a functional landscape of resistance to mek1/2 andcdk4/6inhibitioninnras-mutantmelanoma ·...

Translational Science

A Functional Landscape of Resistance to MEK1/2and CDK4/6 Inhibition in NRAS-Mutant MelanomaTikvah K. Hayes1,2, Flora Luo1,2, Ofir Cohen1,2, Amy B. Goodale3, Yenarae Lee3,Sasha Pantel3, Mukta Bagul3, Federica Piccioni3, David E. Root3, Levi A.Garraway4,Matthew Meyerson1,2, and Cory M. Johannessen2

Abstract

Combinatorial inhibition of MEK1/2 and CDK4/6 iscurrently undergoing clinical investigation in NRAS-mutantmelanoma. To prospectively map the landscape of resis-tance to this investigational regimen, we utilized a seriesof gain- and loss-of-function forward genetic screens toidentify modulators of resistance to clinical inhibitors ofMEK1/2 and CDK4/6 alone and in combination. First, weidentified NRAS-mutant melanoma cell lines that weredependent on NRAS for proliferation and sensitive toMEK1/2 and CDK4/6 combination treatment. We then useda genome-scale ORF overexpression screen and a CRISPRknockout screen to identify modulators of resistance to eachinhibitor alone or in combination. These orthogonal screen-ing approaches revealed concordant means of achieving

resistance to this therapeutic modality, including tyrosinekinases, RAF, RAS, AKT, and PI3K signaling. Activated KRASwas sufficient to cause resistance to combined MEK/CDKinhibition and to replace genetic depletion of oncogenicNRAS. In summary, our comprehensive functional geneticscreening approach revealed modulation of resistance to theinhibition of MEK1/2, CDK4/6, or their combination inNRAS-mutant melanoma.

Significance: These findings reveal that NRAS-mutantmelanomas can acquire resistance to genetic ablation ofNRASor combination MEK1/2 and CDK4/6 inhibition by upregu-lating activity of the RTK–RAS–RAF and RTK–PI3K–AKT sig-naling cascade.

IntroductionThere is a lack of effective therapies for NRAS-mutant melano-

ma, which accounts for 20% to 30% of all melanomas (1).Preclinical studies have demonstrated that oncogenic NRAS dys-regulates the MAPK signaling cascade, creating a dependency incell lines that can be exploited with inhibitors of MEK1/2 (2, 3).However, in clinical trials, single-agent MEK inhibitors (4) had amodest impact on progression-free survival (PFS; NCT01763164,ref. 5), suggesting that MEK as a single agent was insufficient toachieve durable responses.

The preclinical observation that CDK4/6 inhibition couldattenuate NRAS oncogenic signaling when combined with MEKinhibition supported the use of combined MEK1/2 and CDK4/6inhibitor combination in NRAS-mutant melanoma (6). A clinicaltrial (NCT01781572) designed to evaluate this combination inNRAS-mutant melanoma patients revealed multiple partialresponses (7, 8) and is under clinical evaluation in KRAS-mutant

colon (NCT02703571), lung (NCT03170206), and pancreatic(NCT02703571) cancer. Thus, understanding the resistance land-scape to MEK/CDK4/6 inhibition will be imperative for improv-ing long-term patient responses.

We used genome-wide functional genetic screening approachestomap the landscape of resistance toMEK1/2 inhibition, CDK4/6inhibition, and their combination in NRAS-mutant melanoma.Our analyses revealed that RTK–PI3K–AKT and RTK–RAS–RAFsignaling cascades were sufficient to drive resistance to combina-tion MEK1/2 and CDK4/6 inhibition. Our study provides aninitial description of the resistance landscape to MEK1/2 andCDK4/6 combination treatment in NRAS-mutant melanoma.

Materials and MethodsCell lines and reagents

Cells were maintained in DMEM (Hs936T, Hs944T; Gibco),RPMI1640 (MELJUSO, SKMEL30, IPC298; Gibco), or EMEM(SKMEL-2; Gibco) supplemented with 10% FBS (Sigma), andincubated at 37�C in 5% CO2 per ATCC guidelines.

Western blot reagentsCells were lysed in RIPA buffer (25 mmol/L Tris*HCl pH 7.6,

150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%SDS; phosphatase and protease inhibitors) and resolved by Tris-Gycline SDS-PAGE. To determine the levels of activated proteins,immunoblot analyses were done with phospho-specific antibo-dies to AKT(S473), MEK1/2(S217/S221), RB1 (S807/811), andERK1/2 (T202/Y204), S6 (235/236) with antibodies recognizingtotal AKT, RB, ERK1/2, and S6 to control for total protein expres-sion (Cell Signaling Technologies). Antibodies to EGFR, PI3K,CCNB1, CCND1, and CCNE2 (Cell Signaling Technologies), and

1Department of Medical Oncology, Dana-Farber Cancer Institute & HarvardMedical School, Boston, Massachusetts. 2Cancer Program, The Broad Instituteof M.I.T. and Harvard, Cambridge, Massachusetts. 3Genetic Perturbation Plat-form, The Broad Institute of M.I.T. and Harvard, Cambridge, Massachusetts. 4EliLilly Oncology, Eli Lilly Company, Indianapolis, Indiana.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Cory M. Johannessen, Broad Institute of Harvard andMIT, 7 Cambridge Center, Room 4023A, Cambridge, MA 02142. Phone: 617-388-2684; Fax: 617-714-7610; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-2711

�2019 American Association for Cancer Research.

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NRAS and phospho-AKT (Santa Cruz) were used tomonitor totalprotein expression. Antibody to KRAS4B was obtained fromCalbiochem. Antibodies for cleaved PARP (Cell Signaling Tech-nologies) were used to monitor apoptosis. Antibody for b-actin(Sigma AC15) was used to verify equivalent loading of totalcellular protein.

Small molecule inhibitorsTrametinib and palbociclib were purchased from Selleckchem.

Trametinib was dissolved in DMSO and stored at stock concen-trations of 10 mmol/L at �20�C. Palbociclib was dissolved inwater and stored at stock concentrations of 10 mmol/L at�20�C.

siRNA transfectionssiRNA silencer select oligonucleotides against scrambled and

NRAS sequences were obtained from Invitrogen and transfectedinto cells by using Lipofectamine RNAiMAX, according to themanufacturer's instructions.

Lentiviral expression vector infectionsThe pLX317 GFP, AKT1, AKT2, AKT3, PI3K H1047R, PI3K

E545K, EGFR L858R, NRAS Q61L, NRAS Q61K, KRAS WT, orKRAS G13D puromycin lentivirus vector were provided byThe Broad Institute Genetic Perturbation Platform, and weretransiently transfected into 293T cells with a D8.9 and VSV-Gpackaging system using XtremeGENE9. Infection of melano-ma cell lines was done in growth media supplemented with5 mg/mL polybrene and selected with 2 mg/mL of puromycinfor 72 hours.

Lentiviral CRISPR vector infectionsThe pLC_V2 GFP and sgRNA RB blasticidin lentivirus vector

were constructed and were transiently transfected into 293T cellswith a D8.9 and VSV-G packaging system using XtremeGENE9.Infection of melanoma cell lines was done in growth mediasupplemented with 5 mg/mL polybrene and selected with 2 mg/mLof blasticidin for 72 hours. Cells were allowed to grow for 7 dayspostinfection before initiation of experiments.

Anchorage-dependent growth assaysTo monitor proliferation, cells were plated into 96-well

plates at a density of 1 � 103 (MELJUSO, Hs944T, Hs936T,and IPC298) and 2 � 103 (SKMEL30 and SKMEL2) cells perwell. To quantitate cell number, after 6 days cells were stainedwith 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) and absor-bance was measured at 490 nm. We also performed a secondproliferation assay to monitor clonogenic growth. Cells wereplated at 5 � 103 (MELJUSO, Hs944T, and Hs936T) and 10 �103 (SKMEL30) cells per well, but in 12-well plates. After 7 to 10days, paraformaldehyde-fixed cells were stainedwith crystal violetto visualize and quantitate colony growth.

Determination of GI50To determine the 50% growth inhibitory concentration (GI50),

1 to 2 � 103 cells were seeded in 96-well plates and treated withconcentrations of trametinib (from 3 nmol/L to 1 mmol/L) orpalbociclib (from 30 nmol/L to 10 mmol/L). After 6 days oftreatment, cell proliferation was analyzed with MTS and read atan absorbance of 490nm.GI50 valueswere calculatedwith PRISMsoftware.

Cell-cycle assayCell-cycle progression was quantified by flow cytometry as

described (Nusse and colleagues 1990). Cells were treated at GI50or siRNA for 96 hours and then washed with PBS, fixed in cold70% ethanol, and then stained with propidium iodide (PI).Modfit software was used for analyses.

Caspase-Glo 3/7 assayApoptosis assays were performed according to the manufac-

turer's instructions. Staurosporine was used at 1mmol/L for 4hours as a positive control.

STRING analysisORF and CRISPR candidates were collectively uploaded

to https://string-db.org/. Clusters reflect high confidenceinteractions.

Trametinib and palbociclib titration for ORF and CRISPRscreens

The doses of trametinib, palbociclib, or their combination touse in these screens were determined by propagating cells indifferent concentrations of trametinib, palbociclib, and theircombination to determine the effect on cell proliferation. Inparallel, the level of phospho-ERK or phospho-RB depletion incells treated with different concentrations of trametinib, palbo-ciclib, or their combination was determined. For the proliferationassay, 1� 106MELJUSOcells were seeded in 10 cmplateswithoutdrug. MELJUSO cells were allowed to adhere for 24 hours, afterwhich fresh media containing different concentrations of trame-tinib, palbociclib, or their combination was added. MELJUSOcells were passaged or media was refreshed every 3 days, and cellswere counted at each passage. For immunoblots, MELJUSO cellswere treated with DMSO or the indicated concentrations oftrametinib, palbociclib, or their combination for 48 hours. Phos-pho-ERK or phospho-RB levels were assessed by immunoblotanalysis.

Human ORFeome and avana CRISPR lentiviral librariesHuman ORFeome barcoded library was cloned in the pLX317

barcoded vector and contains 17,255ORFsoverexpressing 10,135distinct human genes with at least 99% nucleotide and proteinmatch (Yang and colleagues 2011). The creation, cloning,sequencing, and production of the Avana library have beenpreviously described (Doench and colleagues 2016). The Avanalibrary contains 73,687 barcoded guide RNAs (sgRNAS) targeting18,675 genes and 1,000 nontargeting guides (Doench and col-leagues 2016).

Genome scale ORF resistance screenTo titer the ORF library in MELJUSO cells, 3 � 106 cells were

seeded per well in a 12-well plate and were infected with differentamounts of virus (0, 50, 100, 150, 200, 400 mL), with a finalconcentration of 4 mg/mL polybrene. Cells were spun for 2 hoursat 2,000 rpm at 30�. Approximately 6 hours after infection,100,000 cells from each infection were seeded into duplicatewells in a six-well plate. Twenty-four hours after infection, onewell was treated with puromycin and one withmedia alone. After2 to 3 days of selection, cells were counted to determine theamount of virus that resulted in 30% to 50% infection efficiency,and this amount of virus was used in the screen. For theMELJUSOORF resistance screen, 1 � 108 cells were infected per replicate

Resistance to MEK and CDK Inhibition in NRAS-Mutant Melanoma

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with 40% infection efficiency, in order to obtain 1,000 cells perORF after selection (�2 � 107 surviving cells containing17,255 ORF clones). A total of 3 � 106 cells per well wereseeded in 12-well plates and were infected with the amount ofvirus determined during optimization, with a final polybreneconcentration of 4 mg/mL. Plates were spun for 2 hours at2,000 rpm at 30�. Approximately 24 hours after infection, allwells within a replicate were pooled and were split into T225flasks. Forty-eight hours after infection, cells were selected in 2mg/mL puromycin for 5 days and expanded in puromycin-freemedia for 4 days (MELJUSO). For MELJUSO, 4.0 � 107 cellswere seeded in T225 flasks with 10 nmol/L trametinib, 2mmol/Lpalbociclib, and 10 nmol/L trametinib/2mmol/L palbociclib onDay 0. Cells were passaged in drug or fresh media containingtrametinib, palbociclib, or trametinib/palbociclib was addedevery 3 to 4 days. Cells were harvested 21 days after initiationof trametinib, palbociclib, or trametinib/palbociclib treatment.Genomic DNA was extracted using the Qiagen Blood and CellCulture DNA Maxi Kit according to the manufacturer's protocol.PCR of gDNA and pDNA (ORF plasmid pool used to generatevirus) was performed as previously described (Doench and col-leagues 2016).

Genome scale CRISPR resistance screenTo generate cell lines stably expressing Cas9, cells were infected

with the Cas9 expression vector pXPR_BRD111 and selected with10 mg/mL blasticidin for 4 to 7 days. Cas9-expressing cells weremaintained in 2 to 5 mg/mL blasticidin. To titer the Avana libraryin Cas9-expressing cells, 3 � 106 cells were seeded per well in a12-well plate andwere infectedwith different amounts of virus (0,50, 100, 150, 200, 400 mL), with a final concentration of 4 mg/mLpolybrene. Cells were spun for 2 hours at 2,000 rpm at 30�.Approximately 6 hours after infection, 100,000 cells from eachinfection were seeded into duplicate wells in a six-well plate.Twenty-four hours after infection, one well was treated withpuromycin and one with media alone. After 2 to 3 days ofselection, cells were counted to determine the amount of virusthat resulted in 30% to 40% infection efficiency, and this amountof virus was used in the screen. For theMELJUSOAvana resistancescreen, 1�108 cells were infected per replicatewith 40% infectionefficiency, in order to obtain 500 cells per sgRNA after selection(4.0� 107 surviving cells containing�77,000 sgRNAs). A total of3 � 106 cells per well were seeded in 12-well plates and wereinfected with the amount of virus determined during optimiza-tion, with a final polybrene concentration of 4 mg/mL. Plates werespun for 2 hours at 2,000 rpm at 30�. Approximately 6 hours afterinfection, all wells within a replicate were pooled and were splitinto T225 flasks. Twenty-four hours after infection, cells wereselected in 2 mg/mL puromycin for 5 days and expanded inpuromycin-free media for 4 days (MELJUSO). For MELJUSO,4.0 � 107 cells were seeded in T225 flasks with 10 nmol/Ltrametinib, 2 mmol/L palbociclib, and 10 nmol/L trametinib/2mmol/L palbociclib onDay 0. Cells were passaged in drug or freshmedia containing trametinib, palbociclib, or trametinib/palbo-ciclib was added every 3 to 4 days. Cells were harvested 21 daysafter initiation of trametinib, palbociclib, or trametinib/palboci-clib treatment. Genomic DNA was extracted using the QiagenBlood and Cell Culture DNA Maxi Kit according to the manu-facturer's protocol. PCR of gDNA and pDNA (sgRNA plasmidpool used to generate virus) was performed as previouslydescribed (Doench and colleagues 2016).

Analysis of ORF screenThe log2 (fold-change) in ORF representation between cells

treatedwith trametinib, palbociclib, or trametinib/palbociclib for21 days and the initial early time point of ORFs plasmid used togenerate virus was calculated. Then a Z-score was calculated and aORF was considered significant if the Z-score �2 (See Supple-mentary Table S1).

Analysis of CRISPR screenThe log2 (fold-change) in sgRNA representation between cells

treatedwith trametinib, palbociclib, or trametinib/palbociclib for21 days and the initial pool of sgRNAs plasmid used to generatevirus was calculated. Then a Z-score (x� 2) was calculated. A genewas considered significant if at least half of the sgRNAs targetingthe gene became enriched in the screen. STAR values were alsocalculated for each gene (see Supplementary Table S1).

ResultsNRAS-mutant melanoma cell lines are NRAS dependent

To determine the relationship between NRAS mutation andNRAS dependency in melanoma cell lines, we analyzed shRNAand CRISPR dependency data from the Broad Institute Depen-dency Map (https://depmap.org/portal/; refs. 9, 10). Theseanalyses revealed that five of five NRAS-mutant melanoma celllines were dependent on NRAS compared with NRAS wild-typecell lines (Fig. 1A). To validate this observation, we suppressedNRAS expression in three out of four cell lines identified in theBroad Institute Dependency Map using two NRAS targetingsiRNAs. After 48 hours, we observed a greater than 90% NRASknockdown compared with the siRNA control (Fig. 1B). NRASdepletion reduced cell viability after 4 days in an MTS assay and10 days in an anchorage-dependent clonogenic growth assay(Fig. 1C and D). We observed that siNRAS #1 was mostlyineffective in the MELJUSO cell line, which harbors bothactivating NRAS and HRAS mutations. After performing qPCRto evaluate mRNA levels of HRAS and NRAS, we found thatsiNRAS #1 targets only NRAS, whereas siNRAS #2 targets bothNRAS and HRAS.

To assess the mechanism(s) through which NRAS suppres-sion impairs growth, we investigated the modulation of cleavedPARP a well-established marker of cell death. We observed anincrease in cleaved PARP total protein levels after 96 hoursunder NRAS siRNA targeting conditions in Hs936T andHs944T, but not MELJUSO and SKMEL30 (Supplementary Fig.S1A). In addition, we observed increased activity of caspase-3and caspase-7, known markers of apoptosis, in both Hs936Tand Hs944T after 96 hours of NRAS suppression (Supplemen-tary Fig. S1B).

Upon NRAS suppression, a subpopulation of cells under-went programmed cell death, while a remaining population didnot. To explore this observation, we evaluated the effect ofNRAS depletion after 96 hours on cell-cycle progression using aPI stain. Our analysis revealed a significant increase in G1

arrested cells upon loss of NRAS (Supplementary Fig. S1C).This was also accompanied by a reduction in the number ofcells in both S-phase and G2–M. Consistent with our PI stainingresults, we observed a reduction in the steady-state proteinlevels of phospho-RB, cyclin B1, cyclin D1, and cyclin E2,markers of cell-cycle progression (Supplementary Fig. S1D).Thus, although populations of NRAS-mutant melanoma cell

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Figure 1.

NRAS-mutant melanoma cell lines are NRAS-dependent and sensitive to trametinib and palbociclib. A, Dependency scores (CRISPR vs. shRNA) identify NRAS-dependent melanoma cell lines. Red circles, NRASmutant; black circles, NRASWT (19, 20). B, Representative immunoblots displaying the effect of NRASknockdown after 48 hours on total protein levels of NRAS. Vinculin immunoblotting was used to determine equivalent loading. C, CellTiter MTS tetrazoliumassay displayingmean cell viability of MELJUSO, SKMEL30, Hs936T, and Hs944Tmelanoma cell lines with or without knockdown of NRAS (six replicates), eachfrom three independent experiments. D, Representative images of crystal violet–stained clonogenic growth assays with MELJUSO, SKMEL30, Hs944T, andHs936T melanoma cell lines with or without knockdown of NRAS. E,MTS assays showing mean cell viability of MELJUSO, Hs944T, SKMEL30, and Hs936Tmelanoma cell lines after 144 hours of combination trametinib and palbociclib treatment, each from three independent experiments performed in triplicate.F, Normalized cell viability score where DMSOwas 100% cell viability as measured by MTS in MELJUSO, Hs944T, SKMEL30, Hs936T, IPC298, and SKMEL2.G, Representative images of crystal violet–stained clonogenic growth assays with MELJUSO and Hs944Tmelanoma cell lines after 10 days of trametinib andpalbociclib treatment, each from three independent experiments, performed in duplicate. H, Representative immunoblots displaying the effect of trametiniband palbociclib treatment in MELJUSO and Hs944T melanoma cell lines after 48 hours on phosphorylated and total protein levels of RB, AKT, and ERK. b-Actinimmunoblotting was used to determine equivalent loading.






lines are dependent on NRAS for growth, their response toNRAS depletion is heterogeneous, with a subset undergoing cellarrest, and others undergoing apoptosis.

NRAS-dependent melanoma cell lines are sensitive topharmacologic MEK and CDK inhibition

Next, we evaluated whether our panel of NRAS-mutantmelanoma cell lines were sensitive to pharmacologic inhibitionof MEK1/2 and CDK4/6. To test this, we used two FDAapproved inhibitors, trametinib and palbociclib and deter-mined whether our panel of cell lines was sensitive to theseagents. NRAS mutant melanoma cell line sensitivity, after 72and 144 hours, to each single agent varied ranging from 1 to 10nmol/L GI50 for trametinib and 1 to 10 mmol/L GI50 forpalbociclib (Supplementary Fig. S1E).

Next, we evaluated the effect of combination trametinib andpalbociclib treatment in our panel. We performed an MTSassay, where we treated each cell line with increasing doses ofeach drug and measured cell viability after 144 hours. Weobserved that each cell line was sensitive to each drug individ-ually as well as in combination (Fig. 1E; Supplementary Fig.S1F). By averaging the percent cell viability count of each wellcompared with the vehicle control, we calculated a NormalizedCell Viability Score to represent dual inhibitor sensitivity(Fig. 1F). Based on the Normalized Cell Viability Score, ourpanel of NRAS mutant melanoma cell lines are differentiallysensitive to dual inhibition of MEK1/2 and CDK4/6. To elu-cidate the effect of combination trametinib and palbociclibtreatment on anchorage-dependent colony formation, we trea-ted MELJUSO, SKMEL30, Hs936T, and Hs944T with varyingdoses of trametinib (10 and 20 nmol/L) and palbociclib (100nmol/L, 0.5 mmol/L, and 1 mmol/L) as single agents as well asthe combination for 10 days (Fig. 1G). Consistent with ourprevious observations, each cell line was sensitive to both singleagents as well as the combination, although the dose–responsevaried between cell lines.

In parallel, we assessed the levels of pathway inhibition aftertreatment with trametinib and palbociclib, alone, and in combi-nation after 48 hours. Treatment with single agent trametinibreduced steady-state levels of phospho-ERK and phospho-RB inall cell lines tested (Fig. 1H; Supplementary Fig. S1G). It has beenpreviously shown thatMAPK pharmacologic inhibition can resultin changes to steady-state levels of phospho-AKT; in contrast, inour model, phospho-AKT levels remained constant after treat-ment with trametinib.

Next, we evaluated the effect of single-agent palbociclib treat-ment in our panel of cell lines.Weobservedpalbociclib inhibitionled to a reduction in steady state levels of phospho-RB (Fig. 1H;Supplementary Fig. S1G and S1H) and an increase in CCND1(Supplementary Fig. S1H) total protein levels, both of which areconsistent with previous studies (7, 11). Palbociclib inhibitiondid not alter the MAPK signaling components RAF and MEK,however we did observe an increase in phospho-ERK levels(Fig. 1H).

Finally, we examined the effects of combination trametinib andpalbociclib treatment on pathway inhibition. We observed areduction in steady-state levels of phospho-ERK and phospho-RB; however, unexpectedly, combination treatment did not sig-nificantly further reduce phospho-RB levels, in three of fourNRAS-mutant melanoma cell lines, with Hs936T being inconclu-sive (Fig. 1H).

To assess the mechanism(s) through which trametinib andpalbociclib, alone, and in combination, impair growth in ourpanel of NRAS-mutant melanoma cell lines, we examined levelsof cleaved PARP and cleaved caspase-3. We did not observechanges to cleaved PARP or cleaved caspase-3 total protein levelsafter either 48 or 96 hours of inhibitor treatment. This is incontrast to previous studies, where cell line models of NRAS-mutant melanoma underwent programmed cell death after treat-ment with either a MEK inhibitor alone or in combination with aCDK4/6 inhibitor (6). However, this could be a result of usingdistinct melanoma cell lines, as our data support the hypothesisthat NRAS-mutant melanoma cell lines are heterogeneous.

To understand the mechanisms of growth impairment follow-ing combination drug treatment, we evaluated the effect oftrametinib and palbociclib, alone, and in combination on cell-cycle progression.We detected a significant increase in G1 arrestedcells and a reduction in the number of cells in both S phase andG2–M after 96 hours of inhibitor treatments (Supplementary Fig.S1I). This observation is consistent with other studies evaluatingthe effect of trametinib and palbociclib on cell-cycle progression.

Next, we examined changes to cell-cycle markers after combi-nation treatment. Protein levels of phospho-RB, cyclin B1, andcyclin D1 were reduced after inhibitor treatments at 96 hours(Supplementary Fig. S1H). Given our observations with bothgenetic NRAS-depletion and pharmacologic inhibition ofMEK1/2 and CDK4/6 in NRAS mutant melanoma cell lines, weconclude these perturbations share a similarmechanism of actionas it relates to cell-cycle progression. However, it is clear that asubset of cell lines undergo programmed cell death upon NRASdepletion,which isnot the casewithpharmacological inhibitionofMEK1/2 and CDK4/6 in our model. Together, our results supportthe hypothesis that NRAS-dependent melanoma cell lines aresensitive to pharmacologic inhibition of MEK1/2 and CDK4/6.

A functional landscape of resistance to pharmacologicinhibition of MEK1/2 and CDK4/6 in NRAS-mutant melanomausing an ORF expression library

To elucidate the resistance landscape to MEK1/2 and CDK4/6inhibition in NRAS mutant melanoma, we performed a neargenome-wide pooled ORF overexpression screen to identifygenetic modulators of resistance (12–16). We performed athree-armed screen in NRAS mutant MELJUSO cell lines usingtrametinib (10 nmol/L), palbociclib (2 mmol/L), and trametinib/palbociclib (10 nmol/L/2 mmol/L; Fig. 2A; SupplementaryFig. S2A, S2B, and S2C; ref. 17).

Our screening results revealed distinct ORFs whose overexpres-sion caused resistance to trametinib and/or palbociclib (Fig. 2B–D). In the trametinib arm, we identified several genes involved intheMAPK signaling cascade, consistentwith previously publishedscreens using MAPK inhibition (Fig. 2B; refs. 12, 18, 19). In thepalbociclib arm, we observed a significant enrichment of genesinvolved in the PI3K–AKT signaling cascade (Fig. 2C). In thecombination trametinib/palbociclib arm, activated KRAS ORFswere the top scoring candidates (Fig. 2D). We observed somecandidate overlap between trametinib and palbociclib singleagents in theDNA and RNAbinding proteins, G-protein–coupledreceptors (GPCR), tyrosine and serine/threonine protein kinases,and transcription factors (Fig. 2E and F). In the combination,network analysis of resistance candidates using the STRINGprotein–protein interaction platform identified a Ras signalinginteractome and a GPCR interactome (Supplementary Fig. S2D).

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Figure 2.

Systematic genetic ORF expression screen to understand resistance to trametinib and palbociclib in NRAS-mutant melanoma cell lines. A, Screening schematicsand timelines. B–D,MELJUSO cell line stably expressing the Human ORFeome Pooled Library Version 8 was treated for 21 dayswith trametinib (B), palbociclib(C), and trametinib/palbociclib combination (D). E, Venn diagram of screening candidates with a z-score greater than 2, trametinib (125), palbociclib (125), andtrametinib/palbociclib (125). F, Heatmap displaying the overlap of candidates identified in each arm of the screen based on the z-score.G and H, Identification ofco-occuring mutations in NRAS-mutant melanoma from patient samples obtained from cBioPortal (www.cbioportal.org).





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As there are limited clinical samples available for the evaluationof resistance to this combination (7, 8), we used cBioPortal(http://www.cbioportal.org/) to determine whether proteinsidentified in the ORF overexpression screen were altered eitherthrough point mutation or amplification in NRAS-mutant mel-anoma. We observed that ARAF, KRAS, AKT1, PIK3CA, RAF1,BRAF, and AKT3 were either mutated (hotspot) or amplified inNRAS-mutant melanoma (Fig. 2G and H), suggesting that co-occurrence of these alterationswithNRASmutation is plausible innature.

A functional landscape of resistance to pharmacologicinhibition of MEK1/2 and CDK4/6 in NRAS-mutant melanomausing a CRISP knockout library

Next, we performed a CRISPR knockout screen and assessedresistance to MEK1/2 and CDK4/6 inhibition in NRAS-mutantmelanoma. We performed a three-armed screen in MELJUSOcell lines using trametinib (10 nmol/L), palbociclib (2 mmol/L),and trametinib/palbociclib (10 nmol/L/2 mmol/L; Fig. 3A;Supplementary Fig. S3A and S3B; refs. 20, 21). In the trame-tinib arm, we observed an enrichment in negative regulators ofboth the EGFR-PI3K and EGFR-Ras signaling cascades. We alsoidentified several PPP family phosphatases, transcription fac-tors, E3 ligases, and epigenetic regulators. Overall, our CRISPRscreening results support mechanisms of resistance identifiedin the trametinib arm of the ORF overexpression screen(Fig. 3B).

Two independent studies evaluated the resistance landscapeto trametinib in KRAS mutant (PDAC and LUAD) and NRASmutant (LUAD) cancers (22, 23). After analyzing all fourscreens, we found a subset of candidate genes associated withresistance to trametinib in two or more contexts, whereas amajority of candidates were unique to each cellular context(Supplementary Fig. S3C). The comparison of the melanomaNRAS screen and the PDAC KRAS screen appears informativebecause these screens use the same CRISPR libraries revealingCIC, KEAP1, and PPP6C to modulate MEK inhibitor sensitivity(Supplementary Fig. S3D). From these data, we conclude thereare likely resistance mechanisms to MEK inhibition that spandisease and lineage boundaries.

The top scoring guides from the palbociclib arm of theCRISPR screen were RB1 targeting guides, which is consistentwith the known mechanism of action for palbociclib (Fig. 3C;refs. 24, 25). We utilized two sgRNAs that efficiently knockedout RB1 as measured by protein expression (SupplementaryFig. S3E) and led to palbociclib resistance (SupplementaryFig. S3F). In addition, we identified multiple guides targetingNF2, PTEN, RBL2, and RALGAPB, all of which are regulatorycomponents of the PI3K–AKT–mTOR signaling cascade (26)and are consistent with our results from the ORF expressionscreen.

Similar to our observations in the ORF screen, there were farfewer genes whose deletion could drive resistance to combinationMEK/CDK inhibition relative to those discovered for single agents(Fig. 3D and E). In addition, many of the top scoring genes onlyhad a single scoring guide in the presence of the combination,technically limiting our ability to identify genes that drive resis-tance to the combination. However, we did observe RB1, whichhas been linked to Ras signaling and has been implicated inresistance to CDK4/6-directed therapeutics. Together with theORF screening data, these findings suggest that few genes beyond

Ras, itself, can overcome combination trametinib/palbociclibtreatment.

Similar to the ORF overexpression screen, we observed sev-eral CRISPR candidates that were either truncated or deleted inpatient samples (NF2, TSC1, FBXW7, RB1, PTEN, TSC2, PPP6C,and TP53; Fig. 3F), again suggesting that co-occurrence withNRAS is feasible in human tumors (cbioportal). Together theORF and CRISPR screening results highlight modulators toMAPK and PI3K signaling cascades that are clinically plausible(Fig. 3G).

After performing both the ORF overexpression and the CRISPRdepletion screens, we gathered potential candidates based on theaforementioned statistical measurements and conducted aSTRING analysis, which is an unbiased pathway network analysistool. From these analyses, we found that the RTK–RAS–RAF andRTK–PI3K–AKT pathways were among the most highly enrichedpathways (Supplementary Fig. S3G). Given the tractable clinicaltargets, we focused on validating various members of these twosignaling cascades.

EGFR–PI3K–AKT signalingmodulates resistance to trametinib/palbociclib treatment in NRAS-mutant melanoma cell lines

Next,we validated if the EGFR–PI3K–AKT signaling cascade canimpact response to trametinib and/or palbociclib. The ORF over-expression screen revealed thatwild-typeAKT, activated PI3K, andactivated EGFR all conferred resistance, thus we chose validationconstructs representing this observation (Supplementary TableS1). We infected MELJUSO, Hs944T, SKMEL30, and Hs936Tcell lines with GFP, AKT1, AKT2, PI3KH1047R, PI3KE545K, andEGFRL858R, and then treated them with trametinib and/or palbo-ciclib in a 6 days cell viability assay. Consistent with our previousobservation that NRAS mutant melanoma cell lines are hetero-geneous, our screening validation data highlight distinct mod-ulators of resistance amongst the cell lines assayed. We observedthat AKT1, AKT2, PI3KH1047R, PI3KE545K, or EGFRL858R expressionled to resistance to trametinib and/or palbociclib in MELJUSOand Hs936T (Fig. 4A and B; Supplementary Fig. S4A and S4B),whereas only PI3KH1047R or PI3KE545K mediated resistance inSKMEL30 and Hs944T (Fig. 4C and D; Supplementary Fig. S4Cand S4D). AKT3 caused resistance to both inhibitors alone and incombination in the MELJUSO, SKMEL30, and Hs936T cell lines(Supplementary Fig. S4E and S4F).

Next, we determined the effect of the EGFR–PI3K–AKT signal-ing cascade on clonogenic growth. InMELJUSO, we observed thatexpression of AKT1, AKT2, PI3KH1047R, and PI3KE545K causedresistance to trametinib (Fig. 4E), whereas EGFRL858R had amodest effect. Notably, PI3KH1047R and PI3KE545K only mediatedtrametinib resistance in Hs944T (Fig. 4F). Expression of AKT1alone in the Hs944T cell line caused an unexpected, yet repro-ducible, growth defect. In MELJUSO and Hs944T, expressingAKT1 (only MELJUSO), AKT2, PI3KH1047R, PI3KE545K orEGFRL858R drove resistance to palbociclib (Fig. 4E and F). Weobserved a similar pattern of insensitivity in cells treated with thecombination, though resistance phenotypes were reduced forAKT2 and EGFR. These results are consistent with our cell viabilityfindings. Overall, our data suggest that the EGFR–PI3K–AKTsignaling cascade can modulate resistance to trametinib and/orpalbociclib, although the strength of resistance is cell linedependent.

Next, we assessed pathway inhibition in the MELJUSO,SKMEL30, Hs936T, and Hs944T cell lines expressing AKT1, PI3K,

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http://www.cbioportal.org/


Figure 3.

Systematic CRISPR knockout screen to evaluate resistance to trametinib and palbociclib in NRAS-mutant melanoma cell lines. A, Screening schematics andtimelines. B–D,MELJUSO cell line stably expressing the Avana Pooled Library was treated for 21 dayswith trametinib (B), palbociclib (C), and trametinib/palbociclib combination (D). E, Venn diagram of screening candidates with a z-score and guide number greater than or equal to 2, trametinib (150), palbociclib(150), and trametinib/palbociclib (150). F, Identification of co-occuring mutations in NRAS-mutant melanoma from patient samples obtained from cBioPortal(www.cbioportal.org). G, EGFR signaling pathway displaying candidates from both the CRISPR and ORF screens.





www.cbioportal.org


Figure 4.

EGFR–PI3K–AKT signaling modulates resistance to trametinib/palbociclib treatment in NRAS-mutant melanoma cell lines. A and C, CellTiter MTS tetrazoliumassays showing mean cell viability of MELJUSO (A) and Hs944T (C) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR after 144 hours of combinationtrametinib and palbociclib treatment, each from three independent experiments performed in triplicate. B and D, Normalized cell viability score where DMSOwas100% cell viability as measured by MTS in MELJUSO (B) and Hs944T (D). E and F, Representative images of crystal violet–stained clonogenic growth assays withMELJUSO (E) and Hs944T (F) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR after 10 days of trametinib, palbociclib, and trametinib/palbociclibcombination treatment, each from three independent experiments performed in triplicate. G and H, Representative immunoblots displaying the effect oftrametinib, palbociclib, and trametinib/palbociclib combination treatment in MELJUSO (G) and Hs944T (H) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFRafter 48 hours on phosphorylated and/or total protein levels of RB, AKT, ERK, S6, CCNB1, CCND1, and CCNE2. b-Actin immunoblotting was used to determineequivalent loading.

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or EGFR constructs to identify potential mechanisms overcom-ing the inhibitor block. First, we checked total protein levels ofour AKT, PI3K, and EGFR constructs in our panel of cell lines(Fig. 4G and H; Supplementary Fig. S4G–S4I). We then treatedeach cell line with single-agent trametinib and/or palbociclibfor 48 hours. We observed a reduction of phospho-ERK(trametinib and combination) and phospho-RB (trametinib,palbociclib, and combination) in our GFP expressing cell lines,suggesting target inhibition. In MELJUSO, SKMEL30, andHs936T expressing AKT, PI3K, and EGFR, we did not observeany changes to phospho-ERK after inhibitor treatments, sug-gesting that these genes drive resistance independent of MAPKsignaling (Fig. 4G; Supplementary Fig. S4H and S4I). This wasin contrast to the Hs944T cells, where we observed that expres-sion of AKT1, AKT2, and PI3KH1047R lead to increased levels ofphospho-ERK (Fig. 4H).

Next, we examined changes to levels of phospho-RB. InSKMEL30 and Hs936T cell lines expressing our AKT, PI3K, andEGFR constructs, we observed a significant rebound of phospho-RB in the presence of trametinib and/or palbociclib (Supplemen-tary Fig. S4H and S4I). In MELJUSO cells expressing the sameconstructs there was a slight rebound of phospho-RB in thepresence of trametinib and the combination, but not uniformlyin the presence of palbociclib (Fig. 4G). This was in contrast toHs944T cells, where expression of PI3KH1047R and PI3KE545K

resulted in slight rebounding of phospho-RB in the presence oftrametinib and/or palbociclib (Fig. 4H). These data indicate thatresistance to trametinib and/or palbociclib in NRAS mutantmelanoma cell lines is partially associated with re-activation ofphospho-RB, which is consistent with the notion of pathway re-activation.

Next, we assessed phospho-S6 levels, as they have beenpreviously associated with resistance to trametinib and/orpalbociclib treatment in other NRAS mutant melanoma mod-els. Treatment with trametinib alone and in combination withpalbociclib reduced phospho-S6 levels in all cell lines exam-ined (Fig. 4G and H; Supplementary Fig. S4H and S4I). In thepalbociclib arm, we observed a slight decrease in phospho-S6levels. In MELJUSO, SKMEL30, and Hs936T cell lines expres-sing our AKT, PI3K, and EGFR constructs we observed a sig-nificant rebounding of phospho-S6 levels in the presence oftrametinib alone or in combination (Fig. 4G; SupplementaryFig. S4H and S4I). However, only in the Hs944T cells expressingeither PI3KH1047R, PI3KE545K, or EGFRL858R caused a rebound ofphospho-S6 levels (Fig. 4H). These data suggest that reengage-ment of mTOR signaling is associated with resistance to tra-metinib and palbociclib treatment in NRAS mutant melanoma,which is consistent with known in vivo and clinical mechanismsof inhibitor resistance (7, 11).

Next, we investigated the effect of trametinib and/or palboci-clib treatment on levels of cell-cyclemarkers. In theMELJUSOandHs944T cell lines expressing AKT, PI3K, and EGFR constructs, wefound that cyclin E2 levels were restored in the presence oftrametinib and/or palbociclib,which correlatedwith theobservedresistance phenotype (Fig. 4G and H). In Hs944T cells expressingeither PI3KH1047R or PI3KE545K, we observed that cyclin B1 andcyclin D1 levels rebounded in the presence either/both drugs.Together, these results suggest that NRAS mutant melanomacell lines can overcome inhibitor sensitivity to trametinib and/orpalbociclib by increasing the levels of phospho-RB and phospho-S6, as well as cell-cycle markers.

KRAS signaling modulates resistance to trametinib andtrametinib/palbociclib treatment in NRAS-mutant melanomacell lines

Alleles of activated KRASwere among the top scoring hits in thetrametinib single-agent screen and comprised the top two scoringORFs identified in the combination arm. We expressed GFP oractivated KRASG13D in the MELJUSO, SKMEL30, Hs936T, andHs944T cell lines and assayed cell viability and clonogenicgrowth. Similar to our screening results, we observed that acti-vatedKRAS caused strong resistance to trametinib and trametinib/palbociclib in all cell lines evaluated in cell viability and clono-genic growth (Fig. 5A–F; Supplementary Fig. S5A–S5D). How-ever, KRAS WT could not overcome sensitivity to trametinib/palbociclib treatment (Supplementary Fig. S5D–S5I).When thesecell lines were treated with palbociclib alone, we observed min-imal outgrowth in the MELJUSO, Hs936T, and Hs944Tcell lines,but not the SKMEL30 cell line, where there was no measurableadvantage to having activated KRAS mutations.

NRAS clones are not present in theORFeome Library used here.Thus, we investigated whether overexpression of activatedNRASQ61L/K could drive resistance to trametinib and/or palboci-clib in NRAS-mutant cells. As observed with KRAS, mutant NRASdrove resistance to trametinib and trametinib/palbociclib, withminimal impact on palbociclib resistance (Fig. 5A–F; Supplemen-tary Fig. S5A). Together, these data imply that activated Rasisoforms are sufficient to drive resistance to overcome MEK1/2and CDK4/6 inhibition.

Consistent with the resistance phenotypes observed withexpression of activated Ras isoforms, expression of mutant NRASor KRAS led to an increase in cyclin D1 levels and a rebound ofphospho-RB and phospho-S6 levels in the presence of trametinib,alone, and in combination with palbociclib, whereas no reboundof phospho-RB levelswas seenwithpalbociclib alone (Fig. 5GandH; Supplementary Fig. S5J). Additionally, phospho-ERK levelswere elevated in the presence of trametinib and the combinationin cell lines expressing activated KRAS and NRAS. Together thesedata support the idea that activated RAS is the strongest driver ofresistance to combination trametinib and palbociclib in NRASmutant melanoma cell lines.

EGFR–PI3K–AKT signaling differentially modulates sensitivityofNRAS-mutantmelanoma cell lines to genetic loss of activatedNRAS

Ourdata suggest that expressionof single components found inthe EGFR–PI3K–AKT signaling cascade can partly overcome sen-sitivity to trametinib and/or palbociclib. This led us to evaluatewhether any of these components could overcome genetic abla-tion of activated NRAS. To address this question, we expressedAKT1, AKT2, PI3KH1047R, PI3KE545K, or EGFRL858R in MELJUSOand Hs944T cell lines, treated with siRNAs and assessed cellviability and clonogenic growth. Expression of AKT1, PI3K, orEGFR was sufficient to partially rescue NRAS depletion inMELJUSO (Fig. 6A and B; Supplementary Fig. S6A). However,rescue was muchmore pronounced in the Hs944T cell line in cellviability and clonogenic assays (Fig. 6C and D; SupplementaryFig. S6A). Expression of AKT1, PI3K, or EGFR constructs inSKMEL30 and Hs936T was unable to overcome genetic ablationof NRAS (Supplementary Fig. S6A–S6G). These differences didnot appear to be a consequence of the expression levels ofcomponents of the EGFR–PI3K–AKT pathway or the result ofco-occurring mutation. These data highlight the heterogeneity of






Figure 5.

RAS signaling modulates resistance to trametinib/palbociclib treatment in NRAS-mutant melanoma cell lines. A and C, CellTiter MTS tetrazolium assay showingmean cell viability of MELJUSO (A) and Hs936T (C) cell lines expressing GFP, KRAS, or NRAS after 144 hours of combination trametinib and palbociclibtreatment, each from three independent experiments performed in triplicate. B and D, Normalized cell viability score where DMSOwas 100% cell viability asmeasured by MTS in MELJUSO (B) and Hs936T (D). E and F, Representative images of crystal violet–stained clonogenic growth assays with MELJUSO (E) andHs936T (F) cell lines expressing GFP, KRAS, or NRAS after 10 days of trametinib, palbociclib, and trametinib/palbociclib combination treatment, each from threeindependent experiments performed in triplicate. G and H, Representative immunoblots displaying the effect of trametinib, palbociclib, and trametinib/palbociclib combination treatment in MELJUSO (G) and Hs936T (H) cell lines expressing GFP, KRAS, or NRAS after 48 hours on phosphorylated and/ortotal protein levels of NRAS, RB, AKT, ERK, S6, CCND1, and KRAS. b-Actin immunoblotting was used to determine equivalent loading.

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NRAS-mutantmelanoma cell lines and suggest thatmutant NRASablation in some contexts may not be enough to prevent re-occurrence.

Next, we assessed the effects of genetic loss of NRAS onsignaling output. Genetic loss of NRAS resulted in a reductioninphospho-ERK andphospho-RB levels in theGFP control,which

Figure 6.

EGFR–PI3K–AKT signaling cascade differentially affects loss of genetic NRAS in NRAS-mutant melanoma cell lines. A and B, CellTiter MTS tetrazolium assaydisplayingmean cell viability of MELJUSO (A) and Hs944T (B) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRAS (sixreplicates) after 96 hours. C and D, Representative images of crystal violet–stained clonogenic growth assays with MELJUSO (C) and Hs944T (D) cell linesexpressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRAS (duplicate), each from three independent experiments. E and F, Representativeimmunoblots displaying the effect of MELJUSO (E) and Hs944T (F) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRASafter 96 hours on phosphorylated and/or total protein levels of NRAS, EGFR, PI3K, RB, AKT, ERK, and CCND1. b-Actin immunoblotting was used to determineequivalent loading.






Figure 7.

Active KRAS can replace loss of genetic NRAS. A and B, CellTiter MTS tetrazolium assay displayingmean cell viability of MELJUSO (A) and Hs936T (B) cell linesexpressing GFP, KRAS, or NRASwith or without knockdown of NRAS (six replicates) after 96 hours, each from three independent experiments. C and D,Representative images of crystal violet stained clonogenic growth assays with MELJUSO (C) and Hs936T (D) cell lines expressing GFP, KRAS, or NRAS with orwithout knockdown of NRAS (duplicate) after 10 days, each from three independent experiments. E and F, Representative immunoblots displaying the effect ofMELJUSO (E) and Hs936T (F) cell lines expressing GFP, KRAS, or NRAS with or without knockdown of NRAS after 96 hours on phosphorylated and/or totalprotein levels of NRAS, RB, ERK, CCND1, and KRAS. Vinculin immunoblotting was used to determine equivalent loading.


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could not be rescued by expression of AKT1, PI3K, or EGFRconstructs in MELJUSO and SKMEL30 cells (Fig. 6E; Supplemen-tary Fig. S6F and S6H). The Hs944T cell line expressing GFP didnot yield a reduction in levels of phospho-ERK after NRAS siRNAtreatment, whereas phospho-RB levels displayed minimalchanges. Consistent with their ability to rescue NRAS ablationin Hs944T, PI3KH1047R, PI3KE545K, or EGFRL858R expressioncaused an increase in cyclin B1 and cyclin D1, suggesting thesecell lines can rescue NRAS ablation via re-engaging the cell cycle(Fig. 6F; Supplementary Fig. S6I). These data imply that singlecomponents in the EGFR–PI3K–AKT signaling cascade may par-tially, but not fully replace genetic loss of NRAS.

Activated KRAS can compensate for genetic loss of activatedNRAS

Next, we examined whether activated KRAS could compensatefor genetic loss of NRAS. We first expressed activated KRASconstructs in the MELJUSO, SKMEL30, Hs936T, and Hs944T celllines. Next, we treated cell lines expressing activated KRAS withNRAS-targeted siRNA and control siRNA. After 96 and 144 hourspost-siRNA treatment, we assessed cell viability and observed thatcell lines expressing activated KRAS grew equally well in thepresence of NRAS-targeted siRNA compared with control siRNA(Fig. 7A and B; Supplementary Fig. S7A–S7C). This observationwas further extended in clonogenic growth assays, where activatedKRAS uniformly restored growth to the same levels as the control(Fig. 7C and D; Supplementary S7D and S7E). When treated withNRAS-targeted siRNA, cell lines expressing activated NRAS wereable to grow to similar levels as cell lines treated with the siRNAcontrol. This observation suggests that our siRNAs in fact targetNRAS.

Next, we next examined how activated KRAS and NRAS wereable to overcome NRAS-targeted siRNA treatment. We observed agreater than 50% knockdown in cell lines treated with NRAS-target siRNA compared with control siRNA (Fig. 7E and F; Sup-plementary Fig. S7F and S7G). Our analyses revealed a significantrebound in phospho-ERK and phospho-RB levels (Fig. 7E and F).We did not observe a change in the levels of phospho-AKT ineither KRAS or NRAS expressing cell lines, suggesting that in thiscontext both NRAS and KRAS signal through the MAPK cascade.Consistent with our growth phenotype, we observed a rebound inlevels of the cell-cycle markers cyclin B1, cyclin D1, and cyclin E2in activated KRAS and NRAS expressing cell lines (Fig. 7E and F;Supplementary S7H and S7I). Based on these data, it seems thatre-engagement of the cell cycle is the mechanism by whichactivated NRAS and KRAS overcome genetic loss of activatedNRAS in NRAS-mutant melanoma.

DiscussionThere is no Ras-specific approved therapy for the treatment of

advanced NRAS-mutant melanoma; however, there are severaltherapeutic avenues under preclinical and clinical evaluation.The modest-but-clear clinical effectiveness of MEK inhibition asa single agent in NRAS-mutant melanoma drove interest indesigning rational therapeutic combination strategies toenhance MEK inhibition (NCT01763164; refs. 4, 27–29). Thisidea resulted in the identification of CDK4/6 as a modulator ofMEK inhibition in NRAS-mutant melanoma. Consequently,this combination is under clinical evaluation in several Ras-mutant cancers.

In our study, we present the first resistance landscape toMEK1/2 and CDK4/6 single agent and combination treatmentin NRAS-mutant melanoma. Using both ORF gain-of-functionand CRISPR loss-of-function screens, we provide evidence thatmodifying levels of components of the RTK–PI3K–AKT andRAS-RAF signaling cascades alters sensitivity to trametiniband/or palbociclib. These discoveries are particularly relevantfor the rational design of future combinatorial therapeutics forpatients with NRAS-mutant melanoma and other RAS-drivencancers. One clinical trial is evaluating binimetinib/LEE011(NCT01781572), and enrolled patients have experienced eithera partial response (34%), stable disease (52%) or progressivedisease (14%; ref. 8).

Our screening results are highly complementary to recentstudies, as we identified both ORFs and CRISPRs that representcomponents of the RTK–PI3K–AKT–mTOR signaling cascade thatmodulate sensitivity to trametinib and/or palbociclib (Figs. 2and 3; refs. 7, 11). These findings will be of particular significanceinformation regarding trametinib/palbociclib efficacy in Ras-mutant cancer in clinical trials becomes available. Together, ourfindings highlight resistance mechanisms to single agent andcombination MEK1/2 and CDK4/6 in NRAS-mutant melanomaand reveal insights into RAS biology.

Disclosure of Potential Conflicts of InterestF. Luo is a Technology Specialist at Wolf Greenfield & Sacks, P.C. L.A.

Garraway is a Sr. VP Oncology R&D at Eli Lilly and Company; was atHoward Hughes Medical Institute; was equity consultant at FoundationMedicine; reports receiving commercial research grant from Novartis andAstellas; and has ownership interest (including stock, patents, etc.) in TangoTherapeutics. L.A. Garraway is a co-founder and equity holder of TangoTherapeutics and was a consultant for Foundation Medicine and heldequity, some of which was sold to Roche. M. Meyerson is a scientificadvisory board member for OrigiMed and receives consulting fees for thisrole; was a consultant for Foundation Medicine and held equity, some ofwhich was sold to Roche; receives research support from Bayer and is aninventor of several joint patents and patent applications, none of whichhave been licensed at the time of this disclosure (12-17-18); is an inventoron several patents on EGFR mutations in lung cancer diagnosis, licensed toLabCorp, for which he receives royalties; is an inventor on several patentapplications on Fusobacterium, none issued or licensed; the inventor on apatent for pathogen discovery, not licensed, for none of which he receivesroyalties. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: T.K. Hayes, F. Luo, F. Piccioni, C.M. JohannessenDevelopment of methodology: T.K. Hayes, F. Piccioni, D.E. Root,C.M. JohannessenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.K. Hayes, S. Pantel, F. Piccioni, L.A. GarrawayAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.K. Hayes, O. Cohen, F. Piccioni, C.M. JohannessenWriting, review, and/or revision of the manuscript: T.K. Hayes, F. Luo,D.E. Root, M. Meyerson, C.M. JohannessenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructingdatabases):T.K.Hayes, F. Luo, A.B.Goodale, Y. Lee,M. BagulStudy supervision: T.K. Hayes, D.E. Root, L.A. Garraway, C.M. Johannessen

AcknowledgmentsThis workwas conducted as part of the Slim Initiative for GenomicMedicine,

a project funded by the Carlos Slim Foundation in Mexico. This work wassupported by a Career Development Award from the Melanoma ResearchFoundation (C.M. Johannessen). We acknowledge additional supportfrom the NCI to L.A. Garraway (3R35CA197737) and M. Meyerson






(1R35CA197568) andDamonRunyonCancer Research Foundation award to T.K.Hayes (David Livingston Fellow).M.Meyerson is anAmericanCancer SocietyResearch Professor.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 28, 2018; revised January 8, 2019; accepted February 25,2019; published first February 28, 2019.

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Hayes et al.




2019;79:2352-2366. Published OnlineFirst February 28, 2019.Cancer Res Tikvah K. Hayes, Flora Luo, Ofir Cohen, et al. Inhibition in NRAS-Mutant MelanomaA Functional Landscape of Resistance to MEK1/2 and CDK4/6

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