function-blocking monoclonal antibody huHER3-8 can inhibit neuregulin-1 activation of ERBB3 and downstream signaling in RAF-inhibited melanoma cells. Targeting mutant BRAF in combination with huHER3-8 decreased cell proliferation and increased cell death in vitro, and decreased tumor burden in vivo, compared with targeting either mutant BRAF or ERBB3 alone. Furthermore, the likelihood of a durable tumor response in vivo was increased when huHER3-8 was combined with RAF inhibitor PLX4720. Together, these results offer a preclinical proof of concept for the application of ERBB3-neutralizing antibodies to enhance the efficacy of RAF inhibitors in melanoma to delay or prevent tumor regrowth. As ERBB3 is often upregulated in response to other kinase-targeted therapeutics, these findings may have implications for other cancers as well.

Of Mice and Melanoma: PDX System for Modeling Personalized Medicine

Targeted therapies have advanced the treatment options for cutaneous melanoma, but many patients will progress on drug. Patient-derived xenografts (PDX) can be used to recapitulate therapy-resistant tumors. Furthermore, PDX modeling can be utilized in combination with targeted sequencing and phosphoproteomic platforms, providing preclinical basis for second-line targeted inhibitor strategies. Clin Cancer Res; 22(7); 1550–2. ©2016 AACR. See related article by Krepler et al., p. 1592

A STATement on Vemurafenib-Resistant Melanoma

Summary Despite recent advancements in the treatment of late-stage mutant BRAF V600E/K melanomas, a major hurdle continues to be acquired resistance to BRAF inhibitors such as Vemurafenib. The mechanisms for resistance have proven to be heterogeneous, emphasizing the need to utilize broad therapeutic approachs. The present study, “Stat3 targeted therapies overcome the acquired resistance to vemurafenib in melanomas” by Liu et al., proposes that STAT3-PAX3 signaling may be a mechanism that is utilized by melanomas to resist RAF inhibitors.

Response and Resistance to Paradox-Breaking BRAF Inhibitor in Melanomas In Vivo and Ex Vivo

FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K–mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox-breaking RAF inhibitor (PLX8394) has been designed. Here, we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice variant–expressing tumors and reduced splice variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test the efficacy of therapeutic agents. Mol Cancer Ther; 17(1); 84–95. ©2017 AACR.

αC IN, αC OUT-that's what it's all about.

Despite recent successes in drug development, metastatic melanoma remains a challenging disease. Efforts to design new targeted therapies as well as improve efficacy with combinations of existing inhibitors have yielded increases in progression free and overall survival. However, as with many targeted therapies across the cancer spectrum, habitual treatment often leads to drug resistance and progression necessitating alternative treatment regimens. This article is protected by copyright. All rights reserved.

Abstract A31: 14-3-3 binding sites in full-length and splice variant forms of BRAF V600E modulate the response to RAF inhibitors

The serine/threonine kinase BRAF is mutated in 7% of human cancers, the most frequent alteration being a valine to glutamic acid substitution at amino acid 600 (V600E). BRAF V600E melanomas are highly sensitive to RAF inhibitors but the expression of aberrantly spliced BRAF V600E (BRAF V600E ΔEx) isoforms are associated with resistance in 13-30% of progressing patients. Compared to full-length BRAF V600E, BRAF V600E ΔEx exhibit enhanced dimerization and signaling via ERK1/2 during RAF inhibitor therapy; however, much remains unknown regarding their mechanism of action. The 14-3-3 protein binding sites, serine 365 (S365) and serine 729 (S729), play a complex role in regulating the activity and dimerization of RAF isoforms. All reported BRAF V600E ΔEx isoforms lose the N-terminal 14-3-3 binding site (S365) but retain the C-terminal site (S729); therefore, we analyzed the involvement of 14-3-3 binding sites in response to RAF inhibitor therapy. In full-length BRAF V600E, mutation of S365 to alanine promotes elevated MEK-ERK1/2 signaling and growth in the presence of RAF inhibitor. BRAF V600E ΔEx exhibits increased phosphorylation on S729 during RAF inhibitor therapy. Mutation of S729 to alanine renders the BRAF V600E ΔEx isoforms sensitive to RAF inhibitor, measured by reduced ERK1/2 phosphorylation and cell growth in vitro and in vivo. These data highlight the importance of BRAF V600E 14-3-3 binding sites in mediating targeted therapy resistance in mutant BRAF-driven melanoma. Citation Format: Michael J. Vido, Kaitlyn Le, Justin Rock, Edward J. Hartsough, Neda Dadpey, Andrew E. Aplin. 14-3-3 binding sites in full-length and splice variant forms of BRAF V600E modulate the response to RAF inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A31.