the cdk4/6 inhibitor ly2835219 overcomes vemurafenib ... · developed an in vivo...

Small Molecule Therapeutics

The CDK4/6 Inhibitor LY2835219 Overcomes VemurafenibResistance Resulting fromMAPKReactivation and Cyclin D1Upregulation

Vipin Yadav1, Teresa F. Burke1, Lysiane Huber1, Robert D. Van Horn1, Youyan Zhang1, Sean G. Buchanan1,Edward M. Chan2, James J. Starling1, Richard P. Beckmann1, and Sheng-Bin Peng1

AbstractB-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for

melanomapatientswithB-RAFV600Emutation.However,most patients treatedwith vemurafenib eventually

develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including

MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to

vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of

MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective

inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma

A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we

developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375

xenografts. Consistently,we found thatMAPK is reactivated and cyclinD1 is elevated invemurafenib-resistant

tumors, aswell as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor

growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced

apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily

mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced

significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin

D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting

cyclin D1–CDK4/6 signaling by LY2835219 is an effective strategy to overcomeMAPK-mediated resistance to

B-RAF inhibitors in B-RAF V600E melanoma. Mol Cancer Ther; 13(10); 2253–63. �2014 AACR.

IntroductionB-RAF is the most commonly mutated driver onco-

gene in melanoma, with activating mutations in codon600 occurring in almost 50% of the patients (1, 2).Treatment with B-RAF selective inhibitors, such asvemurafenib or dabrafenib, has demonstrated signifi-cant benefit in melanoma patients with B-RAF V600Emutation, with extended patient progression-freesurvival and median overall survival compared withchemotherapy (3–6). However, these responses wererelatively short-lived, and drug resistance generally

developed within 5 to 7 months (4, 6). Thus, the emer-gence of resistance remains a considerable therapeuticchallenge to achieve durable responses and prolongedsurvival in these patients.

A variety of molecular mechanisms are identified to beinvolved in resistance to B-RAF inhibition. The mostcommon resistant mechanism is MAPK pathway reacti-vation, which is caused by genetic mutation of MEK ordifferent Ras isoforms (7–9), upstream activation of recep-tor tyrosine kinases (RTK) such as FGFR3 and c-Met (10,11), expression of B-RAFV600E splice variants that dimer-ize inpresence of theB-RAF inhibitor (12), amplification ofB-RAF (13, 14), and upregulation ofMAP3Ks such as COTor C-RAF (15, 16). Alternatively, activation of MAPK-redundant pathways such as PI3K/Akt as a consequenceof PTEN loss (17) or overexpression of RTKs such asPDGFRb and IGF1R have also been reported to induceresistance in B-RAF V600E melanoma (7, 18, 19). In addi-tion, secretion of growth factors such as HGF or FGF hasalso been implicated in resistance to B-RAF inhibition (10,11, 20, 21). Although the resistant mechanisms are fre-quently associated with MAPK reactivation, treatmentwith the MEK inhibitor trametinib, or with trametinibplus dabrafenib, has not been very effective in patients

1Oncology Discovery Research, Lilly Research Laboratories, Indianapolis,Indiana. 2Oncology Business Unit, Eli Lilly and Company, Indianapolis,Indiana.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Authors: Richard P. Beckmann, Oncology DiscoveryResearch, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,IN 46285. Phone: 317-276-4285; Fax: 317-651-6346; E-mail:[email protected]; and Sheng-Bin Peng. Phone: 317-433-4549; Fax: 317-276-1414; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0257

�2014 American Association for Cancer Research.

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Therapeutics

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on April 8, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst August 13, 2014; DOI: 10.1158/1535-7163.MCT-14-0257

http://mct.aacrjournals.org/

who have previously failed B-RAF inhibitor (22), suggest-ing that subsequent targeting of MAPK signaling alone isnot sufficient. Therefore, despite recent advances in theclinic, drug resistance upon selective B-RAF inhibitionremains a considerable therapeutic challenge in clinic.

Constitutive activation of cyclin-dependent kinases(CDK) and deregulation of cell cycle are common featuresacross several cancer types, including melanoma.P16INK4a, a tumor suppressor gene and a negative regu-lator of CDK4, is deleted in 38% of melanoma (2, 23). Inaddition, germline mutations and amplification of CDK4gene have been identified in melanoma, which leads tounrestricted CDK4 activity and increased cell prolifera-tion (2, 24). In general, regulation of cell-cycle entry inproliferating adult mammalian cells is controlled by D-cyclins which bind and activate CDK4 and CDK6 topromote phosphorylation of retinoblastoma (Rb) proteinand G1 to S transition (25). The RAS–MAPK pathway isknown to control cell-cycle entry via upregulation ofcyclin D1 in several cell types (26, 27). Inhibition ofMAPKsignaling by B-RAF inhibitors decreases cyclin D1 expres-sion and upregulates CDK inhibitor p27KIP1 levels, thusblocking cell-cycle entry in B-RAF V600E melanoma(26, 27).Overexpression of cyclinD1 is linked to resistanceto B-RAF inhibition (28). CCND1 is amplified in 11% ofmelanoma, including 17% of B-RAF V600E melanoma,thus suggesting a potential role of cyclin D1 in intrinsicresistance to B-RAF inhibitors (2, 28). However, the role ofcyclin D1 in acquired resistance to vemurafenib has notbeen described, and the therapeutic value of targetingcyclin D1/Rb axis to overcome vemurafenib resistancehas not been explored.

In this study, we have generated multiple in vitro celllines and an in vivomodel of resistance to vemurafenib anddiscovered that MAPK reactivation and cyclin D1 eleva-tion are common in these resistant models. We describethat cyclin D1 is an important mediator of vemurafenibresistance and provides a potential therapeutic target toovercome resistance to B-RAF inhibition in B-RAF V600Emelanoma. Using these in vitro and in vivo models, weshow that cyclinD1 is generally elevatedand functions as acritical node for the survival of vemurafenib-resistant cells.We further demonstrate that LY2835219, a selective dualCDK4/6 inhibitor currently in phase II clinic studies (29),can overcome vemurafenib resistance in these resistantmodels. Altogether, this study sheds new light onmechan-isms of resistance to B-RAF inhibition, identifies cyclin D1elevation concurrent with MAPK reactivation as a com-mon resistant mechanism, and proposes targeting cyclinD1 through CDK4/6 inhibition by LY2835219 as an effec-tive therapeutic strategy to overcome B-RAF resistance.

Materials and MethodsCell culture, reagents, and transfections

A375, SH4, and A2058 cells were obtained from ATCCon May 7, 2012, July 2, 2012, and June 27, 2006, respec-tively. M14 cells were purchased from NCI on March 10,2005. Cells were stored within a central cell bank that

performs cell line characterizations. All these cells werepassaged for fewer than 2 months after which time newcultures were initiated from vials of frozen cells. Charac-terization of the cell lines was done by a third-partyvendor (RADIL, which included profiling by PCR) forcontamination by various microorganisms of bacterialand viral origin. As a result, no contamination wasdetected. The samples were also verified to be of humanorigin without mammalian interspecies contamination.The alleles for 9 different genetic markers were used todetermine that the banked cells matched the geneticprofile that has been previously reported.

All cells weremaintained inDMEM (Thermo Scientific)supplementedwith 10%FBS (Invitrogen). B-RAFselectiveinhibitor vemurafenib and CDK4/6 dual inhibitorLY2835219 were synthesized by Eli Lilly and Company.The mesylate salt of LY2835219 (LY2835219.CH4O3S) wasused in all the in vitro studies. All siRNAs were obtainedfromDharmacon (OnTargetPlus SiRNA). siRNAtransfec-tions were performed using Lipofectamine RNAiMAXtransfection reagent (Invitrogen) as per manufacturer’sinstructions. Cyclin D1 and nonspecific control shRNAlentiviral particles were obtained from Sigma-Aldrich(Mission shRNA). Cells were transduced with the virusfor 72 hours as per manufacturer’s instructions. Antibo-dies against phospho-ERK1/2 (4370), phospho-Akt1(S473) (4060), phospho-MEK (9154), cyclin D1 (2978),cyclin D2 (3741), phospho-Rb (S807/811) (8516), p27(2552), cleaved PARP (9541), and cleaved caspase-3(9664) were purchased from Cell Signaling Technology.Antibodies against B-RAF (sc-166, Santa Cruz), GAPDH(Santa Cruz), tubulin (ab7291, AbCam), phospho-histoneH3 (S10) (Millipore), and phospho-Rb (S780) (BD Bio-sciences) were obtained from the indicated companies.

Generation of vemurafenib-resistant cell linesA375R1, A375R3, M14R, and SH4R models with

acquired resistance to vemurafenib were generated bytreating respectiveparental cellswithgradually increasingconcentrations of vemurafenib, up to 2 mmol/L, as previ-ously described (10). A375R2 cells were generated bytreating A375 cells with a high concentration of vemur-afenib (2 mmol/L) for 1 week, followed by culturing withvemurafenib (1 mmol/L) for up to 20 passages. Uponestablishment of resistance, the inhibitor was withdrawnand all of the cell lines were maintained in regular mediafor subsequent passages. ForA375R4 cells, theNRasQ61Kwas stably transfected intoA375 cells, and single-cell clonewas selected and characterized for this study. All of themodels retained resistance to vemurafenib for up to 20passages (data not shown). In this study, all resistant celllineswith less than10passageswereused for experiments.

Cell proliferation assayCells (1 � 103 to 3 � 103) were normally plated in 96-

well plates (BD Biosciences). Cells were treated the nextday for 96 hours, and then assessed for viability usingCellTiter Glo (Promega), as per manufacturer’s

Yadav et al.

Mol Cancer Ther; 13(10) October 2014 Molecular Cancer Therapeutics2254




instructions and a luminescence plate reader (Victor,Perkin Elmer). GraphPad Prism 4 software was used togenerate sigmoidal dose-response curves and calculatethe proliferation IC50.

Caspase-3/7 activity assayCells (5 � 103) were plated in 96 well plates (BD

Biosciences). Cells were treated the next day for 24 to48 hours and then assessed for caspase-3 activity byCaspase-Glo-3/7Assay (Promega), as permanufacturer’sinstructions and a luminescence plate reader (Victor,Perkin Elmer).

Preparation of cell lysates and immunoblottingCells and tumor tissues were lysed using either RIPA

lysis buffer (Bio-Rad; cell lines) or 1% SDS solution(tumor lysates), containing 1� phosphatase and pro-teinase inhibitor cocktail (Pierce). Tumor lysates wereprepared with freshly frozen tumor samples. Approx-imately 200 mg of tumor tissue was homogenized andlysed using 0.5 mL of lysis buffer. The protein concen-tration of individual samples was determined with DCProtein Assay Kit (Bio-Rad). SDS-PAGE was performedon cell lysates containing 20 mg of total protein using 4%to 20% Novex tri-glycine gradient gels (Invitrogen).Protein was transferred onto 0.2-mm nitrocellulosemembranes using Trans-Blot Turbo Transfer system(BioRad) as per manufacturer’s instructions. Proteinswere detected using the Odyssey Infrared ImagingSystem (Li-COR Biosciences).

Flow cytometryCell pellets were fixed in 70% ethanol for 30 minutes at

�20�C and then washed with PBS. Fixed cells werestainedwith propidium iodide (PI)/Triton X-100 stainingsolution and incubated for 30 minutes at room tempera-ture. Fixed cells were then subjected to flow cytometricanalysis on the Beckman Coulter FC 500 Cytomics flowcytometer.Datawere analyzedwithModFit LT 3.0 (VerityHouse Software).

In vivo experiments and drug administrationAll animal studies were performed in accordance with

American Association for Laboratory Animal Care insti-tutional guidelines. The Eli Lilly and Company AnimalCare and Use Committee approved all the experimentalprotocols. Athymic nude female mice were inoculatedwith 0.2 mL of 1 � 107 A375 cells, prepared in a 1:1Matrigel to media mixture, in the hind flank region. Atotal of 60 mice, 8 to 10 in each group, were used forcompound treatments and vemurafenib-resistant modeldevelopment in each study. Vemurafenibwas formulatedbydissolving inDMSO inavolumeequivalent to 5%of thefinal formulation volume, and then the remaining volumewas added to the solution of 1% methylcellulose in dis-tilled water. CDK4/6 inhibitor LY2835219 was formulat-ed in 1% HEC in 20 mmol/L phosphate buffer, pH 2.0.Treatment was administered orally (gavage) with the

dose schedules described in each study. Tumor growthand body weight were monitored over time to evaluateefficacy.

Generation of cell lines from xenograft tumorsTumors were aseptically removed from the animals,

washed with cold PBS, then minced, and trypsinized in10-cm petri dish containing 5 mL of TrypLE reagent(Invitrogen) for 15 minutes at 37�C. Dislodged cellswere collected, resuspended, and plated in DMEM(Thermo Scientific) supplemented with 50% FBS (Invi-trogen) and 10% penicillin/streptomycin (Invitrogen).After 1 week, cells were switched to regular media(DMEM þ 10% FBS).

ResultsDevelopment of vemurafenib-resistant cell lines

To study the resistant mechanisms of B-RAF inhibition,we generated several vemurafenib-resistant cells outlinedin Table 1. A375R1, A375R3, M14R, and SH4R cells weregenerated by treating their respective parental cells withgradually increasing concentrations of vemurafenib, up to2 mmol/L as described previously (10). The resistantmechanism of A375R1, A375R3, and M14R cells wasassociated with RTK/RAS activation and MAPK reacti-vation (10). MAPK reactivation was also observed in SH4cells, but the underlying mechanism has not been fullycharacterized. A375R2 cells were generated by treatingA375 cells with a constant 2 mmol/L vemurafenib asdescribed (12). Consistent with the earlier reports, theresistance of A375R2 cells to vemurafenib is conferred byexpression of B-RAF splice variant (Supplementary Fig.S1A). The A375R4 cells were generated through stabletransfection of NRas Q61Kmutant and single clone selec-tion, which resulted in vemurafenib resistance (Supple-mentary Fig. S1B).

MAPK reactivation and cyclin D1 elevation invemurafenib-resistant cells and their sensitivity toCDK4/6 inhibitor LY2835219

As shown in Table 1, all in vitro generated resistantcell lines, A375R1-R4, M14R, and SH4R, demonstratedresistance to vemurafenib. Importantly, these resistantcells showed enhanced MAPK activation and cyclin D1elevation (Fig. 1). MAPK pathway activation and loss ofcell-cycle control are generally the hallmarks of mela-noma (2). Therefore, we used a selective CDK4/6 dualinhibitor LY2835219 and evaluated its growth-inhibito-ry effects in a panel of melanoma cell lines that are eithersensitive or resistant to vemurafenib mediated bydiverse mechanisms (Table 1). Interestingly, B-RAFV600E melanoma cells that are either sensitive tovemurafenib, such as A375, M14, and SH4, or resistantto vemurafenib, such as A375R1-4, M14R, and SH4R,demonstrated comparable sensitivity to LY2835219 withIC50 values ranging from 0.3 to 0.6 mmol/L (Table 1). Onthe contrary, B-RAF V600E mutant A2058 cells with denovo resistance to vemurafenib via MAPK-independent

Target Vemurafenib Resistance by the CDK4/6 Inhibitor LY2835219

www.aacrjournals.org Mol Cancer Ther; 13(10) October 2014 2255




mechanism (i.e., PTEN deletion) were relatively insen-sitive to LY2835219 (Table 1).

Antitumor effects of vemurafenib, LY2835219, andtheir combination in A375 xenograft model

In vitro analysis revealed that parental B-RAF V600Emelanoma cells, such as A375 cells, were sensitive to bothvemurafenib and LY2835219. To compare their activitiesin vivo, we tested their antitumor effects as single agents orin combination in an A375 xenograft model. As demon-strated in Fig. 2A, vemurafenib treatment at 15 mg/kgtwice daily induced significant tumor growth regressionin the first 2 weeks of treatment. Tumor growth was alsoinhibited by LY2835219 in a dose-dependent fashion (Fig.2B). Statistically significant tumor growth inhibition byLY2835219 was observed at 45 or 90 mg/kg once daily

dose schedule. Furthermore, analysis of tumor lysatesshowed that LY2835219 treatment significantly reducedpS780-Rb and pS10-Histone H3 levels, indicating inhibi-tion of cell cycle and adecrease in proliferating tumor cellsas a result of CDK4/6 inhibition (Fig. 2C).When xenografttumors were treated with a combination of vemurafeniband LY2835219, an additive antitumor growth effect wasobserved (Fig. 2D).

Development of in vivo vemurafenib-resistantmodeland efficacy of CDK4/6 inhibitor LY2835219 in thisresistant model

To evaluate the efficacy of LY2835219 in the vemura-fenib-resistant tumors, we developed an in vivo vemur-afenib-resistant model as shown in Fig. 3A. Following theestablishment of tumors, vemurafenib at 15 mg/kg wasadministered orally twice a day. Consistent with theprevious observations in thismodel, vemurafenib-treatedmice demonstrated significant regression in tumor vol-ume initially. We continued dosing with vemurafenibuntil resistance was evident. Approximately 40 to 45 daysafter dosing, many animals relapsed and resistant tumorsstarted to emerge. When vemurafenib-resistant tumorsreached sizes approximately 600 to 1,000 mg, the animalswere randomized into 2 groups, each 7 to 8 animals.One group continued to be dosed with vemurafenib,and the other group was switched to CDK4/6 inhibitorLY2835219 treatment alone at 90mg/kg once daily sched-ule. As demonstrated in Fig. 3A, LY2835219-treated micedemonstrated significant tumor growth regression,whereas the tumors in vemurafenib-treated mice contin-ued to grow. Furthermore, LY2835219-mediated tumorgrowth inhibition was maintained upon cessation of thetreatment. To rule out the possibility that the tumors haddeveloped a dependence on vemurafenib for continuedgrowth as previously described (14), we repeated the

Table 1. Overview of drug sensitivities, mutational status, andmechanisms of resistance of melanoma celllines tested in this study

IC50, nmol/L

Vemurafenib LY2835219

Cell line MutationSensitivity tovemurafenib Resistance mechanism Average SD Average SD Apoptosis

A375 B-RAFV600E Sensitive — 125 35 412 103 NoA375-R1 B-RAFV600E Resistant FGF-mediated ERK activation 4,541 939 407 54 YesA375-R2 B-RAFV600E Resistant p61–B-RAF splicing 3,364 1,449 361 149 YesA375-R3 B-RAFV600E Resistant FGF-mediated ERK activation 3,035 1,781 555 132 YesA375-R4 B-RAFV600E Resistant NRas Q61K 8,214 2,640 468 82 —

M14 B-RAFV600E Sensitive — 303 97 843 224 NoM14-R B-RAFV600E Resistant FGF-mediated ERK activation 5,337 1,190 538 171 YesSH4 B-RAFV600E Sensitive — 489 131 379 45 —

SH4-R B-RAFV600E Resistant Not characterized 7,775 827 374 33 —

A2058 B-RAFV600E Resistant PTEN inactivation 3,586 115 1,729 237 —

A375-RV1 B-RAFV600E Resistant p61–B-RAF splicing 4,413 1,808 221 67 YesA375-RV2 B-RAFV600E Resistant Not characterized 3,619 2,553 368 188 Yes

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Figure 1. MAPK reactivation and cyclin D1 elevation in vemurafenib-resistant melanoma cell lines.

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Figure 2. Antitumor activity of vemurafenib and LY2835219 in A375 xenograft model. A, antitumor activity of vemurafenib in A375 xenografts.Mice bearing subcutaneous A375 tumors were dosed with vehicle (n ¼ 8) or 15 mg/kg vemurafenib (n ¼ 10) twice daily. The y-axis is mean tumorvolume � SEM. B, antitumor activity of CDK4/6 inhibitor LY2835219 in A375 xenograft model. Mice bearing subcutaneous A375 tumors weredosed with vehicle (n ¼ 7), 22.5 mg/kg (n ¼ 5), 45 mg/kg (n ¼ 5), or 90 mg/kg (n ¼ 5) LY2835219 once daily for 21 days. C, dose-dependent reductionof CDK4/6 activity in A375 xenograft tumors by LY2835219. Mice bearing subcutaneous A375 tumors were dosed with vehicle, 22.5, 45, or 90 mg/kgof LY2835219 once daily. Tumors were collected 24 hours after the third dose. Tumor lysates were prepared and analyzed by immunoblottingusing indicated antibodies. D, activities of LY2835219, vemurafenib, and their combination in A375 xenograft model. Mice bearing subcutaneousA375 tumors were dosed for 21 days with either vehicle (n ¼ 8), 45 mg/kg LY2835219 (n ¼ 8), 10 mg/kg vemurafenib (n ¼ 8), or combinationof 45 mg/kg LY2835219 and 10 mg/kg vemurafenib (n ¼ 8). LY2835219 was dosed once daily and vemurafenib was dosed twice daily The brown linebelow the x-axis indicates the dosing (Rx) period in all studies. The y-axis is mean tumor volume � SEM. The pairwise comparisons of eachtreatment versus control are all statistically significant (P < 0.001). The tests between the combination group and each single agent group are alsostatistically significant (P < 0.001). The P value for the 2-way interaction to determine whether combinations are different from additive is notstatistically significant (P ¼ 0.657), thus indicating that the combination of these 2 agents is additive rather than synergistic.






same experiment with a vehicle control arm in addition tothe continued vemurafenib arm and the LY2835219 treat-ment group. In this study, both the vehicle control groupand the vemurafenib group showed indistinguishableand continued growth after vemurafenib withdrawal(Supplementary Fig. S2). These results suggest thatCDK4/6 inhibitor LY2835219 as a single agent is effectivein overcoming vemurafenib resistance in this in vivomodel.We further investigated themolecularmechanismbehind increased sensitivity of resistant tumors toLY2835219. We found hyperelevated levels of phospho-ERK, phospho-MEK, cyclin D1, and phospho-Rb (S780,S807, S811) in these vemurafenib-resistant tumors, indi-cating upregulation of MAPK and CDK activity (Fig. 3B).This observation is consistent with previous findings thatupregulation of cyclin D1 was associated with enhancedsensitivity of cancer cells to CDK4/6 inhibitors (30, 31).Interestingly, analysis of whole-exome sequencing dataderived from parental A375 and vemurafenib-resistantA375R1 cells revealed a copy number gain of 11q13 regionin theA375R1 cells and amplification of genes in the 11q13locus including CCND1, FGF3, FGF4, and FGF19 (Supple-mentary Table S1). Copy number variations ofCCND1 are

currently being evaluated in other resistant cell lines.Taken together, our results indicate that MAPK reactiva-tion and upregulation of cyclin D1 are associated withvemurafenib resistance and sensitivity to CDK4/6inhibition.

Cyclin D1 upregulation and vemurafenib resistancein cells generated from resistant xenograft tumors

To further understand the mechanism of vemurafenibresistance and increased sensitivity to LY2835219, wegenerated 2 cell lines from vemurafenib-resistanttumors, hereafter referred them to as A375RV1 andA375RV2 cells. Genotype analysis [short tandem repeat(STR)] confirmed that A375RV1 and A375RV2 have thesame genotypes of parental A375 cells. Compared withA375 cell line (vemurafenib IC50: 102 nmol/L),A375RV1 and A375RV2 cell lines maintained resistanceto vemurafenib with IC50 of 1,520 and 1,095 nmol/L,respectively (Fig. 4A and Table 1). We further charac-terized these cells and found that phospho-MEK andphospho-ERK levels remained high in the resistant cellsin the presence of vemurafenib concentrations as highas 3 mmol/L compared with parental A375 cells where

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Figure 3. Development of in vivovemurafenib-resistant A375xenograft model and activity ofCDK4/6 inhibitor LY2835219 inthe resistant model. A, efficacyof LY2835219 in an in vivomodelof acquired resistance tovemurafenib. Mice bearingsubcutaneous A375 tumorswere dosed with vehicle or15 mg/kg vemurafenib twicedaily after tumors wereestablished. Dosing wascontinued until the resistanttumors were evident. Uponemergence of resistant tumorswith size 600 to 1,000 mg, micewere randomized and dosedwith 90 mg/kg LY2835219 oncea day, or continued dosing with15 mg/kg vemurafenib twicedaily. The blue or red line underthe x-axis indicates the dosing(Rx) period of vemurafenibor LY2835219, respectively.The y-axis is mean tumorvolume � SEM. B, cyclinD1 elevation in vemurafenib-resistant tumors. Tumorlysates were analyzed byimmunoblotting using indicatedantibodies.

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phospho-MEK and p-ERK were diminished at concen-trations as low as 500 nmol/L (Fig. 4B and Supplemen-tary Fig. S3). In addition, we also detected expression ofp61-B-RAF splice variant in the A375RV1 cells (Fig. 4B),suggesting that one mechanism of resistance to vemur-afenib in this cell line is likely due to alterations in B-RAF splicing. Consistent with the observations from theanalysis of the tumor lysates, the cell lines derived fromthe resistant tumors also showed enhanced expressionof cyclin D1 (Fig. 4B and Supplementary Fig. S4C).

LY2835219 inducesG1 arrest in the parental cells butapoptosis in vemurafenib-resistant cells derivedfrom tumorsWe tested whether the vemurafenib-resistant cells

derived from tumors were sensitive to LY2835219 incell culture and found that LY2835219 inhibited theproliferation of the parental A375 and resistantA375RV1 and A375RV2 cells with similar potencieswith IC50 values of 395, 260, and 463 nmol/L, respec-tively (Fig. 5A). This is consistent with the responses ofother in vitro vemurafenib-resistant cells. It is wellestablished that inhibition of cyclin D1–CDK4/6 axis,and subsequent inactivation of Rb, arrests proliferatingcells in G1 stage of cell cycle. We performed PI stainingand FACS analysis of cell-cycle distribution to study the

mechanism of antiproliferative effects of LY2835219 inparental and vemurafenib-resistant cells. As expected,92% of parental A375 cells arrested in the G1 phase uponLY2835219 treatment (Fig. 5B). However, LY2835219treatment induced cell death in the resistant cells, upto 90% in the A375RV1 and 69% in A375RV2 cells, in 48hours as indicated by the presence of a subdiploid peakin the cytometry histograms. Similarly, LY2835219arrested 75% of parental M14 cells in the G1 stage butinduced cell death in 70% of vemurafenib-resistantM14R cells (Supplementary Fig. S4A). Thus, antitumoreffects of LY2835219 are differentially mediated inparental and resistant cells, although the IC50 valuesare similar in CellTiter Glo proliferation assay. Wefurther investigated whether LY2835219-induced celldeath in the resistant cells is mediated by apoptosis.As demonstrated in Figs. 5C and 6A, LY2835219 treat-ment significantly elevated caspase-3/7 activity inA375RV1 and A375RV2 cells and cleaved PARP activityin A375RV1 cells in a concentration-dependent fashion.Similarly, LY2835219 induced higher caspase-3/7 activ-ity and PARP cleavage in other vemurafenib-resistantA375R1 and M14R cells in a concentration-dependentmanner (Supplementary Fig. S4B), suggesting thatLY2835219 induces apoptosis preferably in the vemur-afenib-resistant cells.

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Figure 4. Cell lines derivedfrom vemurafenib-resistanttumors maintain resistance tovemurafenib. A, sensitivity of A375and tumor-derived resistantA375RV1 and A375RV2 cells tovemurafenib. Cell viability wasassessed using CellTiter Glo. B,cyclin D1 elevation and MAPKpathway reactivation in tumor-derived A375RV1 cells in thepresence of vemurafenib. Cellswere treated with indicatedconcentrations of vemurafenibfor 24 hours. Cell lysates wereanalyzed by immunoblottingusing indicated antibodies.






Upregulation of cyclin D1 is associated withLY2835219-induced apoptosis and important for thesurvival of vemurafenib-resistant tumor cells

Treatment with LY2835219 caused a concentration-dependent inhibition of phospho-Rb (S780, S807/S811)with similar potencies in parental and resistant cells,

indicating inhibition of CDK4/6 activity and inactivationof Rb function. Furthermore, LY2835219 treatmentinduced a concentration-dependent enhancement ofcyclin D1/D2 proteins in A375 cells, indicative of growtharrest in these cells (Fig. 6A). Consistent with the in vivodata, cyclin D1 levels remained high in vemurafenib-resistant tumor cells (Fig. 6A and B and SupplementaryFig. S4C). We explored whether vemurafenib-resistantcells are dependent on cyclin D1 for their survival. Weperformed shRNA-mediated selective knockdown ofcyclin D1 in the parental and vemurafenib-resistant cellsand found that the loss of cyclin D1 decreased phospho-Rb levels in both A375 and A375RV1 cells but inducedsignificantly higher levels of cleaved PARP and cleavedcaspase-3 fragments in A375RV1 cells (Fig. 6B). Similarly,specific knockdown of cyclin D1 via siRNA also inducedPARP cleavage in A375RV1 and other vemurafenib-resis-tant cells including A375RV2 and A375R1 cells (Supple-mentaryFig. S4C), indicating that loss of cyclinD1 inducesapoptosis in vemurafenib-resistant cells and not in theparental cells. Cyclin D1 knockdown did not affect phos-pho-ERK levels (Fig. 6B) or levels of other D-type cyclins,such as cyclin D2 (Supplementary Fig. S4C). Thus, weshow that vemurafenib-resistant cells are dependent oncyclin D1 for their survival.

DiscussionIn this study, we generated multiple in vitro cell lines

and an in vivo model resistant to vemurafenib anddiscovered that MAPK reactivation and cyclin D1 ele-vation are associated with acquired resistance in thesemodels. We further describe that inhibition of cyclinD1–CDK4/6 signaling by CDK4/6 inhibitor LY2835219is an effective therapy to overcome resistance. Expres-sion of B-RAF V600E splice variants, RTK/Ras activa-tion, NRas mutation, and B-RAF amplification are thepredominant clinical mechanisms of resistance tovemurafenib that have been observed to date, and allof these resistant mechanisms together with B-RAFmutation induce hyperactivation of the MAPK pathway(7, 12, 13). We found that the cells resistant to vemur-afenib with hyperactivation of the MAPK pathway haveelevated cyclin D1 expression and that they were sen-sitive to CDK4/6 inhibitor LY2835219. We subsequentlydeveloped a vemurafenib-resistant model in vivo andevaluated the antitumor effect of LY2835219 in thismodel. Consistent with the in vitro findings, phospho-ERK and cyclin D1 were elevated in these resistanttumors (Fig. 3B). More importantly, LY2835219 inducedregression of these vemurafenib-resistant tumors (Fig.3A). These results suggest that CDK4/6 inhibitionrepresents an effective therapeutic strategy to overcomevemurafenib resistance due to MAPK reactivation andassociated with cyclin D1 elevation.

To investigate the mechanism of LY2835219 sensitivityand vemurafenib resistance, we generated resistant cellslines from vemurafenib-resistant xenograft tumors.Molecular analysis of the tumor-derived resistant cell

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%G1= 69.39%S = 24.04%G2–M = 6.58

%G1= 92.57%S = 3.70%G2–M = 3.73

%G1= 82.81%S = 8.31%G2–M = 8.88

%G1= 58.08%S = 30.76%G2–M = 11.16

%G1= 58.33%S = 20.20%G2–M = 21.47

%G1= 46.14%S = 26.95%G2–M = 26.91

%G1= 54.68%S = 31.39%G2–M = 13.92

%G1= 53.07%S = 17.33%G2–M = 29.60

%G1= 42.48%S = 39.67%G2–M = 17.84%Debris = 88.77%Debris = 90.72

%Debris = 69.93%Debris = 37.41

Figure 5. CDK4/6 inhibitor LY2835219 is active against and inducesapoptosis in tumor-derived and vemurafenib-resistant cells. A, sensitivityof A375, A375RV1, and A375RV2 cells to LY2835219. Cell viabilitywas assessed using CellTiter Glo. B, cell-cycle analysis of A375 andA375RV1 cells treated by LY2835219. Cells were treated with indicatedconcentrationof LY2835219 for 48hours andsubjected toPI stainingandsubsequent FACS analysis of cell-cycle distribution. Dead cells areindicated as debris. C, caspase-3/7 activities in LY2835219-treatedA375, A375RV1, and A375RV2 cells. Cells were treated with increasingconcentrations (0, 0.3, 1, 3.3, and 5 mmol/L) of LY2835219 for 24 hours,and caspase-3/7 activity was determined using Caspase-Glo-3/7 Assay.

Yadav et al.





lines revealed hyperactivation of theMAPKpathway andan increase in cyclin D1 expression just as had beenobserved in the in vitro resistant cells. Similar to thexenograft studies in mice, resistant cells derived fromtumors retained resistance to vemurafenib, as well assensitivity to LY2835219 (Figs. 4A and 5A). We furtherexamined the mechanism of antiproliferative effects ofLY2835219 in parental and resistant cells. As expected,LY2835219 arrested parental cells in the G1 stage (Fig. 5Band Supplementary Fig. S4A), consistent with the role ofCDK4/6 function in progression of cells from G1 to Sstage of cell cycle (25). However, treatment of resistantcells with LY2835219 induced apoptosis within 24 to 48hours in a concentration-dependent fashion (Figs. 5Band C and 6A and Supplementary Fig. S4A and S4B).The surprising pro-apoptotic effects of LY2835219 wereobserved across a variety of vemurafenib-resistant celllines, including cells derived from resistant xenografttumors. Thus, vemurafenib-resistant B-RAF V600E mel-anomas with hyperactivated MAPK signaling andenhanced cyclin D1 expression are prone to apoptosisupon CDK4/6 inhibition by LY2835219, suggesting thatthese resistant cells are more dependent on cyclin D1–CDK4/6 signaling for survival.Both vemurafenib and LY2835219 were demonstrated

to inhibit tumor growth of B-RAF V600E melanoma insingle agent, and combination of these 2 resulted in anadditive tumor growth inhibition (Fig. 2). This suggeststhat upfront combination of CDK4/6 and B-RAF inhibi-tors may be more efficacious than single-agent therapy.However, a more robust tumor growth regression wasobserved when CDK4/6 inhibitor LY2835219 was usedfor the treatment of xenograft tumors acquired resistanceto vemurafenib (Fig. 3A). These results suggest thatvemurafenib followed by LY2835219 treatment schedulemight be a more effective approach than upfront combi-

nation of these 2 agents in delaying and overcomingresistance. Additional studies in defining these dose sche-dules are ongoing in preclinical models.

Previous studies revealed that cyclin D1–overexpres-sing cells demonstrate constitutive CDK activity andincreased sensitivity to CDK4/6 inhibitors (30, 31). CyclinD1was alsodemonstrated to be implicated in resistance toinhibitors of ERBB2, EGFR, and ER signaling (32, 33).Recent studies suggest that cyclin D1 overexpressionmaybe sufficient to render B-RAF V600E melanoma cellsresistant to B-RAF inhibition (28). CCND1 is amplified in11% of melanoma (2), including 17% of B-RAF V600Emelanoma (28), indicating that it could be a mechanismof de novo resistance to B-RAF inhibitors. Molecular anal-ysis of our vemurafenib-resistant tumors revealed signif-icant upregulation of cyclin D1 levels relative to parentaltumors (Fig. 3B). Consistently, cyclin D1 was also upre-gulated across most vemurafenib-resistant cell lines,including cell lines derived from resistant xenografttumors. The cyclin D1 upregulation could simply resultsfromMAPK reactivation, as it is a downstream effector ofMAPK signaling. To date, we have not fully characterizedthe cyclin D1 amplification in every resistant cell line.However, in at least A375R1 case, we found that thevemurafenib resistance was associated with 11q13 copynumber gain (Supplementary Table S1). This chromosom-al region includes the genes for FGF ligands as well asCCND1, potentially explaining both the cyclin D1 upre-gulation andCDK4/6 dependence described here, aswellas the previously reported FGF pathway activation (10).Analysis of cyclin D1 amplification in other resistant cellsis ongoing. We further investigated whether cyclin D1 isrequired for proliferation and survival of vemurafenib-resistant cells. Consistent with the pro-apoptotic effects ofLY2835219, knockdown of cyclin D1 by shRNA alsoinduced higher levels of apoptosis in vemurafenib-

+ ++ +

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BAFigure 6. Cyclin D1 and CDK4/6signaling is required for the survivalof vemurafenib-resistant B-RAFV600E melanoma cells. A, cellsignaling analysis in A375 andA375RV1 cells treated withCDK4/6 inhibitor LY2835219. Cellswere treated with indicatedconcentrations of LY2835219 for48 hours. Cell lysates wereanalyzed by immunoblotting usingantibodies indicated. B, cyclin D1knockdown by shRNA inducedapoptosis in vemurafenib-resistantcells. Cells were transduced withlentivirus encoding either control orcyclin D1 shRNA. Cell lysates werecollected 72 hours postinfectionand analyzed by immunoblottingusing indicated antibodies. Celllysates were collected 72 hoursposttransfection and analyzed byimmunoblotting using indicatedantibodies.






resistant versus parental cells (Fig. 6B and SupplementaryFig. S4C). However, these data do not entirely exclude thepossibility that either off-target effects of LY2835219 orinhibition of Rb-independent pathways by cyclin D1knockdown might also contribute to the induction ofapoptosis observed selectively in vemurafenib-resistantcells. Altogether, we demonstrate that cyclin D1 is impor-tant for survival of vemurafenib-resistant cells withhyperactivation of the MAPK pathway and cyclin D1upregulation and propose LY2835219, a CDK4/6 dualinhibitor, as a potential therapy to overcome suchresistance.

Disclosure of Potential Conflicts of InterestT.F. Burke, L. Huber, E.M. Chan, and R.P. Beckmann have ownership

interest (including patents) in Eli Lilly and Co. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: V. Yadav, S.G. Buchanan, E.M. Chan,R.P. Beckmann, S.-B. PengDevelopment of methodology: V. Yadav, T.F. Burke, L. Huber, R.D. VanHorn, Y. Zhang, E.M. Chan, R.P. Beckmann, S.-B. Peng

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Yadav, T.F. Burke, L. Huber, R.D. Van Horn,Y. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):V. Yadav, T.F. Burke, Y. Zhang, S.G. Bucha-nan, E.M. Chan, R.P. Beckmann, S.-B. PengWriting, review, and/or revision of themanuscript:V. Yadav, T.F. Burke,S.G. Buchanan, E.M. Chan, J.J. Starling, R.P. Beckmann, S.-B. PengAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. Yadav, S.-B. PengStudy supervision: V. Yadav, E.M. Chan, R.P. Beckmann, S.-B. Peng

AcknowledgmentsThe authors thankDr. Philip J. Elbert forDNAsequence andmutational

analysis of A375- and A375R1-resistant cell lines.

Grant SupportThis work was supported by Eli Lilly and Company.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch 25, 2014; revised July 30, 2014; accepted August 1, 2014;published OnlineFirst August 13, 2014.

References1. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al.

Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54.

2. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP,et al. A landscape of driver mutations in melanoma. Cell 2012;150:251–63.

3. Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, et al. Clinicalefficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 2010;467:596–9.

4. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J,et al. Improved survival with vemurafenib in melanoma with BRAFV600E mutation. N Engl J Med 2011;364:2507–16.

5. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA,et al. Inhibition of mutated, activated BRAF in metastatic melanoma.N Engl J Med 2010;363:809–19.

6. LongGV, TrefzerU,DaviesMA,KeffordRF, AsciertoPA,ChapmanPB,et al. Dabrafenib in patientswith Val600Glu or Val600LysBRAF-mutantmelanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial. Lancet Oncol 2012;13:1087–95.

7. Nazarian R, Shi H,Wang Q, Kong X, Koya RC, Lee H, et al. Melanomasacquire resistance to B-RAF(V600E) inhibition by RTK or N-RASupregulation. Nature 2010;468:973–7.

8. Su F, Bradley WD, Wang Q, Yang H, Xu L, Higgins B, et al. Resistanceto selective BRAF inhibition can be mediated by modest upstreampathway activation. Cancer Res 2012;72:969–78.

9. WagleN, EmeryC,BergerMF, DavisMJ, Sawyer A, PochanardP, et al.Dissecting therapeutic resistance to RAF inhibition in melanoma bytumor genomic profiling. J Clin Oncol 2011;29:3085–96.

10. Yadav V, Zhang X, Liu J, Estrem S, Li S, Gong XQ, et al. Reactivation ofmitogen-activated protein kinase (MAPK) pathway by FGF receptor 3(FGFR3)/Ras mediates resistance to vemurafenib in human B-RAFV600E mutant melanoma. J Biol Chem 2012;287:28087–98.

11. Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J,et al. Tumour micro-environment elicits innate resistance to RAFinhibitors through HGF secretion. Nature 2012;487:500–4.

12. Poulikakos PI, Persaud Y, JanakiramanM, Kong X, Ng C, Moriceau G,et al. RAF inhibitor resistance is mediated by dimerization of aberrantlyspliced BRAF(V600E). Nature 2011;480:387–90.

13. Shi H, Moriceau G, Kong X, Lee MK, Lee H, Koya RC, et al. Melanomawhole-exome sequencing identifies (V600E)B-RAF amplification-

mediated acquired B-RAF inhibitor resistance. Nat Commun 2012;3:724.

14. Das Thakur M, Salangsang F, Landman AS, Sellers WR, Pryer NK,Levesque MP, et al. Modelling vemurafenib resistance in melanomareveals a strategy to forestall drug resistance. Nature 2013;494:251–5.

15. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L,Johnson LA, et al. COT drives resistance to RAF inhibition throughMAP kinase pathway reactivation. Nature 2010;468:968–72.

16. Montagut C, Sharma SV, Shioda T, McDermott U, UlmanM, Ulkus LE,et al. Elevated CRAF as a potential mechanism of acquired resistanceto BRAF inhibition in melanoma. Cancer Res 2008;68:4853–61.

17. Paraiso KH, Xiang Y, Rebecca VW, Abel EV, Chen YA,MunkoAC, et al.PTEN loss confers BRAF inhibitor resistance to melanoma cellsthrough the suppression of BIM expression. Cancer Res 2011;71:2750–60.

18. Villanueva J, Vultur A, Lee JT, SomasundaramR, Fukunaga-KalabisM,Cipolla AK, et al. Acquired resistance to BRAF inhibitors mediated by aRAF kinase switch in melanoma can be overcome by cotargetingMEKand IGF-1R/PI3K. Cancer Cell 2010;18:683–95.

19. Shi H, Kong X, Ribas A, Lo RS. Combinatorial treatments that over-come PDGFRbeta-driven resistance of melanoma cells to V600EB-RAF inhibition. Cancer Res 2011;71:5067–74.

20. Olson OC, Joyce JA. Microenvironment-mediated resistance to anti-cancer therapies. Cell Res 2013;23:179–81.

21. Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, et al.Widespread potential for growth-factor-driven resistance to antican-cer kinase inhibitors. Nature 2012;487:505–9.

22. Kim KB, Kefford R, Pavlick AC, Infante JR, Ribas A, Sosman JA, et al.Phase II study of the MEK1/MEK2 inhibitor Trametinib in patients withmetastatic BRAF-mutant cutaneousmelanomapreviously treatedwithor without a BRAF inhibitor. J Clin Oncol 2013;31:482–9.

23. Hacker E, Muller HK, Irwin N, Gabrielli B, Lincoln D, Pavey S, et al.Spontaneous and UV radiation-induced multiple metastatic mela-nomas in Cdk4R24C/R24C/TPras mice. Cancer Res 2006;66:2946–52.

24. Hayward NK. Genetics of melanoma predisposition. Oncogene 2003;22:3053–62.

25. MalumbresM,BarbacidM. Tocycleor not to cycle: a critical decision incancer. Nat Rev Cancer 2001;1:222–31.


Yadav et al.




26. Smalley KS, Haass NK, Brafford PA, Lioni M, Flaherty KT, Herlyn M.Multiple signaling pathways must be targeted to overcome drugresistance in cell linesderived frommelanomametastases.MolCancerTher 2006;5:1136–44.

27. Bhatt KV, Spofford LS, Aram G, McMullen M, Pumiglia K, Aplin AE.Adhesion control of cyclin D1 and p27Kip1 levels is deregulated inmelanoma cells through BRAF-MEK-ERK signaling. Oncogene 2005;24:3459–71.

28. Smalley KS, Lioni M, Dalla Palma M, Xiao M, Desai B, Egyhazi S, et al.Increased cyclin D1 expression canmediate BRAF inhibitor resistancein BRAF V600E-mutated melanomas. Mol Cancer Ther 2008;7:2876–83.

29. Gelbert LM, Cai S, Lin X, Sanchez-Martinez C, Del Prado M, LallenaMJ, et al. Preclinical characterization of the CDK4/6 inhibitorLY2835219: in-vivo cell cycle-dependent/independent anti-tumor

activities alone/in combination with gemcitabine. Invest New Drugs.2014 Jun 13. [Epub ahead of print].

30. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, et al. PD0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibitsproliferation of luminal estrogen receptor-positive human breast can-cer cell lines in vitro. Breast Cancer Res 2009;11:R77.

31. Lapenna S, Giordano A. Cell cycle kinases as therapeutic targets forcancer. Nat Rev Drug Discov 2009;8:547–66.

32. Musgrove EA, Sutherland RL. Biological determinants of endocrineresistance in breast cancer. Nat Rev Cancer 2009;9:631–43.

33. Kalish LH, Kwong RA, Cole IE, Gallagher RM, Sutherland RL, Mus-grove EA. Deregulated cyclin D1 expression is associated withdecreased efficacy of the selective epidermal growth factor receptortyrosine kinase inhibitor gefitinib in head and neck squamous cellcarcinoma cell lines. Clin Cancer Res 2004;10:7764–74.






2014;13:2253-2263. Published OnlineFirst August 13, 2014.Mol Cancer Ther Vipin Yadav, Teresa F. Burke, Lysiane Huber, et al. UpregulationResistance Resulting from MAPK Reactivation and Cyclin D1 The CDK4/6 Inhibitor LY2835219 Overcomes Vemurafenib

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