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Supplementary Figure S4. PC9 GR4 xenograft response to W+T treatment. Mice were treated with two doses (A) or four doses (B) of WZ4002, trametinib or a combination thereof. Tumors were lysed, subjected to immunoblot and phosphorylation of EGFR, AKT, ERK1/2 and S6 were assessed. Hsp90 was used as a loading control. (C) Mice were photographed 31 days following cessation of drug treatment.

Supplementary Figure S2. Comparison of WZ4002 + crizotinib and WZ4002 + trametinib combinations in TKI resistant cell lines. HCC827 GR6 and HCC827 + HGF were treated with DMSO control (Media), 100 nM WZ4002, 30 nM trametinib, 100 nM crizotinib or WZ4002 combinations weekly. Positive wells were tallied weekly (mean +/- SD, n = 3 biological replicates of 60 wells each). Arrow indicates that confluence was not reached at 9 weeks.

Supplementary Table 1. List of cell lines used in this study.

Supplementary Figure S1: Representative H&E, phospho-ERK and FISH images of pre-dabrafenib (3) and post-dabrafenib (4) tumors. Supplementary Figure S2: WES analysis of copy number variation in pre- and postdabrafenib tumors. Supplementary Figure S3: Whole copy number profiles from WES of pre- and posttreatment tumors. Supplementary Figure S4: Homology alignment of BRAF p. L514 with residues in other tyrosine kinases. Supplementary Figure S5: Relative frequency of BRAF variant alleles in SK-BT-DR cells determined by individual clone sequencing. Supplementary Figure S6: BRAF V600E/L514V reduces dabrafenib sensitivity in NIH- 3T3 cells, related to Figure 2. Supplementary Figure S7: BRAF V600E/L514V confers biochemical resistance to dabrafenib over a time course, related to Figure 2G. Supplementary Figure S8: Comparison of IC50, IC75 and IC90 of dabrafenib against A375 cells expressing BRAF V600E and BRAF V600E/L514V, related to Figure 2H. Supplementary Figure S9: The BRAF V600E/L514V double mutant promotes homodimerization, related to Figure 3A and B. Supplementary Figure S10: BRAF L514V alone is hypoactive and associated with decreased ERK signaling that is not sensitive to dabrafenib. Supplementary Figure S11: BRAF V600E/L514V is inhibited by dabrafenib in a purified kinase assay, indicating that it is not a gatekeeper mutation. Supplementary Figure S12: Quantitation of p-MEK and p-ERK immunoblots, related to Figure 4B. Supplementary Figure S13: Comparison of IC50, IC75 and IC90 of trametinib against A375 expressing BRAF V600E and BRAF V600E/L514V, related to Figure 4C. Supplementary Figure S14: The BRAF V600E/L514V mutant mediates resistance to dabrafenib that cannot be completely overcome by trametinib or dabrafenib plus trametinib, related to Figure 4D. Supplementary Figure S15: V5, p-MEK, total MEK immunoblots, and quantitation of p-ERK immunoblots, related to Figure 5A. Supplementary Figure S16: Novel RAF dimer inhibitors, MEK inhibitor and ERK inhibitor equipotently inhibit cell growth in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S17: Novel RAF dimer inhibitor, MEK inhibitor and ERK inhibitor equipotently inhibit ERK signaling in BRAF V600E and V600E/L514V expressing cells. Supplementary Figure S18: BGB3245 binds mutant BRAF V600E monomer and second site of V600E/L514V dimer with similar affinity. Supplementary Table S1: Mutations identified by WES of pre-dabrafenib and postdabrafenib tumors. Supplementary Table S2: Secondary mutations associated with acquired resistance and occurring in residues homologous with L514 in BRAF. Supplementary Table S3: BRAF L514V allele frequency determined by ddPCR.

Supplementary Figure S1. Trametinib sensitivity of EGFR mutant NSCLC cell lines and inhibition of ERK dependent genes. (A) Inhibition of ERK1/2 was assessed in each cell line after 8 hours of treatment followed by immunoblot for total and phosphorylated ERK1/2. Hsp90 was used as a loading control. (B) The viability of PC9, HCC4006, HCC2279 and HCC2935 cells was assessed after 72 hours. (C) PC9 GR4 (EGFR Del746_A750/T790M) cells were treated with DMSO (Media), 100 nM WZ4002, 30 nM trametinib or a combination thereof for 8 hours or (D) 24 hours and ERK1/2 dependent gene transcription was assessed by quantitative PCR, asterisk, p < 0.05 by one-tailed t-test.

Supplementary Figure S6. Resistance to W+T combination treatment. (A) Individual tumor volume beyond 20 weeks of WZ4002 + trametinib co-treatment. (B) Representative MRI image of tumor rebound. (C) Immunohistochemical analysis of WZ4002 + trametinib combination resistant tumors demonstrating positive staining for phospho-EGFR, ERK1/2, S6 and 4EBP1 at 50 nM (left panel) and 100 nM (right panel). (D) Immunoblot analysis of EGFR pathway reactivation in WZ4002 + trametinib resistant LT GEMMs. Phospho-EGFR, AKT, ERK1/2, S6 and 4EBP1 were assessed. Lysates from LT GEMMs were treated with vehicle or with WZ4002 for 24 hours were included as positive and negative controls for EGFR pathway activation.

Supplementary Figures 1 - 8, Tables 1 - 3

Supplementary Methods

Supplementary Figure S5. Response to W+T treatment in the L858R/T790M EGFR genetically engineered mouse model (LT GEMM). (A) Immunohistochemical analysis LT GEMMs treated with two doses of WZ4002, trametinib or a combination thereof assessing phosphorylation of EGFR, AKT and ERK1/2 at 100 nM. (B) TUNEL staining analysis of LT GEMMs treated with two doses of WZ4002, trametinib or a combination thereof at the 50 and 100 nM. (C) Tumor volume of individual LT GEMMs treated with 50 mg/kg WZ4002, 2.5 mg/kg trametinib and the combination up to 20 weeks (13). (D) Representative MRI images from each treatment.

Supplementary Figure S3. Biochemical analysis of WZ4002 + trametinib (W+T) resistant and sensitive cell lines. Each cell line was treated with 100 nM WZ4002 (W) or 30 nM trametinib (T) or a combination (C) thereof for 8 hours and total or phospho-EGFR, ERK1/2, AKT and S6 were assessed by western blot. Sensitivity to six week W+T treatment is indicated below the image. Hsp90 was used as a loading control.

Supplementary Figure S7. Effect of Torin2 on PC9 and PC9 derived WZ4002 + trametinib resistant cell lines. (A) Inhibition of S6 was assessed at the indicated concentration by immunoblot. (B) Viability was assessed by MTS assay after 72 hours of the indicated concentration of Torin2 (mean +/- SD, n = 6 technical replicates). (C) PC9 W+T resistant clone was treated with WZ4002 + trametinib for 72 hours and viability was assessed by MTS (mean +/- SD, n = 6 technical replicates).

G(1) cell cycle progression is controlled largely by growth factors in early G(1) indicating that it is appropriate to divide and by nutrients in late G(1) indicating sufficient raw material for cell division. We previously mapped a late G(1) cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G(1) checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G(1) into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [(3) H]-thymidine deoxyribose ([(3) H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase – the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [(3) H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G(1). Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin – the most abundant protein in serum – reflects a broader availability of essential amino acids needed for cell growth.

G1 cell cycle progression is controlled largely by growth factors in early G1 indicating that it is appropriate to divide and by nutrients in late G1 indicating sufficient raw material for cell division. We previously mapped a late G1 cell cycle checkpoint for lipids upstream from a mammalian target of rapamycin complex 1 (mTORC1)-mediated checkpoint and downstream from a mid-G1 checkpoint known as the Restriction point. We therefore investigated a role for lipids in progression through late G1 into S-phase. Quiescent BJ-hTERT human fibroblasts were primed with 10% fetal bovine serum (FBS) for 3.5 h at which time, cells were treated with a mixture of lipids and carrier bovine serum albumin (BSA) along with [3 H]-thymidine deoxyribose ([3 H]-TdR) to monitor progression into S-phase. Surprisingly, BSA by itself was more effective than FBS in promoting progression to S-phase – the lipids had no impact on progression. While insulin strongly stimulated mTORC1 activity, it did not impact on [3 H]-TdR incorporation. Although BSA modestly elevated mTORC1 activity, rapamycin strongly inhibited BSA-induced progression to S-phase. BSA treatment promoted mitosis, but not progression through a second G1. Thus, after priming quiescent cells with FBS, albumin was sufficient to promote progression into S-phase. The BSA was not simply a source of amino acids in that amino acids were present in the culture media. We propose that the presence of albumin – the most abundant protein in serum – reflects a broader availability of essential amino acids needed for cell growth.

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

Recent studies have identified somatic ESR1 mutations in patients with metastatic breast cancer and found some of them to promote estrogen-independent activation of the receptor. The degree to which all recurrent mutants can drive estrogen-independent activities and reduced sensitivity to ER antagonists like fulvestrant is not established. In this report, we characterize the spectrum of ESR1 mutations from more than 900 patients. ESR1 mutations were detected in 10%, with D538G being the most frequent (36%), followed by Y537S (14%). Several novel, activating mutations were also detected (e.g., L469V, V422del, and Y537D). Although many mutations lead to constitutive activity and reduced sensitivity to ER antagonists, only select mutants such as Y537S caused a magnitude of change associated with fulvestrant resistance in vivo. Correspondingly, tumors driven by Y537S, but not D5358G, E380Q, or S463P, were less effectively inhibited by fulvestrant than more potent and bioavailable antagonists, including AZD9496. These data point to a need for antagonists with optimal pharmacokinetic properties to realize clinical efficacy against certain ESR1 mutants. Significance: A diversity of activating ESR1 mutations exist, only some of which confer resistance to existing ER antagonists that might be overcome by next-generation inhibitors such as AZD9496. Cancer Discov; 7(3); 277–87. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 235

BRAF

Irreversible pyrimidine-based EGFR inhibitors, including WZ4002, selectively inhibit both EGFR-activating and EGFR inhibitor–resistant T790M mutations more potently than wild-type EGFR. Although this class of mutant-selective EGFR inhibitors is effective clinically in lung cancer patients harboring EGFRT790M, prior preclinical studies demonstrate that acquired resistance can occur through genomic alterations that activate ERK1/2 signaling. Here, we find that ERK1/2 reactivation occurs rapidly following WZ4002 treatment. Concomitant inhibition of ERK1/2 by the MEK inhibitor trametinib prevents ERK1/2 reactivation, enhances WZ4002-induced apoptosis, and inhibits the emergence of resistance in WZ4002-sensitive models known to acquire resistance via both T790M-dependent and T790M-independent mechanisms. Resistance to WZ4002 in combination with trametinib eventually emerges due to AKT/mTOR reactivation. These data suggest that initial cotargeting of EGFR and MEK could significantly impede the development of acquired resistance in EGFR-mutant lung cancer. Significance: Patients with EGFR-mutant lung cancer develop acquired resistance to EGFR and mutant-selective EGFR tyrosine kinase inhibitors. Here, we show that cotargeting EGFR and MEK can prevent the emergence of a broad variety of drug resistance mechanisms in vitro and in vivo and may be a superior therapeutic regimen for these patients. Cancer Discov; 5(9); 960–71. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 893

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.

Recent studies have identified somatic ESR1 mutations in patients with metastatic breast cancer (MBC) and found some of them to promote estrogen-independent activation of the receptor. The degree to which all recurrent mutants can drive estrogen-independent activities and reduced sensitivity to ER antagonists like fulvestrant is not established. In this report, we characterize the spectrum of ESR1 mutations from over 900 patients. ESR1 mutations were detected in 10%, with D538G being the most frequent (36%), followed by Y537S (14%). Several novel, activating mutations were also detected (e.g. L469V, V422del, Y537D). While many mutations lead to constitutive activity and reduced sensitivity to ER antagonists, only select mutants such as Y537S caused a magnitude of change associated with fulvestrant resistance in vivo. Correspondingly, tumors driven by Y537S, but not D5358G, E380Q or S463P were less effectively inhibited by fulvestrant than more potent and bioavailable antagonists including AZD9496. These data point to a need for antagonists with optimal pharmacokinetic properties to realize clinical efficacy against certain ESR1 mutants.

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.