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BRAFV600E hyperactivates ERK and signals as a RAF inhibitor–sensitive monomer. Although RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAFV600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAFL514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAFV600E/L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Newer RAF dimer inhibitors and an ERK inhibitor are effective against BRAFL514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that, like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence. Significance: In contrast to receptor tyrosine kinases, in which secondary mutations are often responsible for acquired resistance, second-site mutations in BRAF have not been validated in clinically acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confirm functionally that this mutation is responsible for resistance. Cancer Discov; 8(9); 1130–41. ©2018 AACR. See related commentary by Romano and Kwong, p. 1064. This article is highlighted in the In This Issue feature, p. 1047

The clinical significance of BRAF alterations in well-differentiated (WD) metastatic pancreatic neuroendocrine tumor (panNET) is unknown, but BRAF-mutated panNET could represent a subset characterized by an identifiable and clinically actionable driver. Following the identification of two patients with WD metastatic panNET whose tumors harbored BRAF mutations, we queried the MSK-IMPACT series of 80 patients with WD metastatic panNET for additional mutations in BRAF, and in other genes involved in RAS/ RTK/ PI3K signaling pathways. BRAF mutations were identified in six samples (7.5%): two tumors harbored V600E mutations, one tumor each expressed K601E, T599K, and T310I mutations, and one tumor expressed both G596D and E451K BRAF. Few additional actionable driver alterations were identified. To determine the ERK activating capability of four BRAF mutations not previously characterized, mutant constructs were tested in model systems. Biochemical characterization of BRAF mutations revealed both high and low activity mutants. Engineered cells expressing BRAF K601E and V600E were used for in vitro drug testing of RAF and MEK inhibitors currently in clinical use. BRAF K601E demonstrated reduced sensitivity to dabrafenib compared to BRAF V600E, but the combination of RAF plus MEK inhibition was effective in cells expressing this mutation. Herein, we describe the clinical course of a patient with BRAF K601E and a patient with BRAF V600E WD metastatic panNET, and the identification of four mutations in BRAF not previously characterized. The combined clinical and biochemical data support a potential role for RAF and MEK inhibitors, or a combination of these, in a selected panNET population.

BRAF V600E hyperactivates ERK and signals as an RAF inhibitor-sensitive monomer. While RAF inhibitors can produce impressive clinical responses in patients with mutant BRAF tumors, the mechanisms of resistance to these drugs are incompletely characterized. Here, we report a complete response followed by clinical progression in a patient with a BRAF V600E-mutant brain tumor treated with dabrafenib. Whole-exome sequencing revealed a secondary BRAF L514V mutation at progression that was not present in the pretreatment tumor. Expressing BRAF V600E L514V induces ERK signaling, promotes RAF dimer formation, and is sufficient to confer resistance to dabrafenib. A novel class of RAF dimer inhibitors and an ERK inhibitor are effective against BRAF L514V-mediated resistance. Collectively, our results validate a novel biochemical mechanism of RAF inhibitor resistance mediated by a secondary mutation, emphasizing that like driver mutations in cancer, the spectrum of mutations that drive resistance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specific prevalence. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, Sharmeen Uddin, Ira J. Dunkel, Mary Petriccione, Katia Manova, Sofia Haque, Marc Rosenblum, David J. Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A secondary mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3184.

BRAF V600E drives tumors by hyperactivating ERK signaling, is activated in a RAS-independent manner, and signals as a monomer. Selective inhibitors of RAF potently inhibit BRAF V600E and downstream ERK signaling, and have been associated with impressive clinical responses in patients whose tumors harbor V600E mutations in BRAF. Almost all patients, however, ultimately develop resistance. To date, acquired resistance mechanisms validated in patient samples have included splice forms of BRAF, mutations in NRAS, and BRAF amplification, all of which promote formation of RAF dimers, and mutations in MEK. Second-site point mutations in BRAF that promote dimer formation and are associated with acquired resistance have not previously been identified in RAF inhibitor-resistant tumors. Here, we report a complete response followed by clinical progression of a BRAF V600E-mutant pediatric brain tumor treated with the RAF inhibitor dabrafenib. We identified a second-site mutation in BRAF at the time of progression that was not present in the pretreatment tumor. Further study indicated the acquired mutation is in cis with V600E. We demonstrate that the acquired resistance mutation induces RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Moreover, we showed that ERK signaling activated by the double mutation is sensitive to MEK inhibition or to the RAF plus and MEK inhibitor combination. We also find that two novel RAF dimer inhibitors may overcome resistance mediated by the novel mutation in BRAF. This is the first study, to our knowledge, that demonstrates the emergence of a confirmed second-site mutation in BRAF V600E and validates that this mutation is responsible for acquired resistance in an initially responding patient treated with dabrafenib. The mechanism of acquired resistance can be abrogated by the novel RAF dimer inhibitors. Our data confirm that a novel class of RAF dimer inhibitors are active against an acquired resistance mutation, suggesting an improved personalized treatment option for patients who harbor similar second-site mutations in BRAF. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, David Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A129.

FMS-like tyrosine kinase-3 (FLT3) is one of the most commonly mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (FLT3/ITD) is an established driver mutation in AML and results in constitutive activation of the receptor, activating downstream signaling pathways including STAT5, PI3K/AKT, and RAF/MEK/ERK. The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3/ITD AML has been explored as a promising strategy for over a decade, but clinical responses have remained limited. In some cancers driven by mutated oncogenes, small molecule inhibition of the target is known to result in reactivation of downstream signaling pathways. This response has been observed in the context of PI3K/AKT/mTOR and BRAF inhibition, but it remains unclear whether this phenomenon extends to cancers driven by receptor tyrosine kinase (RTK) activation, such as FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response. To test this, we treated FLT3/ITD AML cell lines (Molm14 and MV4;11) with FLT3 TKIs for up to 48 hours. We observed a rebound in ERK phosphorylation beginning as early as 6 hours and continuing for the duration of treatment, while no such rebound was observed in the phosphorylation of downstream targets AKT or STAT5. When these cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed, and the anti-leukemia effects were more pronounced compared to either drug alone, both in vitro and in vivo. In vitro, the addition of low dose MEK inhibitor to FLT3 TKI treatment synergistically increased cell death and decreased cell proliferation. In a xenograft transplant model of leukemia, the addition of low-dose MEK inhibitor to FLT3 TKI treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts (p < 0.05). We next sought to determine whether rebound in ERK signaling following FLT3 inhibition was likely to occur in patients as well. We first examined whether primary leukemic cells from patients with FLT3/ITD AML also exhibit the same phenomenon observed in FLT3/ITD cell lines. After 24 hours of sorafenib treatment, the majority of samples revealed a similar rebound in ERK phosphorylation, despite sustained inhibition of phospho-STAT5. As observed with the cell lines, the addition of a MEK inhibitor to sorafenib treatment resulted in sustained pERK inhibition after 24 hours. Next, we explored whether signaling adaptation observed in culture conditions would be recapitulated by the concentrations of sorafenib present in human plasma from patients being treated with this FLT3 TKI. To replicate these conditions, we cultured Molm14 cells in plasma samples obtained from AML patients treated with sorafenib for a prolonged period. Using a variant of the plasma inhibitory activity assay, Molm14 cells were treated in either normal human plasma for one hour or patient plasma for either one or 24 hours. For all plasma samples, profound pERK rebound was observed by 24 hours after treatment, despite maximal inhibition at one hour and persistent inhibition of FLT3 and STAT5 phosphorylation over the duration. Finally, we sought to determine whether TKI-mediated rebound in ERK activity was restricted to FLT3/ITD AML or may also occur in the context of other RTK-driven cancers, such as those driven by EGFR mutation or HER2 amplification. We treated an EGFR mutant lung cancer (PC-9) and a HER2 amplified breast cancer (SKBR3) cell line with the EGFR/HER2 inhibitor lapatinib. As was seen for FLT3/ITD driven AML, pERK rebound was observed following 24 hours of TKI treatment despite persistent inhibition of the receptor and downstream target AKT. Again, the rebound could be overcome with the addition of low concentrations of MEK inhibitor. Together, these studies reveal that FLT3/ITD and other RTK driven cancer cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to upstream target inhibition. This adaptation limits the efficacy of TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML and possibly other RTK-driven cancer types. Disclosures Levis: Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria.