A Secondary Mutation in BRAF Confers Resistance to RAF ...€¦ · A Secondary Mutation in BRAF...

A Secondary Mutation in BRAF Confers Resistance to RAF Inhibition in a BRAF V600E -Mutant Brain Tumor Jiawan Wang 1 , Zhan Yao 2 , Philip Jonsson 3 , 4 , Amy N. Allen 1 , Alice Can Ran Qin 1 , Sharmeen Uddin 2 , Ira J. Dunkel 5 , 6 , Mary Petriccione 5 , Katia Manova 7 , Sofi a Haque 8 , Marc K. Rosenblum 9 , David J. Pisapia 10 , Neal Rosen 2 , Barry S. Taylor 3 , 4 , 11 , and Christine A. Pratilas 1

RESEARCH ARTICLE

Research. on August 27, 2020. © 2018 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst June 7, 2018; DOI: 10.1158/2159-8290.CD-17-1263

http://cancerdiscovery.aacrjournals.org/

SEPTEMBER 2018 CANCER DISCOVERY | 1131

ABSTRACT BRAF V600E 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 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 suffi cient to confer resistance to dabrafenib. Newer 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 resist-ance to targeted therapy are heterogeneous and perhaps emerge with a lineage-specifi c 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 clini-cally acquired resistance to RAF inhibitors. We demonstrate a secondary mutation in BRAF (V600E/L514V) following progression on dabrafenib and confi rm 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.

1 Division of Pediatric Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland. 2 Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York. 3 Human Oncology and Pathogenesis Program, Memo-rial Sloan Kettering Cancer Center, New York, New York. 4 Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York. 5 Department of Pediatrics, Memorial Sloan Ket-tering Cancer Center, New York, New York. 6 Department of Pediatrics, Weill Cornell Medical College, New York, New York. 7 Developmental Biol-ogy Program, Memorial Sloan Kettering Cancer Center, New York, New York. 8 Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York. 9 Department of Pathology, Memorial Sloan Ketter-ing Cancer Center, New York, New York. 10 Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York. 11 Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memo-rial Sloan Kettering Cancer Center, New York, New York. note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). Corresponding Author: Christine A. Pratilas, The Sidney Kimmel Compre-hensive Cancer Center at Johns Hopkins, 1650 Orleans Street, CRB1 Room 208, Baltimore, MD 21287. Phone: 443-287-8623; Fax: 410-502-7223; E-mail: [email protected] doi: 10.1158/2159-8290.CD-17-1263 ©2018 American Association for Cancer Research.

INTRODUCTION Oncogenic activation of the ERK signaling pathway is com-

mon in human tumors and occurs by multiple mechanisms, including mutational activation of RAS-GTPases, point mutations and fusions in BRAF , dysregulation of receptor tyrosine kinases, and genetic inactivation of the RAS-GTP-ase activating protein neurofi bromin-1 ( NF1 ). These genetic events are drivers of transformation in approximately one third of all human cancers ( 1, 2 ). Consequently, there have been signifi cant efforts to develop small molecules that target ERK signaling. ATP-competitive small-molecule RAF inhibi-tors have been developed and approved for use in patients

with advanced melanoma ( 3, 4 ). Although the downstream effects of RAF and MEK inhibitors in BRAF V600E -mutant melanoma cells are biologically similar ( 5 ), the ability of fi rst-generation RAF inhibitors to inhibit ERK signaling is restricted to tumors harboring mutant BRAF V600E . In tumors with wild-type (WT) RAFs, RAF inhibitors increase MEK and ERK phosphorylation and thus activate signaling ( 5–8 ). This mechanism of so-called paradoxical activation involves RAS-dependent dimerization and transactivation of BRAF molecules ( 6 ).

Molecular events that increase RAS activity and genetic alterations that cause BRAF V600E to dimerize in a RAS-inde-pendent manner prevent pathway inhibition by vemurafenib and dabrafenib ( 9–16 ). BRAF fusions, identifi ed in a subset of pediatric low-grade astrocytomas and other solid tumors, and many non-V600E BRAF mutants, which, despite high BRAF kinase activity, function biochemically as RAF homodimers, demonstrate de novo insensitivity to RAF inhibitors ( 17–19 ). Molecular events associated with acquired resistance have mostly emerged from studies of patients with melanoma and colorectal cancer treated with the RAF inhibitor vemurafenib or from laboratory efforts to generate models of acquired resistance.

Clinical responses to single-agent RAF inhibition have varied among cancer types, suggesting lineage-specifi c dif-ferences in signaling that may drive de novo and acquired resistance ( 14, 20, 21 ). It is currently unknown whether similar lineage-specifi c mechanisms of acquired resistance may also exist, especially in cancer types where RAF inhibi-tors remain under investigation. We therefore hypothesized that acquired resistance may be driven by and specifi c to the tumor type in which BRAF V600E signals and is inhibited, and set out to genomically characterize the longitudinal pre- and postprogression specimens from a patient with a BRAF V600E -mutant anaplastic ganglioglioma who initially responded to, but then progressed on, treatment with dabrafenib.




Wang et al.RESEARCH ARTICLE

1132 | CANCER DISCOVERY SEPTEMBER 2018 www.aacrjournals.org

RESULTSGenomic Characterization of a Dabrafenib-Resistant Pediatric Brain Tumor

We sought to determine the mechanistic basis for an ini-tial dramatic response and subsequent relapse to dabrafenib therapy in a 15-year-old boy diagnosed with a BRAFV600E-mutant left temporal lobe brain tumor. The patient was originally diagnosed at 33 months of age with a low-grade astrocy-toma [World Health Organization (WHO) grade 2]. The patient’s tumor recurred nearly 10 years later with malig-nant progression and was at that time characterized as ana-plastic ganglioglioma (WHO grade 3). He was treated with radiation and temozolomide, and then at second recurrence with lomustine and vincristine. Prospective sequencing of the resected third recurrence revealed a BRAFV600E mutation,

prompting enrollment in the pediatric phase I/II trial of dabrafenib (NCT01677741). A partial response was evident 8 weeks after the initiation of therapy, ultimately evolving into complete radiographic response. However, progressive disease was evident by 40 weeks, when therapy was discon-tinued and the progressing tumor was resected (Fig. 1A). Our review of the pathology then revealed an anaplastic gangli-oglioma with new areas of rhabdomyosarcomatous transfor-mation (Fig. 1A; Supplementary Fig. S1).

We performed whole-exome and transcriptome sequencing (WES and RNA sequencing) on tumor specimens acquired from the third recurrence prior to trial enrollment (pre-dabrafenib) and at the time of progression on therapy (post-dabrafenib). Our analysis of WES data from the pre-dabrafenib tumor reaffirmed the presence of the BRAFV600E mutation along with clonal, likely functional mutations in SMAD4, ATM,

Figure 1. BRAFL514V emerges upon RAF inhibitor response and resistance in a patient with pediatric glioma. A, Timeline of the patient’s clinical course. Significant events are shown along a timeline in weeks. Surgical resection was performed preenrollment on, and postremoval from, dabrafenib treatment. Dabrafenib was administered daily from weeks 0 to 40. Representative MRI images are shown, demonstrating pretreatment tumor (baseline, week 0), complete response of tumor to dabrafenib (week 12), and local tumor recurrence (week 40). Enhancing tumors of interest are marked with arrows. B, The fraction of cancer cells (cancer cell fraction) harboring key mutations in the pre/posttreatment tumors and the SK-BT-DR short-term culture from the postprogression specimen inferred from whole-exome sequencing and IMPACT. C, Three-dimensional structure of BRAFV600E in complex with dabrafenib where the L514 residue is <4 angstroms away from inhibitor binding, in contact with the selectivity pocket. Protein coordinates are from the BRAFV600E/dabrafenib complex (PDB 4XV2).

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Acquired Secondary Mutation in BRAF Following Dabrafenib RESEARCH ARTICLE


and other genes, a focal homozygous deletion of CDKN2A/B, and multiple large-scale DNA copy-number alterations. Only modest genomic evolution was evident from WES of the post-dabrafenib tumor, as it possessed a high degree of concord-ance among the DNA copy-number alterations and somatic mutations present before treatment (Fig. 1B; Supplementary Figs. S2 and S3; Supplementary Table S1). As analysis of the WES data suggested a modest DNA copy-number gain of chromosome 7 harboring BRAF (Supplementary Fig. S2), we performed multicolor FISH on a short-term cell culture [Sloan Kettering, brain-tumor, dabrafenib resistant (SK-BT-DR)] generated from fresh tissue of the progression specimen and sections from the pre- and post-dabrafenib tumor specimens. Subclonal heterogeneity of BRAF copy number existed with distinct cell populations with modest (3–10 copies) and more pronounced (11–16 copies) gains. Nevertheless, FISH con-firmed that these BRAF gains (3–9 copies) preexisted dab-rafenib treatment (Supplementary Fig. S1).

To determine the basis of treatment resistance, we further investigated the WES and transcriptome data for previously characterized mechanisms of resistance to RAF inhibitor ther-apy in other cancer types (9–11, 22–24). Although we found no such alteration, we did identify a subclonal BRAF L514V mutation in the post- but not pre-dabrafenib tumor (Fig. 1B). Similar multimodality sequencing of SK-BT-DR confirmed it also possessed the BRAFL514V mutation and faithfully rep-resented the genomics of postprogression disease in this patient (Fig. 1B).

Although second-site mutations are often acquired and mediate resistance in other commonly mutated oncogenic targets of therapy such as EGFR, KIT, NTRK, and others, this has never been previously validated in BRAF-mutant patients who have responded and relapsed on RAF inhibitor therapy. Reasoning that BRAF L514V might represent such a second-ary mutation acquired in response to dabrafenib therapy, we sought to determine the impact of this allele on BRAF protein structure. We examined the mutant BRAF protein structure bound to dabrafenib, which indicated that the L514 residue is located within the ATP-binding pocket of the αC-β4-loop of the kinase domain (25) and adjacent to the site of drug binding (∼4 angstroms; Fig. 1C). Furthermore, the BRAFL514 residue is homologous to residues in other oncogenically activated kinases that are mutated as a mecha-nism of acquired resistance to targeted therapy (Supplemen-tary Fig. S4; Supplementary Table S2).

The post-dabrafenib tumor retained expression of BRAFV600E, confirmed by targeted mass spectrometry–based genotyp-ing (Sequenom). To determine whether the resistant tumor retained activation of ERK signaling, we assessed ERK phos-phorylation using IHC analysis in both the pre- and post-dab-rafenib tumors. The posttreatment tumor retained activation of ERK signaling as detected by IHC measurement of phos-phorylated ERK (pERK), particularly in areas with astrocytic and sarcomatous morphology (Supplementary Fig. S1).

In order to determine whether a rare BRAF L514V-mutant subclone was present pretreatment but not captured by exome sequencing, we performed droplet digital PCR of BRAF L514V. This confirmed its presence in both the post-dabrafenib tumor and short-term cell culture (SK-BT-DR) at a mutant allele burden consistent with exome sequencing

of the tumor tissue, but the mutant allele was not detectable in either the normal blood or pretreatment tumor speci-mens (sensitivity of 0.016%; Supplementary Table S3). To determine whether BRAF L514V was present in cis with V600E, we randomly screened and sequenced 20 individual bacte-rial colonies transformed by plasmids carrying BRAF cod-ing sequence amplified from SK-BT-DR cells. Although a subset of colonies was WT BRAF, others BRAFV600E, and 25% BRAFV600E/L514V, there was no evidence that BRAF L514V existed in the absence of V600E (Supplementary Fig. S5). Together, these data suggest that BRAF L514V was an acquired secondary mutation on the V600E-mutant allele.

BRAFV600E/L514V Reduces Sensitivity to Dabrafenib by Inducing BRAF Dimerization

To assess whether BRAFL514V was sufficient to drive clin-ical resistance to dabrafenib, we assessed the ability of L514V, alone and in cis with V600E, to activate MEK–ERK signaling compared with BRAFV600E and WT BRAF. We utilized four different cellular contexts: (i) SKBR3 cells in which RAS activation is HER2-dependent and lapatinib treatment can produce low levels of RAS activity to study the effects of transfected BRAF variants without the influ-ence of endogenous RAS activity on RAF dimerization (19); (ii) the BRAFV600E brain tumor cell line DBTRG-05MG; (iii) the immortalized fibroblast cell line NIH-3T3 with WT BRAF; and (iv) a BRAFV600E-expressing, dabrafenib-sensitive melanoma cell line, A375. In the latter model, we stably transduced doxycycline-inducible constructs of BRAFV600E and BRAFV600E/L514V to study the expression of the BRAF double mutant in the context of endogenous BRAFV600E, which recapitulates the BRAF genetics of our patient’s post-dabrafenib tumor. Both BRAFV600E (VE) and BRAFV600E/L514V (VELV) significantly elevated MEK and ERK phosphoryla-tion over baseline levels (Fig. 2A–E and G). The ability of dabrafenib to inhibit pERK was reduced in BRAFV600E/L514V compared with BRAFV600E in all cell types studied here (Fig. 2A–C; Supplementary Fig. S6). BRAFV600E/L514V-associated resistance to dabrafenib was dose-dependent. Expressing higher levels of BRAFV600E/L514V resulted in further increased resistance to RAF inhibition, whereas overexpression of BRAFV600E alone to similar levels resulted in only minimally reduced sensitivity (Fig. 2D). BRAFV600E/L514V-expressing cells were less sensitive to dabrafenib at lower doses than were cells expressing BRAFV600E alone, as measured by a shift to the right of the curve of pERK inhibition (Fig. 2E and F). Furthermore, the levels of pERK recovered more rapidly over time in BRAFV600E/L514V cells following a single dabrafenib treatment than did cells expressing BRAFV600E alone (Fig. 2G; Supplementary Fig. S7). Cell proliferation, as meas-ured by growth in culture over 72 hours, was less sensitive to dabrafenib treatment in BRAFV600E/L514V compared with BRAFV600E cells. Moreover, the ability of BRAFV600E/L514V- expressing cells to form colonies in the presence of dab-rafenib was significantly increased compared with that of BRAFV600E cells (Fig. 2H and I, IC50, IC75, and IC90 provided in Supplementary Fig. S8), consistent with the incomplete pERK inhibition we observed. These data indicate that BRAFV600E/L514V is sufficient to cause acquired resistance to dabrafenib.






Figure 2. BRAFV600E/L514V is an activating mutation and is resistant to dabrafenib. A, V5-tagged BRAF mutants (VE or VELV) were expressed in SKBR3 cells for 24 hours, followed by lapatinib treatment (1 μmol/L for 1 hour), and then followed by treatment with DMSO or dabrafenib (200 nmol/L) for another hour. UT, untransfected. B, DBTRG-05MG cells were transiently transfected with V5-tagged BRAFV600E or BRAFV600E/L514V for 24 hours and treated with DMSO or dabrafenib for 1 hour. C–G, A375 (BRAFV600E melanoma) cells stably transduced with retrovirus carrying doxycycline (dox)-induc-ible GFP-tagged empty vector, or vector encoding V5-tagged BRAF mutants, were treated with doxycycline (150, 200, 150 ng/mL, respectively, in C and E; 50 or 200 ng/mL in D) for 24 hours, followed by treatment with dabrafenib (100 nmol/L in C, D, and G; dose range as shown in E) for 1 hour (C–F) or over time course as shown in G. A–E and G, Expression and/or phosphorylation of the indicated proteins were assessed by immunoblot. F, pERK signal intensity was quantitated using densitometric analysis and graphed as a function of dabrafenib dose. H, A375 cells treated with dabrafenib at doses shown were assessed for cell viability and graphed as percentage relative to control. Doxycycline was replenished at 50% every 24 hours. Data, mean ± SEM. I, A375 cells as in C–H were grown in soft agar with the indicated doses of dabrafenib (30 and 100 nmol/L). The y-axis represents the number of colonies, expressed as an average of four fields per well, three wells per condition, relative to DMSO controls, for each cell line (data, mean ± SEM; ****, P < 0.0001, unpaired Student t test).

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We next sought to determine the mechanism by which BRAFL514V mediates resistance to dabrafenib. BRAFV600E sig-nals as a monomer, which is the mechanistic basis for the selective inhibition of ERK output in BRAFV600E-mutant tumors by existing type I RAF inhibitors such as vemu-rafenib and dabrafenib. Notably, the p61 BRAF splice-forms, which are acquired in BRAFV600E-mutant patients treated with type I RAF inhibitors, non-V600 activating BRAF mutants, and BRAF in-frame deletions (residues L485–P490), confer resistance to these agents by inducing RAF dimer formation (10, 19, 26, 27). We hypothesized that BRAFL514V, acquired in cis with V600E, similarly promotes RAF dimerization as its mechanism of acquired resistance to dabrafenib. To address this question, we cotransfected constructs encoding BRAFV600E and BRAFV600E/L514V with V5 or FLAG tags in SKBR3 cells to detect homodimerization

using an immunoprecipitation (IP) assay. As expected, ectopic expression of p61 BRAFV600E (10) exhibited a strong signal for the BRAF homodimer (Fig. 3A). BRAF homodi-mers were minimal in cells expressing WT BRAF, but ectopic expression of BRAFV600E/L514V promoted a greater degree of BRAF homodimerization than did BRAFV600E alone, regard-less of medium or high levels of BRAF-mutant expression and independent of RAS activity (Fig. 3A and B; Supple-mentary Fig. S9).

We then determined the impact of disrupting dimerization induced by BRAFL514V. Here, we utilized the BRAFR509H tool mutation, which disrupts BRAF dimerization and reduces BRAF kinase activity (6). We introduced the R509H muta-tion to WT BRAF, BRAFV600E, BRAFL514V, and BRAFV600E/L514V. Consistent with our IP results, the activity of BRAFL514V and BRAFV600E/L514V, but not BRAFV600E alone, was reduced by






Figure 3. BRAFV600E/L514V causes resistance to dabrafenib by formation of dimers. A, V5-tagged and FLAG-tagged WT BRAF and the indicated BRAF mutants were coexpressed in SKBR3 cells (lapatinib treated 1 hour prior to collection). The binding of V5-tagged BRAF proteins to FLAG-tagged BRAF proteins was determined by immunoprecipitation followed by immunoblotting analysis. B, Untransfected (UT) and transiently transfected SKBR3 cells expressing V5 or FLAG-tagged BRAF mutants with low (L), medium (M), and high (H) expression levels were treated with lapatinib for 1 hour. Homodi-merization of BRAFV600E and BRAFV600E/L514V was evaluated by immunoblotting following immunoprecipitation. Relative FLAG signal (IP/WCL) in medium and high levels of BRAF mutants was determined by densitometry analysis. WCL, whole-cell lysate. Statistical analysis was performed using unpaired Student t test in replicate experiments. C and D, V5-tagged BRAF [WT; R509H; VE; V600E/R509H (VEH); VELV; or V600E/L514V/R509H (VELVH)] were expressed in SKBR3 cells for 24 hours, followed by lapatinib treatment (1 μmol/L for 1 hour, C and D), then followed by treatment with DMSO (−) or dabrafenib (dab; +; 200 nmol/L) for 1 hour (D only). Relative pMEK and pERK levels were quantitated in replicate experiments and shown as a bar graph with statistical analysis. n.s., not significant. E, A375 (BRAFV600E melanoma) cells stably transduced with retrovirus carrying doxycycline-inducible vector encoding V5-tagged BRAF mutants were treated with doxycycline (dox; 150 or 25 ng/mL) for 24 hours, followed by treatment with dabrafenib (dose range as shown) for 1 hour. Expression and/or phosphorylation of the indicated proteins were assessed by immunoblot. pERK signal intensity was quanti-tated using densitometric analysis, and graphed as a function of dabrafenib dose. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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dimer insufficiency (Fig. 3C; Supplementary Fig. S10A). Although BRAFV600E/R509H does not alter pERK sensitiv-ity to dabrafenib compared with BRAFV600E, pERK levels were reduced to a greater extent in cells expressing dimer-incompetent BRAFV600E/L514V/R509H than those expressing dimerization-competent BRAFV600E/L514V (Fig. 3D and E; Supplementary Fig. S10B). To distinguish this mechanism of action from a conventional gatekeeper mutation, we assessed the BRAF kinase activity of exogenously expressed BRAFV600E and BRAFV600E/L514V with a kinase-dead MEK1 substrate. Indeed, there was no difference in the ability of dabrafenib to inhibit BRAF activity (Supplementary Fig. S11).

MEK Inhibition and RAF Dimer Inhibition Are More Effective against BRAFV600E/L514V-Activated ERK Signaling

Because BRAFV600E/L514V activates dimer-dependent ERK signaling and is insensitive to type I RAF inhibition, we sought alternative small-molecule therapeutic strategies to overcome this acquired resistance. We first assessed the

sensitivity of such cells to MEK inhibition. We treated cells expressing BRAFV600E or BRAFV600E/L514V with a RAF inhibitor (RAFi; dabrafenib), a MEK inhibitor (MEKi; trametinib), or their combination. We tested combination therapy in mul-tiple cellular contexts to exclude the intrinsic sensitivity of BRAFV600E-expressing parental cells to MEK inhibition. Inhi-bition of ERK phosphorylation in BRAFV600E/L514V-expressing cells was more sensitive to MEK inhibition or the combina-tion of RAF and MEK inhibitors than to RAF inhibition alone (Fig. 4A and B; Supplementary Fig. S12). Trametinib alone, or combined with dabrafenib, however, did not equally inhibit cell growth of BRAFV600E and BRAFV600E/L514V mutants in short-term drug incubation (72 hours, corresponding IC50 values in Supplementary Fig. S13) or in long-term colony formation assay (2 weeks; Fig. 4C and D; Supplementary Figs. S13 and S14), even though the therapeutic combina-tion effectively reduced pERK in cells exogenously expressing the double mutant. These data suggest that although cells possessing BRAFV600E/L514V-mediated resistance to dabrafenib retain ERK dependency, this cannot be overcome completely by MEK inhibition.

Figure 4. Dabrafenib plus trametinib more potently inhibits signaling in BRAFV600E/L514V-expressing cells than dabrafenib alone. A, V5-tagged BRAF mutants (VE or VELV) were expressed in SKBR3 cells for 24 hours, followed by lapatinib treatment (1 μmol/L for 1 hour), then followed by treatment with DMSO or dabrafenib (200 nmol/L) and/or trametinib (20 nmol/L) for 1 hour. UT, untransfected. Expression and/or phosphorylation of the indicated proteins were assessed by immunoblot. B, A375 (BRAFV600E melanoma) cells stably transduced with retrovirus carrying doxycycline (dox)-inducible GFP-tagged empty vector or vector encoding BRAF mutants (VE or VELV) were treated with doxycycline (150, 200, 150 ng/mL) for 24 hours, followed by treatment with dabrafenib (doses as shown) and/or trametinib (20 nmol/L) for 1 hour. Expression and/or phosphorylation of the indicated proteins were assessed by immunoblot. C, A375 cells stably expressing GFP, VE, and VELV treated with trametinib at doses shown were assessed for cell viability. Doxycycline was replenished at 50% every 24 hours. Data, mean ± SEM. D, A375 cells as in B and C were grown in soft agar in the presence of doxycycline (150, 200, 150 ng/mL) with the indicated doses of dabrafenib (doses as shown, nmol/L) and/or trametinib (20 nmol/L). The y-axis represents the number of colonies, expressed as an average of four fields per well, three wells per condition, relative to DMSO controls for each cell line (data, mean ± SEM; ****, P < 0.0001, unpaired Student t test).

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LapatinbDabrafenibTrametinib

VE VELV

+

+−

+

−−

+

++

+

−+

+

+−

+

−−

+

++

+

−+

−−+

−−−

UT

ERK

V5

BRAF

pMEK

pERK

D

−3 −2 −1 0 1 2

Log [trametinib], nmol/L

Col

onie

s v.

DM

SO

ctr

l (%

) VEVELV

GFP

**** **** **** **** ****

pERK dark

pERK light

C






To establish a therapeutic modality that is equipotent against BRAFV600E/L514V-mediated resistance, we assessed the efficacy of a novel class of RAF inhibitors shown to inhibit mutant forms of BRAF that promote RAF dimerization (type II αC-IN inhibitors; refs. 10, 28). We therefore tested eight compounds against this mutation including type I αC-OUT/DFG-IN inhibitors (vemurafenib and dabrafenib); type I αC-OUT/DFG-IN paradox breakers (PLX7904 and PLX8394) that suppress mutant BRAF without activating ERK signal-ing in cells bearing upstream activation (29); type II αC-IN/DFG-OUT RAF dimer inhibitors (LY3009120 and TAK-632; refs. 30, 31); and two new RAF dimer inhibitors (BGB3245 and BGB3290; WO 2014206344 A1/US 9670203 B2; ref. 32). We treated both stably transduced A375 and transiently transfected SKBR3 cells with BRAFV600E or BRAFV600E/L514V to compare the relative effectiveness of these inhibitors (Fig. 5A and B; Supplementary Fig. S15). Among these compounds, only BGB3245 and BGB3290 inhibited mono-meric BRAFV600E and dimeric BRAFV600E/L514V with simi-lar potency for both ERK signaling inhibition and cell growth. Conversely, vemurafenib, dabrafenib, PLX7904, PLX8394, LY3009120, and TAK-632 showed potent inhibi-tion of only BRAFV600E and had reduced activity against BRAFV600E/L514V (Fig. 5A–D; Supplementary Figs. S15 and S16). The MEK inhibitor trametinib demonstrated a small differential in potency against BRAFV600E/L514V, whereas the ERK1/2 kinase inhibitor SCH772984 (33) was able to elicit equipotent inhibition of BRAFV600E/L514V compared with BRAFV600E, as demonstrated by inhibition of both pERK

(Supplementary Fig. S17) and cell proliferation (Supple-mentary Fig. S16).

To determine the mechanism of the BGB3245 equipotency we observed for BRAFV600E and BRAFV600E/L514V, we explored its affinity for both the first and second site of the BRAF dimer. We exploited the fact that type I RAF inhibitors bound to the first site in the BRAF dimer reduces its affinity for the second site, which explains how ERK signaling is much more sensitive to these drugs when monomer- rather than dimer-driven (19). To create a condition that only one site of the RAF dimer is occupied by a RAF inhibitor, we pretreated BRAFV600E/L514V-expressing A375 cells with LGX818, a potent type I RAF inhibitor with slow off-rate from the first site and rapid dissociation from the second site in RAF dimers (19). Upon LGX818 washout and incubation with increasing concentrations of dabrafenib or BGB3245, respectively, we found that BGB3245 inhibits the mutant BRAFV600E mono-mer and the second unbound site of the BRAFV600E/L514V dimer with similar potency (Supplementary Fig. S18). These data provide a mechanistic basis for the unique property of BGB3245 in inhibiting activated dimerization-dependent forms of mutant BRAF.

DISCUSSIONMutational activation of BRAF is a frequent oncogenic

event and is described in multiple types of cancer (34–38). ATP-competitive inhibitors of RAF kinase have been shown to be extremely effective in the treatment of melanoma with

Figure 5. RAF dimer inhibitors BGB3245 and BGB3290 overcome L514V-mediated resistance to dabrafenib. A, A375 cells expressing inducible BRAF mutants (VE or VELV) were cultured in doxycycline-containing medium for 24 hours and then treated with increasing concentrations of the indicated RAF inhibitors for 1 hour. Cell lysates were examined by immunoblot using the antibodies indicated. B, VE or VELV were transiently trans-fected in SKBR3 for 24 hours. Then, the cells were treated with 1 μmol/L lapatinib for 1 hour, followed by treatment with DMSO or the RAF inhibi-tors indicated for 1 hour (the dose for vemurafenib, PLX7904, LY3009120, TAK632, BGB3245, and BGB3290 is 1 μmol/L; the dose for dabrafenib and PLX8394 is 300 nmol/L). Untransfected (UT) SKBR3 cells were treated with DMSO or 1 μmol/L lapatinib (lanes 1 and 2). C, SKBR3 cells express-ing VE or VELV were pretreated with lapatinib for 1 hour, followed by treatment with dabrafenib or BGB3245 over a time course. ERK signaling was assessed by immunoblot. D, A375 cells expressing inducible GFP or BRAF mutants (VE or VELV) treated with BGB3245 at concentrations shown were assessed for cell viability and graphed as percentage relative to control. Doxycycline was replenished at 50% every 24 hours. Data, mean ± SEM.

A CDAB BGB3245

VE VELV

0 1 6BRAF

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pMEK

pERK

ERK

h0 1 6 0 1 6 0 1 6

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BRAFV600 mutation (4, 39–42), and these unprecedented clini-cal effects are thought to be due to the unusual mechanism of action of these drugs, which inhibit ERK signaling only in tumors with V600-mutant BRAF. Molecular events that increase intracellular RAS activation or cause RAF to dimer-ize in a RAS-independent manner cause resistance to these drugs. As such, RTK activation, RAS mutation, and aber-rantly spliced RAF isoforms have been shown to confer resistance in model systems, whereas the latter two lesions have been identified in tumors from patients with acquired resistance (9, 10, 43). Published studies have not validated second-site mutations in BRAF as a mechanism of acquired resistance to RAF inhibitor treatment in patients.

In contrast to receptor tyrosine kinases such as EGFR and KIT, in which second-site mutations are responsible for a significant proportion of clinical resistance to targeted therapy (44–47), second-site point mutations in BRAF are not thought to contribute to acquired resistance to the RAF inhibitors vemurafenib and dabrafenib (48). An in vitro mutagenesis screen (49) and two case reports of single-patient tumor evolution studies (50, 51) identified BRAFL505H as a sec-ondary mutation associated with resistance to vemurafenib. In these studies, however, it was either not confirmed that the second-site mutation was absent from the pretreatment specimen (51), or the mutation was known to be present prior to RAF inhibitor exposure (50). Our study is the first, to our knowledge, to unequivocally demonstrate the emergence of a BRAF secondary mutation in a tumor that progressed after initial response to dabrafenib. Rather than BRAF L514V preex-isting in a rare cell population prior to therapy, we identified no evidence of BRAF L514V in the pretreatment sample to a sensitivity of approximately 0.01%.

WES of pre– and post–dabrafenib-treated tumors demon-strated that a single novel point mutation in BRAF emerged throughout the course of the treatment. Our sequencing data, validated by clonal selection of a short-term cell culture derived from the post-dabrafenib tumor and by FISH, sug-gest that V600E was clonal in both the pre- and posttreat-ment specimens, that chromosome 7q encoding BRAF was duplicated, and that V600E was the allele that was gained. Our data further suggest that L514V is subclonal, present in approximately 30% of tumor cells exclusively in the posttreat-ment tumor. Moreover, L514V emerged in cis with V600E, and is therefore also present in at least two copies in the affected cells along with V600E. In addition, no other posttreatment-specific oncogenic alterations were identified. Specifically, loss of CDKN2A, which is a well-described genomic event in BRAFV600E pediatric low-grade glioma/secondary high-grade glioma (sHGG), and is associated with inferior outcomes in these patients and a higher risk of transformation to sHGG (52, 53), was present in both the pre- and post-dabrafenib samples, and therefore did not play an objective role in acquired resistance.

Structurally, the L514 residue is located in the αC-β4-loop of BRAF kinase domain and is involved in the linkage of the αC-helix movements between the α C-helix-in and α C-helix-out conformations within the dimer interface (25). Our results indicate that BRAFL514V induces dimerization of RAF molecules previously capable of signaling as mono-mers, rendering ERK output insensitive to monomer-specific

dabrafenib treatment. BRAFL514V mediates dimerization rather than acting as a conventional gatekeeper mutation, and therefore converges on an established mechanism of type I RAF inhibitor resistance (10, 19), albeit via a novel and perhaps lineage-dependent genetic mechanism. Our work demonstrates that despite the limited activity of the MEK inhibitor trametinib in this resistance model, the newer RAF dimer inhibitors BGB3245 and BGB3290 or the ERK inhibi-tor SCH772984 are active against this acquired mutation, suggesting a potential and improved personalized treat-ment option for patients who harbor this mutation or other acquired mutations in BRAF with a similar function. Finally, our data suggest that a novel, secondary mutation in BRAF represents a mechanism of acquired resistance to currently available RAF inhibitors, and further, that tumors of dif-ferent lineages driven by the same oncogenic mutation and therapeutically targeted with the same drugs may select for acquired resistance in novel ways that biochemically converge on RAF dimerization.

METHODSSample Collection, WES, RNA Sequencing, and Analysis

The patient was treated on the global phase I/II trial of dabrafenib for pediatric patients (NCT01677741) in accordance with the Dec-laration of Helsinki. Tumors were collected preenrollment on and postremoval from (independent of) the trial. Collection and genomic characterization of these samples was conducted under Institutional Review Board (IRB)–approved Research Authorization and Biospeci-men Use protocols (MSKCC IRB #06-107 and #10-130, respectively) with written informed consent on file. For WES and analysis, refer to Supplementary Methods. Integrated mutation profiling of action-able cancer targets (MSK-IMPACT) was performed as described previ-ously (54, 55). The WES data have been deposited in the NCBI dbGaP archive under accession number phs001629.v1.p1.

Cell Lines, Antibodies, and ReagentsThe SK-BT-DR short-term culture was generated from the patient’s

tumor at the time of surgical resection, after progression on dab-rafenib was noted. Collection of the patient’s tumor material and all subsequent analysis of this tumor were performed in accordance with IRB-approved protocols for the collection and use of biospecimens (MSKCC IRB #06-107 and IRB #10-130).

A375, SKBR3, NIH-3T3, and DBTRG-05MG cells were obtained from the ATCC between 2015 and 2018, and verified by short tandem repeat profile for cell line authentication tested after receipt at Johns Hopkins University Core Facility (Baltimore, MD). All these cell lines were grown in the recommended medium, tested negative for Mycoplasma contami-nation, and passaged in vitro for fewer than 2 months after resuscitation. Doxycycline-inducible A375 cells were maintained in DMEM with 10% tetracycline-free FBS, 2 mmol/L -glutamine, 1% penicillin–streptomycin, 50 μg/mL hygromycin, and 0.2 μg/mL puromycin (19).

Antibodies against ERK, pERK, MEK, pMEK, V5, and FLAG were obtained from Cell Signaling Technology, and antibody against BRAF was obtained from Santa Cruz Biotechnology. Vemurafenib, dabrafenib, PLX7904, LY3009120, TAK632, LGX818, trametinib, PD0325901, MEK162, SCH772984, GDC-0994, and lapatinib were purchased from Selleckchem; PLX8394 was obtained from Med-chemExpress; BGB3245 and BGB3290 were provided by BeiGene (MTA to N. Rosen); doxycycline was from Sigma Aldrich; puromycin and hygromycin stock solutions were purchased from Invitrogen. Drugs for in vitro studies were dissolved in DMSO to yield 10 or 1 mmol/L stock solutions, and stored at −20°C.






ImmunoblottingCells were disrupted on ice in NP40 lysis buffer as described

previously (56). Protein concentration was determined with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence with the ECL detec-tion reagents, Immobilon Western HRP substrate Luminol Reagent (Millipore) and Luminol Enhancer Solution (Thermo Fisher Scien-tific). The membranes were imaged on the ChemiDoc Touch Imaging System (Bio-Rad) and relative changes in pERK were quantitated by densitometry analysis using ImageJ as a function of concentration or treatment time. Response curves were generated using Prism 6.

Plasmids and TransfectionsThe pcDNA3.1-BRAF-V5/FLAG and pcDNA3.1-p61 BRAF V600E-

V5/FLAG were constructed as described previously (6). Mutations were introduced using Site-Directed Mutagenesis Kit (Stratagene) and confirmed by Sanger sequencing. Cells were seeded in 6- or 10-cm dishes and transfected the following day using Lipofectamine 2000 (Thermo Fisher Scientific) or NeuroPORTER transfection reagent (Genlantis) according to the manufacturer’s instructions.

Immunoprecipitation and In Vitro Kinase AssayCells were collected and lysed on ice in lysis buffer (25 mmol/L

Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% NP40, 1 mmol/L EDTA, 5% glycerol) supplemented with phenylmethylsulfonyl fluoride (Cell Sig-naling Technology), protease inhibitor cocktail (Millipore), and sodium orthovanadate (New England Biolabs). Exogenously expressed V5-tagged BRAF proteins were immunoprecipitated using a V5-specific antibody agarose conjugate (Sigma-Aldrich). Immunoprecipitations were carried out at 4°C overnight, followed by four washes with ice-cold lysis buffer. Pull-down complexes were eluted with V5 peptide (Sigma-Aldrich) and assayed by immunoblotting. In vitro kinase assays were performed using V5-tagged BRAF proteins isolated from 293H cells and kinase-dead K97R MEK1 (Millipore) as a substrate in the presence of 200 μmol/L ATP. Kinase assays were conducted at 30°C for 15 minutes. MEK phosphoryla-tion was assayed by immunoblotting with phospho-specific antibodies.

Cell Proliferation AssaysCells were seeded in 96-well plates at 1,500 cells per well and cul-

tured in medium containing doxycycline (200 ng/mL for A375-VE and 150 ng/mL for A375-VELV) for 24 hours. A dose range of the compound indicated was prepared by serial dilutions and then added to the dishes containing adherent cells. Cells were incubated for 72 hours, and doxycycline was replenished at 50% every 24 hours. Cell growth was quantitated using the Cell Counting Kit-8 (Dojindo). For each condition, four replicates of each concentration were measured. Relative survival in the presence of drugs was normalized to the untreated controls after background subtraction. Graphs were gener-ated using Prism 6 based on the average of four replicates. IC50, IC75, and IC90 values were calculated using CompuSyn software.

Soft-Agar Colony FormationSoft-agar assay was performed as described previously (56). Briefly,

10,000 cells growing in log phase were mixed with agar (0.33%), treated with either DMSO or compounds indicated in the presence of doxycycline, and plated over a bottom layer of 0.5% agar in 6-well plates. Cells were incubated at 37°C for 2 weeks, with 200 μL of media containing 0.5× doxycycline pipetted over the surface every 24 hours. Colonies were then stained with crystal violet (Sigma-Aldrich) for 1 hour and four random fields per chamber were acquired using Nikon Eclipse Ti inverted microscope (Nikon). The measurement was based on three replicates for each condition.

Disclosure of Potential Conflicts of InterestI.J. Dunkel is a consultant/advisory board member for Apexigen,

Bayer, Bristol-Myers Squibb, Celgene, Eisai, Ipsen, and Pfizer. N. Rosen has ownership interest (including stock, patents, etc.) in Beig-ene and Kura and is a consultant/advisory board member for Chugai, AstraZeneca, Beigene, and Kura. No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: J. Wang, Z. Yao, C.A. PratilasDevelopment of methodology: J. Wang, Z. Yao, K. ManovaAcquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Wang, Z. Yao, P. Jonsson, A.N. Allen, A.C.R. Qin, I.J. Dunkel, M. Petriccione, S. Haque, D.J. Pisapia, N. Rosen, B.S. Taylor, C.A. PratilasAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): J. Wang, Z. Yao, P. Jonsson, A.N. Allen, S. Haque, M.K. Rosenblum, D.J. Pisapia, N. Rosen, B.S. Taylor, C.A. PratilasWriting, review, and/or revision of the manuscript: J. Wang, Z. Yao, P. Jonsson, I.J. Dunkel, K. Manova, M.K. Rosenblum, D.J. Pisapia, B.S. Taylor, C.A. PratilasAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. UddinOther (NP who cared for the patient): M. Petriccione

AcknowledgmentsWe are grateful to the patient described and to his family who

provided authorization for research; to the clinical investigators and Study Sponsor for the Novartis phase I/II trial of dabrafenib in pediatric patients (NCT01677741); to Lusong Luo and BeiGene for provision of compounds; and to Afsar Barlas and Gouri Nanjangud of the Molecular Cytology and Molecular Cytogenetics Core Facili-ties of MSKCC for assistance with pERK IHC and FISH studies, respectively. This work has been funded by the NIH: K08 CA127350 (to C.A. Pratilas), P30 CA008748, U54 OD020355 (to B.S. Taylor), R01 CA204749 (to B.S. Taylor); Giant Food Pediatric Cancer Fund (to C.A. Pratilas); Sontag Foundation (to B.S. Taylor); and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology of MSKCC.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 14, 2017; revised March 22, 2018; accepted June 5, 2018; published first June 7, 2018.

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2018;8:1130-1141. Published OnlineFirst June 7, 2018.Cancer Discov Jiawan Wang, Zhan Yao, Philip Jonsson, et al.

-Mutant Brain TumorV600E BRAFInhibition in a Confers Resistance to RAFBRAFA Secondary Mutation in

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