BRAF Fusion As a Novel Mechanism of Acquired Resistance to Vemurafenib in BRAF V600E Mutant

Author Manuscript Published OnlineFirst on May 24, 2017; DOI: 10.1158/1078-0432.CCR-16-0758 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Title Page

2 Title

3 BRAF fusion as a novel mechanism of acquired resistance to vemurafenib in BRAF V600E mutant

4 melanoma

6 Atul Kulkarni1,2, Husam Al-Hraishawi1, Srilatha Simhadri1,2 , Kim M. Hirshfield1,2, Suzie Chen2,3,

7 Sharon Pine1,2, Chandrika Jeyamohan1, Levi Sokol4, Siraj Ali5, Man Lung Teo6, Eileen White1,

8 Lorna Rodriguez-Rodriguez1,2, Janice M. Mehnert1,2,7, Shridar Ganesan1,2

10 1Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, New Jersey

11 08903 USAiversity60 Frelinghuysen Ro

12 2Department of Medicine, Rutgers Robert Wood Johnson Medical School, Rutgers University,

13 671 Hoes Lane, Piscataway, New Jersey 08854 USA

14 3Rutgers Ernest Mario School of Pharmacy, 160 Frelinghuysen Rd, Piscataway Township, NJ

15 08854 USAUS

16 4Department of Radiology, Rutgers Robert Wood Johnson Medical School, Rutgers University,

17 671 Hoes Lane, Piscataway, New Jersey 08854 USA

18 5Foundation Medicine, Inc. Cambridge, Massachusetts 02141 USA

19 6 Central Comprehensive Cancer Centre, Central Bldg, Central District, 999077, Hong Kong

20 7Developmental Therapeutics/Phase I Program, Rutgers Cancer Institute of New Jersey 08854

21 USA

22 23 Running title

24 BRAF-fusion as mechanism of resistance to BRAF inhibition.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 24, 2017; DOI: 10.1158/1078-0432.CCR-16-0758 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

27 Key words

28 BRAF, melanoma, next-generation sequencing, resistance, MEK

30 Abbreviations

31 CINJ=Rutgers Cancer Institute of New Jersey; CTLA4=cytotoxic T-lymphocyte-associated

32 antigen 4; DIC=differential interference contrast; HIPAA=Health Insurance Portability and

33 Accountability Act; FACS=fluorescence-activated cell sorting; IRB=Institutional Review Board;

34 PCR=polymerase chain reaction; PD-1=programmed cell death-1; PFS=progression-free

35 survival; PI3K=phosphoinositide 3-kinase; OS=overall survival;; RR=response rate

37 Conflict of interest disclosure statements

38 Kim M. Hirshfield receives support from the Ruth Estrin Goldberg Memorial for Cancer

39 Research and the American Hellenic Educational Progressive Association (AHEPA)

40 Foundation. Siraj Ali is an employee of Foundation Medicine, Inc. Atul Kulkarni, Husam Al-

41 Hraishawi, Srilatha Simhadri, Suzie Chen, Sharon Pine, Chandrika Jeyamohan, Levi Sokol,

42 Man Lung Teo, Eileen White, Lorna Rodriguez-Rodriguez, Janice M. Mehnert, and Shridar

43 Ganesan declared no conflict of interest.

45 Corresponding and co-last authors

46 Janice M. Mehnert, MD

47 195 Little Albany Street, New Brunswick, New Jersey 08903 USA

48 Phone: 732-235-8945

49 Fax: 732-235-8094

50 Email: [email protected]

51 Shridar Ganesan, MD, PhD

52 195 Little Albany Street, New Brunswick, New Jersey 08903 USA

53 Phone: 732-235-5211

54 Fax: 732-235-5331

55 Email: [email protected]

78 STATEMENT OF TRANSLATIONAL RELEVANCE

79 In this study, we present evidence that resistance to vemurafenib in a BRAF V600E mutant

80 melanoma is associated with selection for a clone with an AGAP3-BRAF fusion gene. These

81 data demonstrate that BRAF fusions may be an underappreciated mechanism of acquired

82 resistance to BRAF inhibitor therapy. The appearance of AGAP3-BRAF fusion during BRAF

83 inhibitor treatment, and its loss during progression when BRAF inhibitor therapy was

84 discontinued, is consistent with underlying rapid clonal dynamics in response to treatment.

85 These data suggest a rationale for re-challenge with BRAF inhibitors in some clinical settings.

100

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102 ABSTRACT

103 Purpose. Many patients with BRAF V600E mutant melanoma treated with BRAF inhibitors

104 experience a rapid response, but ultimately develop resistance. Insight into the mechanism of

105 resistance is critical for development of more effective treatment strategies.

106 Experimental Design. Comprehensive genomic profiling of serial biopsies was performed in a

107 patient with a BRAF V600E mutant metastatic melanoma who developed resistance to

108 vemurafenib. An AGAP3-BRAF fusion gene, identified in the vemurafenib-resistant tumor, was

109 expressed in BRAF V600E melanoma cell lines and its effect on drug sensitivity was evaluated.

110 Results. Clinical resistance to vemurafenib in a melanoma harboring a BRAF V600E mutation was

111 associated with acquisition of an AGAP3-BRAF fusion gene. Expression of AGAP3-BRAF

112 fusion in BRAF V600E mutant melanoma cells induced vemurafenib resistance; however, these

113 cells remained relatively sensitive to MEK inhibitors. The patient experienced clinical benefit

114 following treatment with the combination of a BRAF and a MEK inhibitor. Rebiopsy of the tumor

115 at a later time point, after BRAF and MEK inhibitors had been discontinued, showed loss of

116 AGAP3-BRAF fusion gene. Mixing experiments suggest that cells harboring both BRAF V600E

117 and AGAP3-BRAF only have a fitness advantage over parental BRAF V600E cells during active

118 treatment with a BRAF inhibitor.

119 Conclusions. We report acquisition of a BRAF fusion as a novel mechanism of acquired

120 resistance to vemurafenib in a patient with melanoma harboring a BRAFV600E mutation. The

121 acquisition and regression of clones harboring this fusion during the presence and absence of a

122 BRAF inhibitor are consistent with rapidly evolving clonal dynamics in melanoma.

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126

127 INTRODUCTION

128 BRAF V600E/K mutations are found in approximately half of malignant melanomas (1-3). These

129 activating mutations in the MAPK/ERK pathway drive tumor progression, survival, and

130 metastasis by promoting cell cycle progression, facilitating escape from apoptosis, and

131 abrogating immune destruction (4). The development of selective BRAF inhibitors, including

132 vemurafenib and dabrafenib, was a landmark in the treatment of melanoma and has led to

133 significant improvements in clinical response rate (RR), progression-free survival (PFS), and

134 overall survival (OS), compared with chemotherapy for patients with BRAF-mutant melanoma

135 (5,6). Although an initial reduction in tumor volume is observed in the majority of patients with

136 BRAF V600E mutant melanoma treated with BRAF inhibitors, clinical resistance associated with

137 progression of disease develops in most patients and limits the long-term utility of these agents.

138 This has led to the clinical investigation and approval of combination BRAF and MEK inhibitor

139 therapy (dabrafenib plus trametinib; vemurafenib plus combimetinib) that is associated with

140 superior RR, PFS, and OS compared with BRAF inhibitor monotherapy (7-9).

141

142 Several mechanisms of acquired resistance to vemurafenib have been identified and these

143 include secondary NRAS mutations (10), mutations in ARAF and CRAF (11), amplification of

144 BRAF (12), activation of other pro-survival signaling pathways including the phosphoinositide-3-

145 kinase (PI3K) pathway (11), and amplification of upstream receptor tyrosine kinases such as

146 MET (13). To date, over a dozen acquired mechanisms of resistance are described and, often,

147 multiple mechanisms of resistance are identified in the same patient (12,14,15). However, in

148 many cases no clear mechanism of acquired resistance to BRAF-inhibitor therapy is identified,

149 even with exome sequencing, although there often is evidence of reactivation of MAPK

150 signaling.

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152 BRAF fusion genes are an alternative mechanism to activate MAPK signaling that was initially

153 described in pediatric gliomas (16) and recently have been found in some melanomas that lack

154 BRAF V600E/K point mutations (17). We now report a case of malignant melanoma in which

155 comprehensive genomic profiling was used to identify the presence of a BRAF fusion in a

156 melanoma with a known BRAF V600E mutation at the time of progression on vemurafenib

157 therapy. This finding suggests that BRAF translocations may be a novel mechanism of acquired

158 resistance to BRAF inhibitors.

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178 MATERIALS AND METHODS

179 Study approval

180 Patient case 1 described in this report was enrolled in an institutional review board (IRB)-

181 approved investigational trial for tumor sequencing (protocol no. 2012002075) conducted at the

182 Rutgers Cancer Institute (CINJ) in New Brunswick, New Jersey, in accordance with the Belmont

183 Report. This patient provided written informed consent prior to study enrollment. Approval for

184 use of de-identified data for patient case 2, including a waiver of informed consent and a HIPAA

185 waiver of authorization, was obtained from the Western IRB (protocol no. 20152817), in

186 accordance with the U.S. Common Rule, by Foundation Medicine, Inc. (Cambridge, MA).

187

188 Tumor sequencing

189 Formalin-fixed tumor samples, and DNA extracted from peripheral blood specimens, were

190 analyzed by a comprehensive genomic profiling assay performed on indexed, adaptor-ligated,

191 hybridization captured libraries targeting all exons of 315 cancer-related genes (FoundationOne)

192 at a commercial CLIA-certified laboratory, Foundation Medicine.

193

194 Construct generation

195 cDNA encoding AGAP3-BRAF fusion gene was synthesized (Life Technologies) and placed in

196 Gateway entry vector. A V5-tagged expression clone (pcDNATM 3.2-V5-DEST) was then created

197 by performing an LR recombination reaction using gateway LR clonaseTM II enzyme mix

198 (Invitrogen Life technologies Cat. No.11791-020) according to the manufacturer’s protocol. The

199 expression of protein was verified by transient transfection of plasmid in 293T cells and Western

200 blot analysis probing with antibody against V5.

201

202 Creation of stable cell line and colony formation assay

203 UACC903 melanoma cells with original BRAF V600E mutations were transfected with either empty

204 vector or the V5-tagged AGAP3-BRAF fusion vector. Cells were then selected on Geneticin

205 (G418)-containing RPMI media. Expression of fusion protein was verified by Western blot. The

206 colony-forming ability of these cells expressing either the empty vector or the AGAP3-BRAF

207 fusion vector was evaluated by plating 300 cells/well in a six-well plate. The cells were allowed

208 to grow for 15 days. The colonies were then stained with crystal violet. To evaluate the effect of

209 BRAF inhibitor (PLX4720) or MEK inhibitor (AZD6244) on the colony-forming ability of cells

210 expressing the AGAP3-BRAF fusion protein, cells were plated in a six-well plate as previously

211 described. Cells were treated with different concentrations of drugs (0, 25nM, 50nM, 75nM,

212 100nM, and 200nM) and allowed to grow for 15 days supplemented with fresh media every 3

213 days. The number of colonies was counted after staining with crystal violet. The percent survival

214 was plotted compared to untreated cells from the data collected from three independent

215 experiments.

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217 Cell proliferation assay

218 To evaluate the effect of a BRAF inhibitor (vemurafenib) and a MEK inhibitor (trametinib) on the

219 proliferation of UACC903 cells harboring either a BRAF V600E mutation or the AGAP3-BRAF

220 fusion gene along with the BRAF V600E mutation, cells were seeded into 96-well plates (4000

221 cells/well). Cells were treated with the indicated concentrations of BRAF inhibitor (vemurafenib)

222 or MEK inhibitor (trametininb) and grown for another 4 days. The proliferation of cells was

223 measured by incubating them with CellTiter 96 Aqueous One Solution reagent (Promega) for 3

224 hours and measuring the absorbance at 490 nm. The values were plotted as an average of

225 three independent experiments.

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227 Western blot analysis

228 UACC903 cells expressing either empty vector or cells expressing the V5-tagged AGAP3-BRAF

229 fusion protein were treated overnight with BRAF inhibitor (1uM), MEK inhibitor (500nM), or the

230 combination of both drugs (500nM each). Protein extracts were prepared using NTEN buffer

231 (Tris pH 8.0, NaCl 150mM, EDTA 1mM, NP40 0.5% and protease and phosphatase inhibitors)

232 and resolved by SDS PAGE. Blots were probed with antibodies against V5 (Bethyl

233 Laboratories), phosphorylated-MEK (pMEK) and total MEK, phosphorylated-ERK (pERK) and

234 total ERK, and BRAF (Cell Signaling Technology).

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236 Cell mixing studies

237 To test if the UACC903 cells expressing the AGAP3-BRAF fusion protein have a survival

238 advantage over the parental UACC903 cells harboring the BRAF V600E mutation alone when

239 treated with the BRAF inhibitor, vemurafenib, the UACC903 parental cells harboring BRAF V600E

240 alone were transfected with pCDNA3-GFP empty vector. Green fluorescent protein (GFP)-

241 positive cells were sorted by fluorescence-activated cell sorting (FACS) (BD Biosciences Influx

242 high speed cell sorter) and grown in RPMI medium. UACC903 parental cells (GFP) and cells

243 expressing the AGAP3-BRAF fusion gene were mixed together in 10:1 ratio (2000:200)/ well in

244 a 12-well plate. The mixed population of cells was either untreated or treated with 500nM of

245 BRAF inhibitor (vemurafenib) for 7 days. The effect of BRAF inhibition on cell growth was then

246 evaluated by imaging for GFP-positive UACC903 parental cells and cells expressing the fusion

247 protein (by differential interference contrast imaging [DIC]) at 10X magnification using a Zeiss

248 Axiovert 200M inverted fluorescence microscope. Twenty images were taken for each

249 treatment. The percentage of GFP positive (UACC903 parental cells) and non-GFP cells

250 expressing the AGAP3-BRAF fusion protein (by DIC) were quantified before and after treatment

251 with vemurafenib and plotted.

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256 RESULTS

257 Patient case 1

258 A 53-year-old man was initially diagnosed 3 years previously with a 0.7 mm thick melanoma on

259 the anterior chest wall that was treated with wide surgical excision and clear margins. The

260 patient did well for 3 years and then presented with an enlarging left axillary mass. A PET-CT

261 scan showed the presence of a PET avid 4.4 cm left axillary mass, as well as PET-avid lesions

262 at T4 and the right ilium, consistent with bony metastatic disease. A fine-needle aspiration

263 biopsy of the T4 lesion was performed with pathologic confirmation of metastatic melanoma. A

264 hotspot sequencing assay showed the presence of BRAF V600E mutation.

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266 The patient was initially treated with the FDA-approved anti-cytotoxic T-lymphocyte associated

267 antigen 4 (CTLA4) antibody therapy, ipilimumab, given the overall low volume of disease;

268 however, he soon developed increasing, intractable back pain. Imaging studies revealed

269 progression of bony metastases, new liver metastases and increasing abdominal and axillary

270 lymphadenopathy (Fig 1A). Ipilimumab was discontinued; the patient received radiation therapy

271 to the spine with subsequent initiation of vemurafenib therapy. He experienced an initial

272 dramatic response to vemurafenib, with decreasing lymphadenopathy and responses in bony

273 and liver metastases (Fig 1A, B). A solitary brain lesion found on restaging was treated with

274 gamma knife therapy approximately 2 months following initiation of vemurafenib. The

275 extracranial disease response was maintained for nearly 4 months at which time the axillary

276 mass, as well as lesions in the liver and spleen, began to enlarge (Fig 1C).

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278 After informed consent, the patient was enrolled in the CINJ Precision Medicine tumor

279 sequencing protocol. The enlarging axillary mass was biopsied while the patient was still

280 receiving vemurafenib, and metastatic melanoma was confirmed on pathologic analysis. This

281 specimen was sent for comprehensive genomic profiling (FoundationOne, Foundation Medicine,

282 Cambridge, MA); which identified both a BRAF V600E mutation and a novel AGAP3-BRAF fusion

283 gene involving the joining of exons 1-9 of AGAP3 in frame to exons 9-18 of BRAF. Also present

284 were a VHL E52* nonsense mutation, CDKN2A/B loss, an alteration in STK11 (STK11 splice site

285 464+1G>C), an ARID1A truncating mutation (ARID1A Y560*) and a subclonal mutation in DNMT3A

286 (DNMT3A R736H) (Table 1). To determine if the AGAP3-BRAF fusion was detectable prior to

287 treatment with vemurafenib, a pre-treatment biopsy specimen was also sent for sequencing

288 using the same assay. The pre-treatment specimen had the BRAF V600E mutation but no BRAF

289 fusion. Identical alterations in VHL, and CDKN2A loss were also present in the pre-treatment

290 biopsy specimen. Intriguingly, the pre-treatment biopsy had focal deletion of STK11 but no

291 evidence of the splice site mutation. The DNMT3A R736H mutation was also present subclonally

292 in the pretreatment specimen (Table 1).

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294 These results were presented at the CINJ Tumor Board. Following panel discussion, it was

295 recommended that the patient be treated with combination BRAF inhibition and MEK inhibition,

296 based on prior disease progression on ipilimumab therapy and the fact that he was not a

297 candidate for any U.S.-based trials of nivolumab or pembrolizumab available at that time due to

298 his history of brain metastases. The patient was treated with the combination of dabrafenib and

299 trametinib and initially experienced marked clinical improvement with significant reduction of

300 right upper quadrant pain and overall improvement in quality of life. However, the patient

301 experienced increasing right-upper quadrant pain accompanied by an increase in the left axillary

302 mass within 2 months, and evidence of disease progression was observed on imaging.

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304 Dabrafenib and trametinib were then discontinued. The patient was subsequently treated with

305 anti-programmed death-1 (PD-1) antibody therapy (pembrolizumab) when it became available

306 as part of an expanded access protocol. However, he again had worsening disease in the same

307 left axillary tumor which became painful within several months of initiating pembrolizumab

308 therapy. The patient underwent palliative resection of this tumor and continued on anti-PD-1

309 therapy. Nevertheless, he soon exhibited clear evidence of disease progression at multiple

310 tumor sites and anti-PD-1 therapy was discontinued. Comprehensive genomic profiling was

311 again performed on the resected left axillary lymph node tumor which had progressed in the

312 absence of BRAF or MEK targeted therapy. Biopsy results from the resected tumor specimen

313 revealed the presence of the BRAF V600E mutation, but no evidence of the AGAP3-BRAF

314 rearrangement (Table 1). Of note, since the region of intron 8 of BRAF harboring the

315 breakpoint of the gene rearrangement was sequenced to >125x depth in all samples (Table 1),

316 the presence of the fusion should have been detected even if present at low frequencies.

317 CDKN2A loss and the VHL E52* and STK11 splice site mutations seen in prior axillary lymph

318 node biopsy specimen were also present.

319

320 Following review of these results at the CINJ Molecular Tumor Board, re-initiation of the

321 combination of dabrafenib and trametinib was recommended. Unfortunately, the patient had

322 progression of brain metastases that required treatment with whole brain irradiation, delaying

323 initiation of this treatment. Therapy with a BRAF inhibitor alone was ultimately initiated, resulting

324 in a mixed response characterized by shrinkage of liver metastases but clear evidence of

325 disease progression at other sites.

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327 AGAP3-BRAF fusion

328 Analysis of the junction sequence of the AGAP3-BRAF fusion showed that intron 8 of BRAF

329 was rearranged to intron 9 of AGAP3, located telomeric to BRAF on chromosome 7, to create

330 an in-frame fusion gene that encodes for a protein containing exons 1-9 of AGAP3 and exons 9-

331 18 of BRAF. This fusion protein includes the full kinase domain of BRAF but lacks the Ras-

332 binding domain and the cysteine-rich domain of the BRAF gene (18). A similar AGAP3-BRAF

333 fusion has been reported in melanoma and colon cancer, but has not been functionally

334 characterized (19). These junction sequences are consistent with a balanced inversion event in

335 chromosome 7 (Fig 2 A,B).

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337 AGAP3 (ArfGAP With GTPase domain, ankyrin repeat and PH domain 3) is a gene encoding for

338 a protein with an N-terminal region containing tandem atypical GTPase domains and paired EF

339 Hands (together comprising a Miro domain), a PH domain, a classical ArfGAP domain, and

340 tandem ankyrin repeats (Fig 2B) (20). The fusion protein includes amino acids 1-407 of AGAP3

341 fused to amino acids 381-766 of BRAF. This fusion protein would include the N-terminal Miro

342 domain and part of the PH domain of AGAP3 fused to the kinase domain of BRAF (Fig 2B).

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344 Functional characterization of AGAP3-BRAF fusion protein

345 The melanoma cell line UACC903 with a known BRAF V600E mutation was stably transfected with

346 either vector alone or vector expressing the AGAP3-BRAF fusion. Expression of the fusion

347 protein was confirmed by Western blotting (Figure 2C). Compared with UACC903 cells

348 expressing empty vector, cells expressing the AGAP3-BRAF fusion gene had similar basal

349 levels of phosphorylated-MEK, but increased basal levels of phosphorylated-ERK (Fig 2C).

350 Treatment with a BRAF inhibitor, PLX4720, for 6 hours abolished MEK and ERK

351 phosphorylation in UACC903 parental cells, but not in UACC903 cells expressing the AGAP3-

352 BRAF fusion gene. Treatment with a MEK inhibitor, AZD6244, for 6 hours was able to

353 significantly reduce MEK and ERK phosphorylation in both parental and UACC903 cells

354 expressing the AGAP3-BRAF fusion gene (Fig 2C). Colony formation assays demonstrated

355 that cells harboring the AGAP3-BRAF fusion, unlike parental UACC903 cells harboring the

356 BRAF V600E mutation alone, were relatively resistant to treatment with a BRAF inhibitor.

357 However, the UACC903 parental cells harboring the BRAF V600E mutation alone, and the cells

358 also expressing the AGAP3-BRAF fusion were similarly sensitive to treatment with a MEK

359 inhibitor (Fig 2D).

360 To complement the colony formation assay, a cell proliferation assay was also performed, in this

361 case using the clinically relevant MEK inhibitor trametinib. In this assay, UACC903 cells

362 harboring both the BRAF V600E mutation and the AGAP3-BRAF fusion gene were significantly

363 less sensitive to BRAF inhibition than parental UACC903 cells (Figure 2E). Both the UACC903

364 parental as well as cells expressing the fusion gene were sensitive to MEK inhibitor (trametinib),

365 however, at higher concentrations (500nM and 1000nM) the cells harboring both the BRAF

366 V600E mutation and the AGAP3-BRAF fusion gene were less sensitive to MEK inhibition

367 compared with parental cells (Fig 2E), Although this relative resistance was seen only at high

368 trametinib concentrations, it suggests that the presence of the BRAF fusion may also induce

369 some partial resistance to MEK inhibition.

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371 Cells expressing the AGAP3-BRAF fusion gene have a growth advantage in the presence

372 of BRAF inhibitor

373 Mixing studies to evaluate the relative growth advantage that the AGAP-BRAF fusion may

374 impart to melanoma cells already harboring a BRAF V600E mutation showed that, in the setting of

375 treatment with vehicle alone, around 90% of the cells were the parental UACC903 cells,

376 retaining the original ratio of mixed cells (Figure 3A). However, when cells were treated with

377 vemurafenib for 7 days, the percentage of GFP-positive cells decreased dramatically, to < 20%

378 of cells. This result demonstrates that, in the absence of a BRAF inhibitor, cells harboring both

379 BRAF V600E and the AGAP3-BRAF fusion gene do not have a significant growth advantage.

380 However, treatment with a BRAF inhibitor resulted in a relative dominance of cells harboring the

381 AGAP3-BRAF fusion. These data suggest that cells harboring both BRAF V600E mutation and the

382 AGAP3-BRAF fusion gene are only more fit than parental cells harboring the BRAF V600E

383 mutation alone when in the presence of BRAF inhibitor treatment.

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386 Patient case 2

387 In addition to the index case, we report on an additional patient case demonstrating the co-

388 occurrence of a BRAF rearrangement with a BRAF V600E mutation in the setting of acquired

389 resistance to vemurafenib and/or MEK inhibitors. Patient case 2 is a 48-year-old man with

390 metastatic melanoma harboring a BRAF V600E mutation who developed progressive disease

391 following treatment with the combination of dabrafenib and trametinib. DNA sequencing of a

392 biopsy of a progressive lesion showed the resistant tumor to harbor both BRAF V600E and a

393 CSTF3-BRAF fusion gene that retained the full kinase domain of BRAF.

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412 DISCUSSION

413 Standard testing for BRAF mutations is usually undertaken by amplicon-based DNA sequencing

414 to identify hotspot BRAF mutations (21). These assays, however, will not detect BRAF fusions,

415 as the coding sequences of individual exons are not altered. Similarly, BRAF fusions are not

416 detectable with standard whole exome sequencing, as the fusion junctions are located in the

417 introns. One approach to identifying rearrangements is through hybrid capture of “hotspot

418 introns”. High-depth sequencing of select introns not only sensitively detect rearrangements but

419 analysis of the junction sequence can identify the partner genes involved. As intron 8 of BRAF is

420 captured in the FoundationOne assay, it can identify novel rearrangements of BRAF involving

421 this region. Other targeted approaches to identify fusion proteins include target enrichment

422 based RNA-seq analysis, where target enrichment can be accomplished by hybrid capture,

423 single primer-extension technology, or the recently described anchored multiplex polymerase

424 chain reaction (PCR) approach (22,23).

425

426 BRAF fusion proteins were initially reported and characterized in pediatric pilocytic

427 astrocytomas (16). The majority of pilocytic astrocytomas harbor translocations which result in

428 an in-frame fusion of the N-terminal exons of KIAA1549 to exons 9-18 of BRAF, leading to a

429 fusion protein that contains the intact wild-type kinase domain of BRAF. The N-terminal region

430 of KIAA1549 is thought to mediate constitutive dimerization of the fusion protein and thus

431 activation of BRAF kinase activity. Introduction of a first-generation BRAF inhibitor, such as

432 vemurafenib, causes paradoxical activation of this fusion protein and increased MEK

433 phosphorylation (24,25). Cells harboring BRAF fusions respond to vemurafenib in a manner

434 similar to cells harboring RAS mutations, through downstream MAPK pathway activation. As

435 such, vemurafenib should be avoided in these settings; however, either MEK inhibitors or

436 second-generation “paradox-breaking” BRAF inhibitors may be effective in the setting of BRAF

437 fusions.

438

439 BRAF fusions have been reported as primary driver mutations in a subset of melanomas without

440 a BRAF V600E mutation (26) and have been shown to be associated with resistance to BRAF

441 inhibitors (19,26). Here, we report an AGAP3-BRAF fusion that was detected in a melanoma

442 with a known BRAF V600E mutation at the time of development of resistance to vemurafenib. The

443 presence of the AGAP3-BRAF fusion was not detected in pre-treatment samples, consistent

444 with this fusion protein representing the mechanism of resistance to vermurafenib. Of note, it

445 was expected that the AGAP3-BRAF fusion harbors a wild-type kinase domain, as seen in other

446 reported cases of BRAF fusion genes, including the prior report of an AGAP3-BRAF fusion.

447 This was confirmed by laboratory studies that demonstrated that the AGAP3-BRAF fusion

448 induces resistance to vemurafenib in cells with pre-existing BRAF V600E mutation, as compared

449 with cells lines harboring a BRAF V600E mutation. Cells harboring both BRAF V600E and AGAP3-

450 BRAF fusion remained sensitive to a MEK inhibitor, suggesting that MEK inhibition can

451 overcome this mechanism of resistance (27,28). Of note, cells harboring both BRAF V600E and

452 AGAP3-BRAF fusion did show some partial resistance to trametinib at high doses in

453 proliferation assays (Figure 2E), suggesting that BRAF fusion genes may also impart some

454 resistance to MEK inhibitors. The case showing development of a CSTF3-BRAF fusion in the

455 setting of acquired resistance to the combination of dabrafenib and trametinib also suggests that

456 some BRAF fusion genes may impart clinical resistance to MEK inhibitors as well. The

457 relatively transient response in the index case also supports this possibility.

458

459 These findings suggest that cells harboring both BRAF V600E and AGAP3-BRAF fusion were

460 present at a very low frequency in the pre-treatment setting, and could not be detected even at

461 relatively high sequencing depth. Treatment with vemurafenib induced selection pressure that

462 allowed preferential growth of cells harboring the BRAF fusion gene, making them the dominant

463 clone and driving tumor resistance and progression. The resistant tumor also had a mutation in

464 ARID1A that was not detected in the pre-treatment biopsy; it is not clear if this mutation plays

465 any role in vemurafenib resistance, especially as the mutant allele frequency was relatively low.

466 Of note, the pre-treatment biopsy had evidence of STK11 deletion; however, neither of the two

467 later biopsies showed evidence of STK11 deletion, but instead had STK11 splice mutations that

468 should lead to loss of function. This finding is consistent with clonal heterogeneity in this patient

469 with a large tumor burden, with convergent evolution for STK11 loss in both clones. The

470 presence of identical BRAF and VHL mutations and CDKN2A deletion in all samples is

471 consistent with evolution from a common ancestral clone.

472

473 Interestingly, the same tumor site noted to have the AGAP3-BRAF fusion and BRAF V600E

474 mutation later progressed after 3 months on anti-PD-1-antibody therapy, during which there was

475 no treatment with either a BRAF or a MEK inhibitor. When this site was excised for palliation,

476 sequencing revealed only the presence of the BRAF V600E mutation, and no evidence of AGAP3-

477 BRAF fusion despite the fact that the region of intron 8 of BRAF that harbored the breakpoint

478 was sequenced to greater than 125x depth. This finding suggests that the clone harboring both

479 the BRAF V600E mutation and the AGAP3-BRAF fusion may have been more fit than clones

480 harboring the BRAF V600E mutation alone only in the setting of ongoing treatment with

481 vemurafenib. However, when selection pressure from BRAF and MEK inhibition was not

482 present, clones harboring BRAF V600E alone may have had a fitness advantage, thereby

483 dominating the recurrent mass. This hypothesis is supported by the reported data from mixing

484 studies showing relative outgrowth of cells harboring both BRAF V600E and AGAP3-BRAF fusion

485 only in setting of BRAF inhibitor treatment (Figure 3A).

486

487 There may be very rapid clonal dynamics under selection for the BRAF inhibitors in malignant

488 melanoma, and dominant clonal populations may rapidly shift during tumor progression when

489 drugs are discontinued (Fig 3B). Similar rapid clonal dynamics have been reported in colon

490 cancer, with transient emergence of clones harboring KRAS mutations emerging during

491 development of cetuximab resistance followed by their regression when cetuxmab is

492 discontinued (29). Re-challenge with cetuximab at the time point when KRAS mutant clones

493 were undetectable led to clinical response (29).

494

495 Due to the possibility of a mixed response, the biopsy showing the presence of the AGAP3-

496 BRAF fusion was obtained when the patient was still receiving vemurafenib therapy. If

497 vemurafenib had been completely discontinued before the biopsy was performed, this

498 mechanism of resistance may not have been detected, as the clone with BRAF V600 alone could

499 have rebounded rapidly. Of note, this patient did experience some clinical benefit, although brief

500 in duration, upon rechallenge with a BRAF inhibitor after progression on immunotherapy. This

501 result suggests that rechallenge with BRAF/MEK inhibitors may be of benefit in the setting of

502 interval progression of kinase inhibitor therapy, as has been reported by others (30-32).

503

504 This is the first report of a BRAF translocation as the mechanism of acquired resistance to

505 vemurafenib in a melanoma with a pre-existing BRAF V600E mutation. It is possible that such

506 BRAF rearrangements may represent an underappreciated mechanism for acquired resistance

507 to BRAF inhibitors. It is hard to estimate the frequency of such events, as post-resistance

508 biopsies using assays that can identify fusion genes are not routinely performed. Given the

509 potential for rapid clonal dynamics, the timing of biopsy of progressive disease to look for

510 mechanisms of resistance may be critical, as there may be rapid rebound of parental clones

511 following discontinuation of kinase inhibitor therapy. Serial biopsies, or serial use of circulating

512 cell-free tumor DNA assays, may identify clinical settings where re-challenge with BRAF

513 inhibitors after progression off therapy may be effective.

514

515

516 ACKNOWLEDGEMENTS

517 This research was supported by the Functional Genomics and Biospecimen Repository Shared

518 Resource(s) of the Rutgers Cancer Institute of New Jersey (P30CA072720) and a generous gift

519 to the Genetics Diagnostics to Cancer Treatment Program of the Rutgers Cancer Institute of

520 New Jersey and RUCDR Infinite Biologics. Funding for this research was also provided by

521 Merck & Co., Inc., Kenilworth, NJ 07033, USA, the Val Skinner Foundation, and Hugs for Brady

522 Foundation. This work is part of the Precision Medicine Initiative at the Rutgers Cancer Institute

523 of New Jersey.

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540

541 REFERENCES

542 1. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF

543 gene in human cancer. Nature 2002;417:949-54.

544

545 2. Hodis E, Watson IR, Kryukov GV, Arold ST,Imielinski M, Theurillat JP et al. A landscape of

546 driver mutations in melanoma. Cell 2012;150:251-63.

547

548 3. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al: Survival in

549 BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med 2012;

550 366:707-14.

551

552 4. Sullivan RJ, Flaherty K: MAP kinase signaling and inhibition in melanoma. Oncogene

553 2013;32:2373-9.

554

555 5. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved

556 survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med

557 2011;364:2507-16.

558

559 6. Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in

560 BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled

561 trial. Lancet 2012; 380:358-65.

562

563 7. Larkin J, Ascierto PA, Dreno B, Atkinson V, Liszkay G, Maio M, et al. Combined vemurafenib

564 and cobimetinib in BRAF-mutated melanoma. N Engl J Med 2014; 371:1867-76.

565

566 8. Long GV, Stroyakovskiy D, Gogas H, Levchenko E, de Braud F, Larkin J, et al. Combined

567 BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N Engl J Med 2014;

568 371:1877-88.

569

570 9. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, et al.

571 Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med

572 2015;372:30-9.

573

574 10. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, et al. Melanomas acquire resistance

575 to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010;468:973-77.

576

577 11. Holderfield M, Deuker MM, McCormick F, McMahon M. Targeting RAF kinases for cancer

578 therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer 2014;14:455-67.

579

580 12. Shi H, Hugo W, Kong X, Hong A, Koya RC, Moriceau G, et al. Acquired resistance and

581 clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov 2014;4:80-93.

582

583 13. Vergani E, Vallacchi V, Frigerio S, Deho P, Mondellini P, Perego P, et al. Identification of

584 MET and SRC activation in melanoma cell lines showing primary resistance to PLX4032.

585 Neoplasia 2011;13:1132-42.

586

587 14. Rizos H, Menzies AM, Pupo GM, Carlino MS, Fung C, Hyman J, et al. BRAF inhibitor

588 resistance mechanisms in metastatic melanoma: spectrum and clinical impact. Clin Cancer Res

589 2014;20:1965-77.

590

591 15. Van Allen EM, Wagle N, Sucker A, Treacy DJ, Johannessen CM, Goetz EM,, et al. The

592 genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma. Cancer

593 Discov 2014;4:94-109.

594

595 16. Jones DT, Kocialkowski S, Liu L, Pearson DM, Bäcklund LM, Ichimura K, Collins VP.

596 Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of

597 pilocytic astrocytomas. Cancer Res 2008;68:8673-7.

598

599 17. Hutchinson KE, Lipson D, Stephens PJ, Otto G, Lehmann BD, Lyle PL, et al. BRAF fusions

600 define a distinct molecular subset of melanomas with potential sensitivity to MEK inhibition. Clin

601 Cancer Res 2013;19:6696-702.

602

603 18. Lavoie H, Therrien M. Regulation of RAF protein kinases in ERK signalling.

604 Nat Rev Mol Cell Biol 2015;16:281-98.

605

606 19. Ross JS, Wang K, Chmielecki J, Gay L, Johnson A, Chudnovsky J, et al. The distribution of

607 BRAF gene fusions in solid tumors and response to targeted therapy. Int. J Cancer 2016;

608 138:881-90.

609

610 20.Oku Y, Huganir RL. AGAP3 and Arf6 regulate trafficking of AMPA receptors and synaptic

611 plasticity. J Neurosci 2013;33:12586-98.

612

613 21. Greaves WO, Verma S, Patel KP, Davies MA, Barkoh BA, Galbincea JM, et al. Frequency

614 and spectrum of BRAF mutations in a retrospective, single-institution study of 1112 cases of

615 melanoma. J Mol Diagn 2013;15:220-6.

616

617 22. Scolnick JA, Dimon M, Wang IC, Huelga SC, Amorese DA. An efficient method for

618 Identifying gene fusions by targeted RNA sequencing from fresh frozen and FFPE samples.

619 PLoS One 2015;10:e0128916.

620

621 23. Zheng Z, Liebers M, Zhelyazkova B, Cao Y, Panditi D, Lynch KD, ,et al. Anchored multiplex

622 PCR for targeted next-generation sequencing. Nat Med 2014;20:1479-84.

623

624 24. Sievert AJ, Lang SS, Boucher KL, Madsen PJ, Slaunwhite E, Choudhari N, et al.

625 Paradoxical activation and RAF inhibitor resistance of BRAF protein kinase fusions

626 characterizing pediatric astrocytomas. Proc Natl Acad Sci U S A 2013;110:5957-62.

627

628 25. Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF

629 dimers and ERK signalling in cells with wild-type BRAF. Nature 2010;464:427–30.

630

631 26. Kim HS, Jung M, Kang HN, Kim H, Park C-W, Kim SM, et al. Oncogenic BRAF fusions in

632 mucosal melanomas activate the MAPK pathway and are sensitive to MEK/PI3K inhibition or

633 MEK/CDK4/6 inhibition. Oncogene 2017 Jan 16 [Epub ahead of print].

634

635 27. Johnson DB, Flaherty KT, Weber JS, Infante JR, Kim KB, Kefford RF, et al. Combined

636 BRAF (dabrafenib) and MEK inhibition (trametinib) in patients with BRAFV600-mutant

637 melanoma experiencing progression with single-agent BRAF inhibitor. J Clin Oncol 2014;32

638 :3697-704.

639

640 28. Ribas A, Gonzalez R, Pavlick A, Hamid O, Gajewski TF, Duad A, et al. Combination of

641 vemurafenib and cobimetinib in patients with advanced BRAFV600-mutated melanoma: a

642 phase 1b study. Lancet Oncol 2014;15:954-65.

643

644 29. Siravegna G, Mussolin B, Buscarino M, Corti G, Cassingena A, Crisafulli G, et al. Clonal

645 evolution and resistance to EGFR blockade in the blood of colorectal cancer patients. Nat Med

646 2015;21:827.

647

648 30. Romano E, Pradervand S, Paillusson A, Weber J, Harshman K, Muehlethaler K, et al.

649 Identification of multiple mechanisms of resistance to vemurafenib in a patient with

650 BRAFV600E-mutated cutaneous melanoma successfully rechallenged after progression. Clin

651 Cancer Res 2013;19:5749-57.

652

653 31. Roux J, Pages C, Malouf D, Basset Seguin N, Madjlessi N, Baccard M, et al. BRAF inhibitor

654 rechallenge in patients with advanced BRAF V600-mutant melanoma. Melanoma Res

655 2015;25:559-63.

656

657 32. Seghers AC, Wilgenhof S, Lebbé C, Neyns B. Successful rechallenge in two patients with

658 BRAF-V600-mutant melanoma who experienced previous progression during treatment with a

659 selective BRAF inhibitor. Melanoma Res 2012;22:466-72.

660

661

662

663

664

665

666

667

668

669 TABLES

670 Table 1: Next-generation DNA sequencing results on serial biopsy specimens

Pre- Axillary Axillary Lymph Treatment Lymph Node Node Biopsy Biopsy Biopsy (Bone) (progressing on (progressing immunotherapy, on off kinase vemurafenib) inhibitors for >3 months) Computational purity estimate 40% 57% 65% BRAF V600E MAF (depth) 30% (685) 54% (819) 51% (208) VHL E52* MAF (depth) 33% (537) 22% (567) 15% (123) ARID1A Y560* (depth) 0% (670) 8% (676) 0% (197) DNMT3A R736H (depth) 1% (561) 2% (642) 0% (132) STK11 splice site 464+1G>C 0% (229) 45% (348) 36% (78) STK11 copy number alteration loss None None CDKN2A/B copy number loss loss loss alteration AGAP3-BRAF fusion absent (0 present with 23 absent (0 supporting supporting supporting reads reads in chimeric read in manual manual pairs confirmation) confirmation) BRAF intron 8 average 546.57 568.21 125.46 coverage 671

672

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681

682 FIGURE LEGENDS

683

684 Figure 1 Representative CT images showing left axillary metastasis and liver metastases

685 A) Prior to vemurafenib treatment B), after 2 months of vemurafenib therapy and C), following

686 development of disease progression in the left axilla on vemurafenib

687

688 Figure 2 Structural and functional characterization of AGAP3-BRAF fusion

689 A) Illustration of BRAF-AGAP3 rearrangement event resulting from an inversion in chromosome

690 7. B) Representation of BRAF and AGAP3 genes and key domains. RBD: Ras binding domain.

691 CR: Cysteine–rich domain. Miro: Mitochondrial Rho protein domain, PH: Pleckstrin homology

692 domain. C) UACC903 cells harboring BRAF V600E expressing either empty vector or V5-tagged

693 AGAP3-BRAF fusion were either untreated or treated with PLX4720 (BRAF inhibitor), AZD6244

694 (MEK inhibitor), or both agents. Cell extracts were processed for Western blotting and probed

695 with the indicated antibodies. D) Parental UACC903 cells and UACC903 cells with stable

696 expression of V5-AGAP3-BRAF were treated with increasing concentrations of BRAF inhibitor

697 (PLX4720) left panel or MEK inhibitor (AZD6244), right panel and colony formation is plotted.

698 E) Parental UACC903 cells and UACC903 cells with stable expression of V5-AGAP3-BRAF

699 were treated with increasing concentrations of BRAF inhibitor (vemurafenib), left panel, or MEK

700 inhibitor (trametinib), right panel, for 4 days and the cell proliferation was measured with Celltiter

701 96 Aqueous One Solution reagent (Promega) and percent cell proliferation is plotted as an

702 average of three independent experiments.

703

704 Figure 3 Mixing studies and proposed rapid clonal dynamics model 705 706 A) UACC903 parental cells stably expressing empty vector GFP and UACC903 cells expressing

707 fusion protein AGAP3-BRAF were mixed in 10:1 ratio and treated with a BRAF inhibitor

708 (vemurafenib) for 7 days. Images were taken for three independent experiments and cell

709 number for each cell type was counted and plotted as percentage of total population. (Yellow

710 arrows indicate UACC903 cells expressing AGAP3-BRAF fusion protein.) B) Depiction of

711 proposed rapid clonal dynamics model. Clones harboring both BRAF V600E and AGAP3-BRAF

712 fusion are more fit than clones harboring BRAF V600E without the AGAP3-BRAF fusion only in the

713 presence of vemurafenib, and expand under vemurafenib treatment, leading to tumor

714 progression. However, when BRAF kinase inhibitor treatment is withdrawn, clones harboring

715 BRAF V600E without the AGAP3-BRAF fusion are now more fit, and rapidly dominate the growing

716 tumor mass.

717

718

BRAF fusion as a novel mechanism of acquired resistance to vemurafenib in BRAF V600E mutant melanoma

Atul Kulkarni, Husam Al-Hraishawi, Srilatha Simhadri, et al.

Clin Cancer Res Published OnlineFirst May 24, 2017.

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