Preexisting MEK1 Exon 3 Mutations in V600E/KBRAF Melanomas Do Not Confer Resistance to BRAF...

Preexisting MEK1 Exon 3 Mutations in V600E/KBRAF Melanomas DoNot Confer Resistance to BRAF Inhibitors

Hubing Shi1, Gatien Moriceau1, Xiangju Kong1, Richard C. Koya3, Ramin Nazarian1,Gulietta M. Pupo9, Antonella Bacchiocchi6, Kimberly B. Dahlman7, Bartosz Chmielowski2,Jeffrey A. Sosman8, Ruth Halaban6, Richard F. Kefford9, Georgina V. Long9, AntoniRibas2,3,4,5, and Roger S. Lo1,4,5

1Division of Dermatology, David Geffen School of Medicine, University of California, Los Angeles,California2Division of Hematology and Oncology, Department of Medicine, David Geffen School ofMedicine, University of California, Los Angeles, California3Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine,University of California, Los Angeles, California4Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University ofCalifornia, Los Angeles, California5Department of Molecular and Medical Pharmacology, David Geffen School of Medicine,University of California, Los Angeles, California6Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut7Departments of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee8Department of Medicine, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee9Melanoma Institute of Australia, Westmead Millennium Institute, Westmead Hospital, Universityof Sydney, New South Wales, Australia

© American Association for Cancer Research

Corresponding Author: Roger S. Lo, 52-121 CHS Department of Medicine/Dermatology, 10833 Le Conte Avenue, Los Angeles, CA90095-1750. Phone: 310-794-6608; Fax: 310-206-9878; [email protected]. Shi and G. Moriceau contributed equally to this work.

Authors’ ContributionsConception and design: H. Shi, G. Moriceau, X. Kong, B. Chmielowski, A. Ribas, R.S. LoDevelopment of methodology: H. Shi, G. Moriceau, X. Kong, R.C. Koya, R. Nazarian, B. Chmielowski, R.S. LoAcquisition of data: H. Shi, G. Moriceau, X. Kong, R.C. Koya, R. Nazarian, G.M. Pupo, K.B. Dahlman, J.A. Sosman, R. Halaban,R.F. Kefford, G.V. Long, A. Ribas, R.S. LoAnalysis and interpretation of data: H. Shi, G. Moriceau, X. Kong, R. Nazarian, R. Halaban, R.F. Kefford, G.V. Long, A. Ribas,R.S. LoWriting, review, and/or revision of the manuscript: R.C. Koya, G.M. Pupo, K.B. Dahlman, B. Chmielowski, J.A. Sosman, R.F.Kefford, G.V. Long, A. Ribas, R.S. LoAdministrative, technical, or material support: X. Kong, R.C. Koya, G.M. Pupo, K.B. Dahlman, G.V. Long, R.S. LoStudy supervision: H. Shi, X. Kong, G.V. Long, A. Ribas, R.S. LoCell lines establishment: H. Shi, A. Bacchiocchi

Note: Supplementary data for this article are available at Cancer Discovery Online (http://www.cancerdiscovery.aacrjournals.org).

Disclosure of Potential Conflicts of InterestA. Ribas and R.S. Lo declare patent application under PCT application serial no. PCT/US11/061552 (Compositions and Methods forDetection and Treatment of BRAF Inhibitor-Resistant Melanomas). K.B. Dahlman, B. Chmielowski, J.A. Sosman, R.F. Kefford, G.V.Long, and A. Ribas have received honoraria from or served as consultants to pharmaceutical firms (Roche-Genentech,GlaxoSmithKline, Illumina). No potential conflicts of interest were disclosed by the other authors.

NIH Public AccessAuthor ManuscriptCancer Discov. Author manuscript; available in PMC 2013 May 01.

Published in final edited form as:Cancer Discov. 2012 May ; 2(5): 414–424. doi:10.1158/2159-8290.CD-12-0022.

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http://www.cancerdiscovery.aacrjournals.org

AbstractBRAF inhibitors (BRAFi) induce antitumor responses in nearly 60% of patients withadvanced V600E/KBRAF melanomas. Somatic activating MEK1 mutations are thought to be rare inmelanomas, but their potential concurrence with V600E/KBRAF may be selected for by BRAFi. Wesequenced MEK1/2 exon 3 in melanomas at baseline and upon disease progression. Of 31baseline V600E/KBRAF melanomas, 5 (16%) carried concurrent somatic BRAF/MEK1 activatingmutations. Three of 5 patients with BRAF/MEK1 double-mutant baseline melanomas showedobjective tumor responses, consistent with the overall 60% frequency. No MEK1 mutation wasfound in disease progression melanomas, except when it was already identified at baseline.MEK1-mutant expression in V600E/KBRAF melanoma cell lines resulted in no significantalterations in p-ERK1/2 levels or growth-inhibitory sensitivities to BRAFi, MEK1/2 inhibitor(MEKi), or their combination. Thus, activating MEK1 exon 3 mutations identified herein andconcurrent with V600E/KBRAF do not cause BRAFi resistance in melanoma.

SIGNIFICANCE—As BRAF inhibitors gain widespread use for treatment of advancedmelanoma, bio-markers for drug sensitivity or resistance are urgently needed. We identify hereconcurrent activating mutations in BRAF and MEK1 in melanomas and show that the presence ofa downstream mutation in MEK1 does not necessarily make BRAF–mutant melanomas resistantto BRAF inhibitors.

INTRODUCTIONThe majority of melanomas harbor an activating mutation in the RAS/RAF/mitogen-activated protein kinase (MAPK) signaling pathway. V600E/KBRAF mutations are found in>50% of melanomas (1), and their “druggability” in humans has been shown using the novelsmall-molecule BRAF inhibitors (BRAFi) PLX4032/vemurafenib and GSK2118436/dabrafenib (2-5). However, acquired resistance to BRAFi is common, and proposedmechanisms include upregulation of MAPK-redundant signaling (via receptor tyrosinekinase overexpression and AKT activation) and MAPK reactivation [via N-RAS mutations,COT expression, V600EBRAF alternative splicing, and V600EBRAF amplification (6-11)]. Amissense MEK1 somatic activating mutation (C121S) in exon 3 has recently been proposedto account for acquired BRAFi resistance in one patient (12). Germline mis-sense MEK1/2mutations have been found in patients with the developmental disorder known as cardio-facio-cutaneous syndrome (13). However, somatic activating MEK1/2 mutations are thoughtto be exceedingly rare among human malignancies (14, 15).

To assess the potential role of MEK1/2 exon 3 mutations in primary (innate) or secondary(acquired) drug resistance to BRAFi therapy, we analyzed samples from 31 patients treatedwith either vemurafenib or dabrafenib from whom there was available baseline (prior toBRAFi treatment) or patient-matched baseline and disease progression [DP (i.e., acquiredBRAFi–resistant)] tissues. Contrary to the expectation, somatic MEK1 exon 3 mutations(P124SMEK1 and I111SMEK1) were found in baseline melanoma tumors concurrentwith V600E/KBRAF mutations. Importantly, the pattern of MEK1 exon 3 mutations could notaccount for either innate or acquired BRAFi resistance. Functional studies using BRAF/MEK1 double-mutant melanoma cell lines show that P124SMEK1 and I111SMEK1 do notdetermine BRAFi sensitivity. This clinical series thus offers important insight into the tumorresponse pattern of BRAF/MEK1 double-mutant melanomas to novel BRAF inhibitors.

RESULTSAmong 31 patients with BRAF–mutant melanomas treated with either the BRAFi,PLX4032/vemurafenib or GSK2118436/dabrafenib, 5 MEK1 exon 3 mutations (P124S in 4,I111S in 1) were detected in baseline melanoma tumors of 5 distinct patients (Table 1). In 4

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of these 5 patients with available normal tissue-derived genomic DNA (gDNA), theseMEK1 mutations were determined to be somatic (Supplementary Fig. S1). No MEK2 exon 3mutation was detected in any baseline melanoma tumor (Table 1). Among 18 of thesepatients whose BRAFi acquired-resistant (DP) tumors were available, MEK1 exon 3mutation was detected in only those patients with preexisting BRAF/MEK1, double-mutantbaseline melanomas and none of the other 23 DP tumors [several patients have multiple DPtissue biopsies (Table 1)]. MEK1 exon 3 mutant alleles were detected in 9 tumor tissues(both baseline and DP) at a ratio of 1:1 to wild-type (WT) MEK1; no homozygous MEK1mutation was detected (Supplementary Fig. S1), suggesting counterselection against MEK1-mutant homozygosity. MEK1 mutations concurred with V600EBRAF in 2 patientsand V600KBRAF in 3 patients (Supplementary Fig. S1).

Four of 5 patients with BRAF/MEK1 double-mutant baseline melanomas displayedobjective responses in the tumors biopsied, and 3 of 5 patients achieved overall partialresponse (by Response Evaluation Criteria in Solid Tumors 1.1) to BRAF inhibition (Table1 and Fig. 1A–C). Both patients who did not achieve objective partial response experiencedclinical response (Table 1 and Supplementary Fig. S2). The mean progression-free survivaland best overall tumor response were not significantly different between BRAF single-mutant versus BRAF/MEK1 double-mutant melanomas (Table 1): mean progression-freesurvival for patients with WT MEK1 melanomas = 182.4 days (SD 84.7) versus mutantMEK1 melanomas = 114 days (SD 25.1), 2-tailed P = 0.09; mean best overall response forpatients with WT MEK1 melanomas = −50% (SD 20.0) versus mutant MEK1 melanomas =41.6 (SD 37.8), 2-tailed P = 0.45. Thus, we have uncovered a concurrence of MEK1 andBRAF mutations in metastatic melanomas prior to the onset of BRAFi selective pressure.The pattern of MEK1 exon 3 mutations and clinical responses could not account for eitherinnate or acquired BRAFi resistance, warranting in vitro validation.

We introduced MEK1 WT and MEK1 P124S into 2 V600EBRAF (Fig. 2) and 1 V600KBRAF(Supplementary Fig. S3) human melanoma cell lines using a lentiviral expression vectorwith a doxycycline-repressible promoter. MEK1 WT or P124S-regulated expression wasachieved with 10 ng/mL (no expression), 0.1 ng/mL (low expression, mimicking a 1:1endogenous vs. exogenous MEK1 expression), and 0 ng/mL (high expression, artificiallymaximizing any observed effect) of doxycycline in the culture media (Fig. 2A). Expressionof exogenous MEK1 WT or P124S was confirmed by a FLAG epitope tag Western blot 2days after doxycycline removal. Additionally, the relative expression of endogenous MEK1versus total levels after exogenous MEK1 WT or P124S mutant induction was shown byMEK1-specific Western blotting. Interestingly, regulated expression of MEK1 WT orP124S at “physiologic” or high levels did not alter the downstream p-ERK levels (Fig. 2A),suggesting that V600EBRAF is dominant over MEK1 WT or P124S mutant in regulatingcellular p-ERK levels. Because 3 of 5 patients’ melanomas harbored MEK1 exon 3mutations concurrent with the V600KBRAF allele, we also showed that MEK1 P124Scoexpression in the V600KBRAF melanoma cell line, YULAC, had no appreciable impact oncellular p-ERK levels and sensitivities to BRAFi or MEK1/2 inhibitor [MEKi(Supplementary Fig. S3A and S3B)]. Moreover, we chose M238, a cell line heterozygousfor V600EBRAF, with regulated expression of MEK1 WT or P124S, to determine a potentialimpact of MEK1 P124S on p-ERK levels under reducing levels of V600EBRAF activity (orincreasing levels of acute vemurafenib treatment for 1 hour). Regulated expression of MEK1WT or P124S in M238 clearly had no significant impact on cellular p-ERK levels modulatedby vemurafenib treatment (Fig. 2B).

We also identified a naturally occurring BRAF/MEK1 double-mutant melanoma short-termculture, YUKSI, and confirmed the RNA expression of the MEK1 P124S mutant allele(Supplementary Fig. S4). The ratio of WT and P124S alleles in this short-term culture is

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approximately 1:1, consistent with that observed in tumor tissues (Supplementary Fig. S1),and these 2 MEK1 alleles appear to be expressed at similar RNA and protein levels, with thelatter extrapolation based on WT and P124S MEK1 having similar protein half-lives(Supplementary Fig. S1; data not shown). We then examined the effect of short hairpinRNA (shRNA)-mediated MEK1 knockdown on the p-ERK level (Fig. 2C). Consistent withthe inducible expression data, stable knockdown of MEK1 did not have an appreciableeffect on the p-ERK level, suggesting that in BRAF–mutant melanoma cell lines the MEK1level or mutational status at P124 is not a limiting modulator of p-ERK output. To examinepotential long-term effects, M238 with regulated expression of MEK1 WT or P124S wasfollowed by cell counting for growth assessment every 4 days over a span of 20 days.Neither WTMEK1 nor P124SMEK1 expression altered the rate of population doublingregardless of expression levels (or doxycycline concentrations), suggesting once againthat P124SMEK1 does not act in a dominant fashion over V600EBRAF in ERKphosphorylation or cell growth regulation in melanoma cell lines (Supplementary Fig. S5Aand S5B).

When transiently overexpressed in a genetic background devoid of mutant BRAF(HEK293T), P124SMEK1, but not MEK1 WT, induced an increased level of cellular p-ERK(Fig. 2D), consistent with P124SMEK1 harboring intrinsically enhanced kinase activitytoward recombinant ERK (Supplementary Fig. S6). On coexpression of V600EBRAF, which,as expected. led to baseline increases in p-MEK and p-ERK levels inHEK293T, P124SMEK1 no longer enhanced the cellular p-ERK level (Fig. 2D andSupplementary Fig. S6). Notably, although P124SMEK1 compared with WT MEK1displayed increased kinase activity toward ERK1/2 in a WTBRAF genetic background, thisdifference was lost in a V600EBRAF background. Taken together, these data support adominant role of V600E/KBRAF over P124SMEK1 in determining the cellular p-ERK outputwhen these mutant alleles coexist in the same melanoma tumor or cell line.

BRAF/MEK1 double-mutant melanoma cell lines were then subjected to vemurafenib (aBRAFi) or AZD6244/selumetinib (a MEKi) titration in survival assays over 3 days (Fig.2E). Regulated expression of P124SMEK1 in the background of V600EBRAF (Fig. 2A)or V600KBRAF (Supplementary Fig. S3B) and stable MEK1 knockdown in the context of anaturally occurring BRAF/MEK1 double-mutant short-term melanoma culture, YUKSI (Fig.2C), consistently produced no significant differences in vemurafenib (Fig. 2E and F, black)or AZD6244 (Fig. 2E and F, red) sensitivities. Similarly, regulated exogenous P124SMEK1expression in the context of V600EBRAF (M238) did not result in relative resistance toBRAF inhibition, compared with vector control or MEK1 WT in a clonogenic assay wherecells were replenished with fresh vemurafenib every 2 days over 12 days (Fig. 2G).Additionally, regulated P124SMEK1 expression at day 8 did not alter cellular p-ERK levelsin a clonogenic assay (Supplementary Fig. S5B). We also performed isobologram analysisof quantitative synergy, additivity, or antagonism using combination treatments withconstant ratios of vemurafenib and AZD6244/selumetenib. Cotargeting of BRAF and MEK1yielded strong synergy (log10 confidence interval from −0.52 to −1.0) in V600EBRAF–mutant melanoma cell lines (Supplementary Fig. S7). However, regulated expressionof P124SMEK1 in BRAF–mutant melanoma cell lines failed to significantly alter the degreeof vemurafenib and AZD6244 synergy and did not confer cross-resistance to BRAFi andMEKi (Supplementary Fig. S7).

Another V600BRAF–mutant concurrent MEK1 exon 3 mutation, found in the baseline tumorand both DP tumors of patient number 27, results in an I111S substitution. In the same cellline and expression system, we compared the signaling and drug sensitivity impact resultingfrom the regulated expression of I111SMEK1 versus C121SMEK1, an exon 3 mutant reportedto mediate acquired vemurafenib resistance (12). In contrast with C121SMEK1, regulated

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expression of I111SMEK1 in the V600EBRAF melanoma cell line M229 did not alter p-ERKlevels (Fig. 3A) or sensitivity to BRAFi (Fig. 3B, left) or to MEKi (Fig. 3B, right).Consistently, exogenous expression of I111SMEK1 did not result in relative p-ERKresistance in response to vemurafenib treatment at increasing concentrations, whereasregulated expression of C121SMEK1 restored the p-ERK levels in the presence ofvemurafenib (Fig. 3C). In a manner similar to P124SMEK1, I111SMEK1 also showedincreased activity toward ERK in a WTBRAF but not in a V600EBRAF background (Fig.3D). In contrast, C121SMEK1 conferred increased phosphorylation of ERK in both WTBRAFand V600EBRAF backgrounds (Fig. 3D), although this latter effect was more robust in amelanoma cell line (Fig. 3A and C).

DISCUSSIONLandmark studies have revealed largely mutually exclusive N-RAS, BRAF, and c-KITactivating mutations driving the MAPK/ERK pathway among human melanomas anddefining therapeutically relevant melanoma subsets (16, 17). We report here the concurrenceof activating mutations in BRAF and MEK1 in human melanomas and the dominant roleof V600E/KBRAF mutants over these somatically acquired MEK1 exon 3 mutants(I111SMEK1 and P124SMEK1) in determining and maintaining a critical level of ERKactivation needed for the growth and survival of melanoma cells. The dominanceof V600E/KBRAF over these MEK1 exon 3 mutants, which preexist prior to BRAFi or MEKitherapy, helps to explain the clear evidence of clinical responses of such double BRAF/MEK1–mutant melanomas to the BRAFi vemurafenib and dabrafenib (approved by the U.S.Food and Drug Administration and in advanced stages of clinical development,respectively). Thus, contrary to logical prediction, the reported MEK1 exon 3 mutations(I111S and P124S) are unlikely to be an important determinant of primary or innatesensitivity to this class of targeted agents.

The relative impact of MEK1 exon 3 mutants (I111S and P124S) versus V600EBRAF on p-ERK levels (Fig. 2D and 3D) may reflect the relative strength of kinase activation/loss ofnegative feedback and suggest a potential dependence of MEK1 exon 3 mutant hyperactivityon adequate upstream activation (i.e., MEK1 exon 3 mutations are not constitutivelyactivating but render MEK1 more readily activated). These intriguing findings on concurrentactivating mutations in BRAF and MEK1 suggest that exon 3 MEK1 mutants may subserveERK-independent effects during melanoma progression and occur also in BRAF WTmelanomas. Additionally, the trend toward a high ratio of double MEK1/V600KBRAFmutations relative to MEK1/V600EBRAF mutations found in this study warrants furthervalidation. In additional tumors we have analyzed from patients with BRAF–mutantmelanoma not treated with BRAFi, we have detected 3 additional MEK1 exon 3 mutants(data not shown). All 3 are P124S substitutions, concurrent with 1 V600RBRAF and2 V600KBRAF mutations.

Aside from the setting of primary or innate drug sensitivity, a P124LMEK1 mutation hasbeen reported to confer acquired resistance in one patient treated with the MEKi AZD6244/selumetinib (18). This P124LMEK1 mutant was proposed to mediate cross-resistance in vitroto PLX4720, a preclinical version of PLX4032/vemurafenib. However, the short-termculture derived from this MEK1 mutant, AZD6244-resistant melanoma, termed M307, waslater found to harbor high levels of COT expression, which has been proposed more recentlyas a mediator of acquired BRAFi resistance in melanoma (6). Thus, it is possible that COToverexpression, rather than MEK1 mutation, accounts for BRAFi resistance in thismelanoma culture. Alternatively, P124LMEK1 may be more catalytically activethan P124SMEK1, with the latter likely being the most common MEK1-mutant alleledetected concurrently with V600BRAF mutants without any prior BRAFi or MEKi exposure

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or selective pressure. Moreover, another MEK1 exon 3 allele, C121S, has been proposed asa determinant of acquired resistance to vemurafenib (12). In vitro data from our studycorroborate this conclusion, although we have not yet detected the C121SMEK1 allele in DPtumors from our patients.

In our clinical series, MEK1 mutations were detected in acquired resistant or DP tumorsonly when the same mutations were also observed in the baseline melanoma tumors. Thus,these preexisting MEK1 mutations cannot account for acquired drug resistance. Notably,none of these patients had been exposed to MEKi prior to starting on the BRAFi therapy.The objective response rate of these 5 patients with preexisting double BRAF/MEK1mutations is in the same range of the 53% objective response rate in a large series of patientstreated with vemurafenib (19).

In our work, regulated expression of P124SMEK1 and I111SMEK1 to mimic endogenousexpression levels (1:1 ratio of mutant to WT MEK1) consistently produced no appreciableeffect on cellular p-ERK levels and vemurafenib sensitivity in several genetic backgroundsharboring either V600EBRAF or V600KBRAF alleles. Outside the context of V600E/KBRAF(e.g., in HEK293T), however, MEK1 P124S and I111S mutants have demonstrativelyhigher levels of kinase activity toward ERK compared with MEK1 WT. Additionally, whendouble BRAF/MEK1-mutant melanoma cell lines were exposed acutely to vemurafenib,resulting in reduced V600EBRAF activity, the activating MEK1 P124S and I111S mutantsstill could not upregulate (or reactivate) cellular p-ERK levels, suggestingcontinued V600E/KBRAF oncogene addiction. Together, these data preclude a critical role ofsomatic MEK1 exon 3 mutations preexisting prior to BRAFi therapy in conferring primaryresistance to BRAF inhibitors. These data also caution against relying on detection of everyMEK1 exon 3 mutant allele as a biomarker of acquired BRAFi resistance.

Emerging clinical data with combined BRAF and MEK inhibitors (20) showing preliminarysafety and a high response rate of BRAF–mutant melanomas suggest improved durability ofclinical response (compared with single-agent therapy). In this context, our data showed astrong degree of BRAFi and MEKi synergy in both BRAF single- and BRAF/MEK1double-mutant genotypes, supporting the utility of such a combinatorial approach as anupfront therapy or a regimen to overcome defined mechanisms of acquired BRAFiresistance (7, 8, 10). The coexistence of BRAF and MEK1-mutant alleles in the same tumorcell and its validation in an additional cohort of metastatic melanoma patients (data notshown) suggest potential ERK-independent roles of activating MEK1 mutants. PotentialERK-independent roles of somatic MEK1 mutants in melanoma pathogenesis would furthersupport the combined BRAF and MEK inhibition therapeutic approach.

In conclusion, we identified a subset of BRAF–mutant melanomas that harbors concomitantMEK1 exon 3 mutations. Although the relevance of these concurrent mutations to multistepmelanoma progression is at present unclear, contrary to expectation, their coexistence cannotpreclude a clinical response to BRAF inhibitors. Data from cell line modeling support theBRAF mutant as dominant over the MEK1 mutants described herein with regard todetermining cellular p-ERK output at a level critical for melanoma growth and survival.Together, the present study helps explain why preexisting MEK1 exon 3 mutations do notdetermine sensitivities of BRAF–mutant melanomas to the BRAF inhibitors vemurafenib/PLX4032 or dabrafenib/GSK2118436.

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METHODSPatients and Samples

Patients participated in the vemurafenib phase I dose-escalation study (NCT00405587),vemurafenib phase II study (NCT00949702), or the dabrafenib phase I/II study(NCT00880321). All patients had V600E/KBRAF mutation-positive, previously treatedmetastatic melanoma (none with MAPK-targeted drugs) and received either 960 or 1120 mgof vemurafenib or 35, 70, or 150 mg of dabrafenib orally twice daily. All consented to thegenetic analysis of their tissue biopsies or samples. The clinical trials included optionalbiopsies at baseline or upon DP, and we selected 31 patients from whom samples from abiopsy at baseline, or both at baseline and DP, were available for analysis.

Genomic DNA and cDNA SequencinggDNAs were isolated using the Flexi Gene DNA Kit (QIAGEN) or the QIAamp DNA FFPETissue Kit. MEK1 exon 3 was amplified using the forward(CCTGTTTCTCCTCCCTCTACC) and the reverse (ACACCCACCAGGAATACTGC)primers. MEK2 exon 3 was amplified using the forward(TTGACCACTGTTGGGAACGCC) and the reverse (TCTGTTCCGTGGAGGCCCTG)primers. Total RNA was extracted using the RiboPure Kit (Ambion), and reverse-transcription reactions were performed using the SuperScript First-Strand Synthesis System(Invitrogen). PCR products were purified using QIAquick PCR Purification Kit (QIAGEN)followed by bidirectional sequencing using BigDye v1.1 (Applied Biosystems) incombination with a 3730 DNA Analyzer (Applied Biosystems).

Cell Culture, Constructs, Infections, and TransfectionsAll cell lines were maintained in DMEM with 10% or 20% heat-inactivated FBS (OmegaScientific), 2 mmol/L glutamine in a humidified, 5% CO2 incubator, and 10 ng/mLdoxycycline and/or puromycin, when applicable. Wild-type and mutant MEK1 were cloned,epitope-tagged, sequence-verified, and cloned into the doxycycline-repressible lentiviralvector pLVX-Tight-Puro (Clontech, Inc.). Knockdown of MEK1 was achieved usingMISSION shRNA lentiviral transduction particles [clone IDs 455, 1163, 612, 1015, 2-753(Sigma)]. V600EBRAF construct was purchased from Addgene. HEK293T cells weretransfected using Lipofectamine2000 (Invitrogen).

Drug Sensitivity, Protein Detection, and Kinase AssayCell proliferation experiments were performed in a 96-well format (5 replicates) and drugtreatments initiated at 24 hours post-seeding for 72 hours. Stocks and dilutions of PLX4032/vemurafenib (Plexxikon) and AZD6244/selumetenib (Selleck Chemicals) were made indimethyl sulfoxide (DMSO). Cells were quantified using CellTiter-GLO Luminescence(Promega) following the manufacturer’s recommendations or by counting of trypan blue–positive cells following trypsinization. Clonogenic assays were performed by plating cells atsingle-cell density in 6-well plates and provided fresh media, doxycycline (if applicable) andPLX4032/vemurafenib (vs. DMSO) every 2 days. Colonies were then fixed by 4%paraformaldehyde and stained with crystal violet at 0.05%. Cell lysates for Western blottingwere made in RIPA (Sigma) with protease (Roche) and phosphatase (Santa CruzBiotechnology) inhibitor cocktails. Western blots were probed with antibodies against p-ERK1/2 (T202/Y204), total ERK1/2, MEK1, FLAG (Cell Signaling Technology), andtubulin (Sigma). In vitro kinase assays were performed as described (7) except recombinantERK is now used as a substrate.

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Study OversightData were generated and collected by the study investigators and analyzed in collaborationbetween the senior authors, who vouch for the completeness and accuracy of the data andanalyses. The corresponding author prepared the initial draft of the manuscript. All theauthors made the decision to submit the manuscript for publication.

Data ProcessingStatistical analyses were performed using InStat 3 Version 3.0b (GraphPad Software);graphical representations using DeltaGraph or Prism (Red Rock Software); and combinationindex calculation using CalcuSyn V2.1 (Biosoft).

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank P. Lin and G. Bollag (Plexxikon, Inc.) for providing PLX4032, A. Villanueva (UCLA) for clinical datamanagement, S. Yashar (UCLA), P. Lyle (Vanderbilt), R. Scolyer (Melanoma Institute of Australia), and R.Sharma (Westmead) for pathology expertise, and K. Carson (Westmead Hospital) and V. Tembe (WestmeadMillennium Institute) for technical assistance.

Grant Support

The study was funded (R.S. Lo) by a Stand Up To Cancer Innovative Research Grant, a Program of theEntertainment Industry Foundation (SU2C-AACR-IRG0611), Burroughs Wellcome Fund, National CancerInstitute (K22CA151638), STOP CANCER Foundation, V Foundation for Cancer Research, Melanoma ResearchFoundation, Melanoma Research Alliance, American Skin Association, Caltech-UCLA Joint Center forTranslational Medicine, Sidney Kimmel Foundation for Cancer Research, Eli and Edythe Broad Center ofRegenerative Medicine and Stem Cell Research, Wesley Coyle Memorial Fund, Ian Copeland Melanoma Fund,Ruby Family Foundation, Louis Belley and Richard Schnarr Fund, and The Seaver Institute. G.M. Pupo, R.F.Kefford, and G.V. Long are supported by Program Grant No. 402761 from the National Health and MedicalResearch Council of Australia, Translational Research Program Grant No. 05/TPG/1-01 from the Cancer InstituteNew South Wales (CINSW), and an infrastructure grant to Westmead Millennium Institute from the HealthDepartment of NSW through Sydney West Area Health Service. Westmead Institute for Cancer Research atWestmead Millennium Institute is the recipient of capital grant funding from the Australian Cancer ResearchFoundation. G.V. Long is also supported by fellowships from CINSW.

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10. Shi H, Moriceau G, Kong X, Lee M-K, Lee H, Koya RC, et al. Melanoma whole-exomesequencing identifies V600EBRAF amplification-mediated BRAF inhibitor resistance. NatureComm. 20126; 3:a724.

11. Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, et al.Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can beovercome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell. 2010; 18:683–95. [PubMed:21156289]

12. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, et al. Dissecting therapeuticresistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol. 2011;29:3085–96. [PubMed: 21383288]

13. Dentici ML, Sarkozy A, Pantaleoni F, Carta C, Lepri F, Ferese R, et al. Spectrum of MEK1 andMEK2 gene mutations in cardio-facio-cutaneous syndrome and genotype-phenotype correlations.Eur J Hum Genet. 2009; 17:733–40. [PubMed: 19156172]

14. Forbes SA, Tang G, Bindal N, Bamford S, Dawson E, Cole C, et al. COSMIC (the Catalogue ofSomatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer.Nucleic Acids Res. 2010; 38(database issue):D652–7. [PubMed: 19906727]

15. Marks JL, Gong Y, Chitale D, Golas B, McLellan MD, Kasai Y, et al. Novel MEK1 mutationidentified by mutational analysis of epidermal growth factor receptor signaling pathway genes inlung adenocarcinoma. Cancer Res. 2008; 68:5524–8. [PubMed: 18632602]

16. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct subtypes ofmelanoma. J Clin Oncol. 2006; 24:4340–6. [PubMed: 16908931]

17. Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of geneticalterations in melanoma. N Engl J Med. 2005; 353:2135–47. [PubMed: 16291983]

18. Emery CM, Vijayendran KG, Zipser MC, Sawyer AM, Niu L, Kim JJ, et al. MEK1 mutationsconfer resistance to MEK and BRAF inhibition. Proc Natl Acad Sci U S A. 2009; 106:20411–6.[PubMed: 19915144]

19. Sosman A, Kim KB, Schuchter LM, Gonzalez R, Pavlick AC, Weber JS, et al. Survival of BRAFV600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012; 366:707–14.[PubMed: 22356324]

20. Infante JR, Falchook GS, Lawrence DP, Weber JS, Kefford RF, Bendell JC, et al. Phase I/II studyto assess safety, pharmacokinetics, and efficacy of the oral MEK 1/2 inhibitor GSK1120212(GSK212) dosed in combination with the oral BRAF inhibitor GSK2118436 (GSK436). J ClinOncol. 2011; 29(suppl) abstr CRA8503.

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Figure 1.Spectrum of clinical responses of V600E/KBRAF/P124SMEK1 double-mutant melanomas toBRAF inhibitors. A, in patient 2, a right axillary nodal melanoma clearly responded tovemurafenib at day 15 [positron emission tomography (PET) scan], at which time a“baseline” biopsy of the same tumor revealed mutations in both BRAF and MEK1. Tumortracking shows timing of baseline and DP tumor biopsies. Best overall responsedemonstrates a partial response, and biopsy locations are indicated. B, in patient 12, a 2-cmright axillary tumor (which was not a chosen target tumor tracked below), from which a 3-mm punch biopsy prior to dabrafenib initiation revealed both BRAF and MEK1 mutations,became nonpalpable within 14 days of starting on dabrafenib. Despite tumor tracking notreaching partial response before DP, initial tumor response in this patient was associated

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with a dramatic increase in right shoulder mobility as well as decreased right breast/axillaryswelling (Supplementary Fig. S2). C, in patient 28, a baseline tumor biopsy in the right sideof the neck taken 4 months and 11 days prior to dabrafenib initiation was shown to harborboth BRAF and MEK1 mutations. PET scans before and after dabrafenib initiation clearlyshowed a rapid metabolic response at day 15 on treatment. Best overall response indicates aPR, and the same neck lesion that responded on day 15 was biopsied when it progressed.

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Figure 2.Regulated P124SMEK1 expression cannot alter p-ERK levels or sensitivity to BRAF or MEKinhibition in V600EBRAF melanoma cell lines. A, doxycycline-repressible expression ofvector (control), FLAG-MEK1 WT, versus FLAG-MEK1 P124S in V600EBRAF melanomacell lines (M229, M238). Protein lysates (48 hours post seeding at 0, 0.1, and 10 ng/mLdoxycycline) were probed by Western blotting for the indicated phospho- and total proteinlevels. Tubulin, loading control. B, dose-dependent suppression of p-ERK levels byvemurafenib (PLX4032) in a V600EBRAF background with or withoutconcurrent P124SMEK1. Indicated M238 stable cell lines were washed free of doxycyclinefor 48 hours, inducing exogenous FLAG-MEK1 WT or FLAG-MEK1 P124S expression,and treated for 1 hour with increasing doses (μM) of vemurafenib: 0 (DMSO), 0.01, 0.1, 1.0,and 10. Cell lysates were then probed for the indicated protein levels. C, stable MEK1knockdown in the naturally occurring V600EBRAF/P124SMEK1 double-mutant melanomashort-term culture (YUKSI). Protein lysates were probed for the indicated protein levels. D,impact of P124SMEK1 on cellular p-ERK levels in BRAF WT versus V600E backgrounds.Indicated FLAG-tagged expression constructs were transiently transfected into HEK293Tcells (BRAF WT) with either pBABE-PURO (empty vector) or pBABE-

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PURO-V600EBRAF. After 72 hours, cell lysates were probed for the indicated protein levelsby Western blotting. E, stable cell lines (M229, top; M238, bottom) were either maintainedwith doxycycline (10 ng/mL) or washed and released incrementally (0.1 or 0 ng/mL) fromdoxycycline-mediated suppression of FLAG-MEK1 WT or FLAG-MEK1 P124S geneexpression for 24 hours prior to treatment with increasing concentrations of vemurafenib/PLX4032 (black) or selumetinib/AZD6244 (red). Survival curves are shown after 72 hoursof drug treatments, and data represent percent surviving cells relative to DMSO-treatedcontrols (mean ± SEM, n = 5). The dashed line corresponds to 50% cell killing. F, theBRAF/MEK1 double-mutant melanoma short-term culture, YUKSI, was infected with eithera control or shMEK1 virus and subjected to PLX4032 or AZD6244 treatments for 72 hours.G, indicated M238 stable cell lines maintained with doxycycline (100 ng/mL) or washedfree of doxycycline, and seeded at single-cell density. At 24 hours after seeding, cells weretreated with indicated concentrations of PLX4032. Cellular colonies were visualized bystaining with crystal violet at 12 days after drug treatments. Photographs are representativeof 2 independent experiments and time points.

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Figure 3.I111SMEK1 expression fails to modulate p-ERK levels or melanoma sensitivity to BRAF orMEK inhibitors in the presence of V600EBRAF. A, doxycycline-repressible expressionvector, FLAG-MEK1 I111S, or FLAG-MEK1 C121S in the V600EBRAF melanoma celllines M229. Protein lysates (48 hours post seeding at 0, 0.1, and 10 ng/mL doxycycline)were probed by Western blotting for the indicated phospho- and total protein levels.Tubulin, loading control. B, M229 stable cell lines were either maintained with doxycycline(10 ng/mL) or washed and released incrementally (0.1 or 0 ng/mL) from doxycycline-mediated suppression of gene expression for 24 hours prior to treatment with increasingconcentrations of vemurafenib/PLX4032 (black) or selumetinib/AZD6244 (red). Survivalcurves are shown after 72 hours of drug treatments, and data represent percent survivingcells relative to DMSO-treated controls (mean ± SEM, n = 5). The dashed line correspondsto 50% cell killing. C, dose-dependent suppression of p-ERK levels by vemurafenib/PLX4032 in a V600EBRAF background with or without concurrent WTMEK1, C121SMEK1,or I111SMEK1 expression. Indicated M229 stable cell lines were treated for 1 hour withincreasing doses (μM) of PLX4032: 0 (DMSO), 0.01, 0.1, 1.0, and 10. Cell lysates werethen probed for the indicated protein levels. D, impact of indicated MEK1 mutants oncellular p-ERK levels in BRAF WT versus V600E backgrounds. Indicated FLAG-tagged

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expression constructs were transiently transfected into HEK293T cells (BRAF WT) witheither pBABE-PURO (empty vector) or pBABE-PURO-V600EBRAF. After 72 hours, celllysates were probed for the indicated protein levels by Western blotting.

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Shi et al. Page 16

Tabl

e 1

Sum

mar

y of

BR

AF,

ME

K1/

2 m

utat

ion

stat

us, a

nd p

atie

nt c

hara

cter

istic

s

Stud

y si

teP

atie

nt n

umbe

rB

RA

F e

xon

15 b

asel

ine

ME

K1

exon

3ba

selin

eM

EK

2 ex

on 3

base

line

ME

K1

exon

3at

pro

gres

sive

dise

ase

Age

and

gen

der

Stag

eD

ose

ofB

RA

Fi (

mg)

Bes

t ov

eral

l res

pons

eP

rogr

essi

on-f

ree

surv

ival

(da

ys)

Site

s of

bas

elin

etu

mor

s bi

opsi

eda

Res

pons

es o

f ba

selin

ea

UC

LA

1V

600E

WT

WT

WT

65 F

M1c

1120

bid

−37

%27

9—

—

WT

WT

2V

600E

P124

SW

TP1

24S

46 M

M1c

960

bid

−32

%14

9A

xilla

ry n

ode

Day

15

PET

upt

ake

decr

ease

(Fig

. 1A

)

3V

600E

WT

WT

WT

66 F

M1b

960

bid

−53

%13

7—

—

WT

WT

4V

600E

WT

WT

WT

48 M

M1c

960

bid

−24

%12

6—

—

WT

5V

600E

WT

WT

WT

54 M

M1c

960

bid

−75

%84

——

6V

600E

WT

WT

WT

65 F

M1c

960

bid

−72

%37

3—

—

WT

7V

600E

WT

WT

WT

51 M

M1c

960

bid

−60

%21

2—

—

WT

8V

600E

WT

WT

WT

47 F

M1b

960

bid

−72

%97

——

9bV

600E

WT

WT

WT

47 F

IIIc

150

bid

−31

%23

8—

—

WT

WT

10V

600E

WT

WT

DP

N/A

52 M

M1c

960

bid

−70

%—

——

11V

600K

P124

SW

TD

P N

/A72

FM

1c96

0 bi

d−

11%

80A

xilla

ry n

ode

Dec

reas

e by

25%

in lo

nges

ttu

mor

dim

ensi

on

12b

V60

0EP1

24S

WT

DP

N/A

48 F

M1c

150

bid

−9%

104

Axi

llary

nod

eN

ot p

alpa

ble

(Fig

. 1B

)

13V

600K

WT

WT

DP

N/A

66 M

M1c

960

bid

−30

%14

0—

—

14V

600E

WT

WT

DP

N/A

57 M

M1c

960

bid

−47

%18

9—

—

15V

600E

WT

WT

DP

N/A

55 M

M1c

960

bid

−50

%30

0—

—

16V

600E

WT

WT

Res

pond

ing

53 F

M1a

960

bid

−91

.7%

——

—

17V

600E

WT

WT

Res

pond

ing

45 F

M1c

960

bid

−73

%—

——


NIH


NIH


NIH


Shi et al. Page 17

Stud

y si

teP

atie

nt n

umbe

rB

RA

F e

xon

15 b

asel

ine

ME

K1

exon

3ba

selin

eM

EK

2 ex

on 3

base

line

ME

K1

exon

3at

pro

gres

sive

dise

ase

Age

and

gen

der

Stag

eD

ose

ofB

RA

Fi (

mg)

Bes

t ov

eral

l res

pons

eP

rogr

essi

on-f

ree

surv

ival

(da

ys)

Site

s of

bas

elin

etu

mor

s bi

opsi

eda

Res

pons

es o

f ba

selin

ea

18V

600E

WT

WT

DP

N/A

29 F

M1c

960

bid

−34

.5%

289

——

19V

600E

WT

WT

Res

pond

ing

74 M

IIIc

960

bid

−20

%—

——

20V

600K

WT

WT

WT

65 M

M1c

960

bid

−22

%16

1—

—

21V

600K

WT

WT

Res

pond

ing

61 M

M1c

960

bid

−34

%—

——

22V

600E

WT

WT

Res

pond

ing

60 F

M1a

960

bid

−42

%—

——

23b

V60

0EW

TW

TD

P N

/A36

FM

1c15

0 bi

d−

66%

85—

—

24b

V60

0EW

TW

TD

P N

/A65

FM

1c15

0 bi

d−

39.5

%76

——

MIA

25b

V60

0KW

TW

TW

T59

MM

1c10

0 bi

d, d

ose

esca

late

d to

100

tid

−14

%22

4—

—

26V

600E

WT

WT

WT

71 M

M1c

960

bid

−53

%23

9—

—

27b

V60

0KI1

11S

WT

I111

S38

FM

1a35

bid

, dos

ees

cala

ted

to10

0 tid

18

Dec

200

9

−10

0%11

9R

ight

ingu

inal

nod

eD

ay 1

5 PE

T u

ptak

e de

crea

sein

sin

gle

mea

sura

ble

lesi

onI1

11S

28b

V60

0KP1

24S

WT

P124

S39

MM

1c15

0 bi

d−

56%

118

Rig

ht n

eck

node

Day

15

PET

upt

ake

decr

ease

(reg

iona

l, Fi

g. 1

C);

SU

Vm

ax r

educ

ed 4

8% o

vera

llm

etas

tase

s

29b

WT

WT

WT

58 F

M1c

70 b

id, d

ose

esca

late

d to

100

tid 2

9D

ec 2

009

−63

%20

9—

—

VI

30V

600E

WT

WT

WT

46 F

M1c

720

−59

%10

7—

—

31V

600E

WT

WT

WT

70 F

M1a

960

−70

%18

3—

—

NO

TE

: Hig

hlig

htin

g, in

form

atio

n fo

r pa

tient

s w

hose

tum

ors

harb

or M

EK

1 m

utat

ions

. Das

hes,

dat

a no

t app

licab

le.

Mea

n pr

ogre

ssio

n-fr

ee s

urvi

val f

or p

atie

nts

with

WT

ME

K1

mel

anom

as 5

182

.4 d

ays

(SD

84.

7) v

ersu

s m

utan

t ME

K1

mel

anom

as 5

114

day

s [S

D 2

5.1,

(2-

taile

d P

= 0

.09)

].M

ean

best

ove

rall

resp

onse

for

pat

ient

s w

ith W

T M

EK

1 m

elan

omas

= −

50%

(SD

20.

0) v

ersu

s m

utan

tM

EK

1 m

elan

omas

= 4

1.6

[SD

37.

8, 2

-tai

led

P =

0.4

5)].

Par

tial r

espo

nse

(PR

) fo

r pa

tient

s w

ith W

T M

EK

1 m

elan

omas

(21

/26

or 8

0%)

vers

us m

utan

t ME

K1

mel

anom

as (

3/5

or 6

0%).

Abb

revi

atio

ns: b

id, t

wic

e a

day;

DP,

dis

ease

pro

gres

sion

; MIA

, Mel

anom

a In

stitu

te, A

ustr

alia

; PE

T, p

ositr

on e

mis

sion

tom

ogra

phy;

SU

Vm

ax, m

axim

um s

tand

ard

upta

ke v

alue

; tid

, 3 ti

mes

a d

ay; U

CL

A, U

nive

rsity

of

Cal

ifor

nia,

Los

Ang

eles

; VI,

Van

derb

ilt-I

ngra

m C

ance

r

Cen

ter

a Info

rmat

ion

only

for

app

licab

le p

atie

nts

with

ME

K1-

mut

ant m

elan

omas

.

b Dab

rafe

nib-

trea

ted

patie

nts.

All

othe

rs w

ere

trea

ted

with

vem

uraf

enib

.


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Preexisting MEK1 Exon 3 Mutations in V600E/KBRAF Melanomas Do Not Confer Resistance to BRAF...

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