Intrinsic Resistance to MEK Inhibition Through BET Protein–Mediated Kinome Reprogramming in NF1-Deficient Ovarian Cancer

Published OnlineFirst May 1, 2019; DOI: 10.1158/1541-7786.MCR-18-1332

Signal Transduction and Functional Imaging Molecular Cancer Research Intrinsic Resistance to MEK Inhibition through BET Protein–Mediated Kinome Reprogramming in NF1-Deﬁcient Ovarian Cancer Alison M. Kurimchak, Claude Shelton, Carlos Herrera-Montavez, Kelly E. Duncan, Jonathan Chernoff, and James S. Duncan

Abstract

Mutation or deletion of Neurofibromin 1 (NF1), an inhib- ic enhancement screens identified BRD2 and BRD4 as integral itor of RAS signaling, frequently occurs in epithelial ovarian mediators of the MEKi-induced RTK signatures. Inhibition of cancer (EOC), supporting therapies that target downstream bromo and extra terminal (BET) proteins using BET bromo- RAS effectors, such as the RAF–MEK–ERK pathway. However, domain inhibitors blocked MEKi-induced RTK reprogram- no comprehensive studies have been carried out testing the ming, indicating that BRD2 and BRD4 represent promising efficacy of MEK inhibition in NF1-deficient EOC. Here, we therapeutic targets in combination with MEKi to block resis- performed a detailed characterization of MEK inhibition in tance due to kinome reprogramming in NF1-deficient EOC. NF1-deficient EOC cell lines using kinome profiling and RNA sequencing. Our studies showed MEK inhibitors (MEKi) were Implications: Our findings suggest MEK inhibitors will likely ineffective at providing durable growth inhibition in NF1- not be effective as single-agent therapies in NF1-deficient EOC deficient cells due to kinome reprogramming. MEKi-mediated due to kinome reprogramming. Cotargeting BET proteins in destabilization of FOSL1 resulted in induced expression of combination with MEKis to block reprogramming at the receptor tyrosine kinases (RTK) and their downstream RAF and transcriptional level may provide an epigenetic strategy to PI3K signaling, thus overcoming MEKi therapy. MEKi synthet- overcome MEKi resistance in NF1-deficient EOC.

Introduction that harbor NF1 defects may be sensitive to targeted PI3K or MEK inhibitors (MEKi; refs. 2, 3, 6). Treatment of epithelial ovarian cancer (EOC) is currently Several highly specific kinase inhibitors targeting MEK1/2 lacking effective targeted therapies (1). Large-scale genomic stud- proteins, including trametinib, have been developed and are ies of EOC tumors have identified frequent deletion/mutation currently being evaluated in clinical trials for a variety of cancers (12%) of the tumor suppressor gene NF1 (Neurofibromin 1), including ovarian cancers (7–9). Although promising, single- which is a Ras-GTPase–activating protein that negatively regulates agent MEKi therapies have shown limited therapeutic benefitin the RAS family of proteins (1). Loss of NF1 promotes activation of the treatment of cancer due to the rapid development of drug several RAS effector pathways including (i) PI3K-mTOR-AKT, (ii) resistance. One mechanism by which tumor cells circumvent the RAF-MEK-ERK, and (iii) RalGDS signaling resulting in oncogenic inhibitory action of single-agent kinase inhibitors is through growth and survival (2–4). Notably, IHC studies have demon- kinome reprogramming, a process characterized by system-wide strated MEK-ERK activation in the majority of NF1-deficient high- changes in kinase networks (10, 11). Recent studies have shown grade serous ovarian cancer tumors, and EOC cell lines harboring that inhibition of key kinase signaling nodes that are critical for NF1 alterations exhibit elevated RAS activity (5). Other studies tumor growth, such as MEK, triggers the rapid activation of have shown NF1-deficient cells and tumors display sensitivity compensatory kinase signaling pathways that facilitates drug toward inhibitors targeting downstream RAS effector pathways resistance. For example, MEK inhibition in triple-negative breast such as PI3K-AKT or MEK-ERK signaling, suggesting EOC tumors cancer (TNBC) resulted in a rapid reprogramming of the kinome via the induced expression and activation of several receptor tyrosine kinases (RTK), including DDR1, PDGFRB, and AXL, that Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania. effectively bypasses the original MEK-ERK inhibition (11). In – Note: Supplementary data for this article are available at Molecular Cancer addition, lung and colon KRAS mutant cancers have been shown Research Online (http://mcr.aacrjournals.org/). to respond to targeted MEKi by increasing the expression and/or activity of ERBB3 and/or EGFR, whereas KRAS-mutant pancreatic The accession number for the mRNA sequencing data reported in this article is GSE127886. cancers overcome MEK inhibition through activation of the RTKs AXL, PDGFRA, HER2, and EGFR (12, 13). Thus, development of Corresponding Author: James S. Duncan, Fox Chase Cancer Center, 333 combination therapies that inhibit, block, or prevent MEKi- Cottman Ave, P3039, Philadelphia, PA 19111. Phone: 215-728-2565; Fax: 215- 214-1623; E-mail: [email protected] induced RTK response and subsequent resistance is a highly tractable goal for the treatment of RAS-altered cancers. Mol Cancer Res 2019;17:1721–34 In this study, we explored the role of kinome reprogram- doi: 10.1158/1541-7786.MCR-18-1332 ming as a resistance mechanism to MEKi in NF1-deficient EOC 2019 American Association for Cancer Research. cells. Through the combined use of multiplexed inhibitor

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beads and mass spectrometry (MIB-MS) and RNA sequencing Compounds (RNA-seq), we demonstrated that resistance to trametinib in BIX02189, bortezomib, GDC-0941, trametinib, LY3009120, NF1-deficient EOC cells is mediated by "adaptive kinome and sorafenib were purchased from Selleckchem, and JQ1 was reprogramming," where inhibition of MEK rapidly triggers the purchased from ApexBio. PP58 (15) and VI16832 (16) were expression and activation of a variety of RTKs and their custom synthesized according to previously described methods downstream RAF and PI3K signaling overcoming MEK inhibi- by The Center for Combinatorial Chemistry and Drug Discovery, tion. We identified the transcriptional coactivators BRD2 and Jilin University (Changchun, China). CTx-0294885 (17) was BRD4 as essential for MEKi-mediated kinome reprogramming purchased from MedKoo. Conjugation of inhibitors to beads was in NF1-deficient EOC. Inhibition of BRD2 and BRD4 by performed by carbodiimide coupling to ECH Sepharose 4B (CTx- genetic interference or with BET bromodomain inhibitors 0294885, VI16832, and PP58) or EAH Sepharose 4B (purvalanol blocked MEKi-induced RTK reprogramming and provided B; GE Healthcare). more durable therapeutic responses, representing a promising combination therapy for NF1-deficient EOC. Western blotting Cell lysates were subjected to SDS-PAGE using 8% gels. For experiments where multiple antibodies are probed, samples in 6 Materials and Methods LSB were prepared in bulk. Twenty micrograms of sample from Cell lines the same batch was run in multiple gels at the same time to ensure Cell lines were verified by IDEXX laboratories and were consistency. Samples were then transferred to polyvinylidene verified to be Mycoplasma negative (January 7, 2019) using the difluoride membranes. Following blocking with 5% milk, mem- Hoechst DNA stain method. A1847 cell lines were obtained branes were cut horizontally to probe multiple proteins of dif- from the Fox Chase Cancer Center Cell Culture Facility ferent molecular weights on the same blot. A list of antibodies (Philadelphia, PA; deposited by Dr. Thomas Hamilton). The used can be found in Supplementary Excel S1. Secondary horse- identity of the cells was authenticated by short tandem repeat radish peroxidase (HRP)-anti-rabbit and HRP-anti-mouse were analysis and comparison to early-passage stocks of the parental obtained from Thermo Fisher Scientific. SuperSignal West Pico cells donated by Dr. T. Hamilton. OVCAR8 cells were main- and Femto Chemiluminescent Substrates (Thermo Fisher Scien- tained in RPMI1640 supplemented with 10% FBS, 100 U/mL tific) were used to visualize blots. Penicillin—Streptomycin, and 2 mmol/L GlutaMAX. A1847 and SKOV3 cell lines were maintained in RPMI1640 supple- RTK arrays mented with 10% FBS, 100 U/mL Penicillin–Streptomycin, Cells were harvested in RTK array lysis buffer containing 20 2 mmol/L GlutaMAX, and 5 mg/mL insulin. SNU-119 cells mmol/L Tris-HCl (pH 8.0), 1% NP-40, 10% glycerol, 137 mmol/L were maintained in RPMI1640 supplemented with 10% FBS, NaCl, 2 mmol/L EDTA, 1 EDTA-free protease inhibitor cocktail 100 U/mL Penicillin–Streptomycin, 2 mmol/L GlutaMAX, (Roche), and 1% each of phosphatase inhibitor cocktails 1 and 2 and 25 mmol/L HEPES. COV362 cells were maintained in (Sigma). After incubating on ice for 20 minutes, cell debris was DMEM supplemented with 10% FBS, 100 U/mL Penicillin— pelleted at 4C. Lysates (500 mg protein) were applied to R&D Streptomycin, and 2 mmol/L GlutaMAX. CAOV3 cells Systems Proteome Profiler Human Phospho-RTK antibody arrays. were maintained in DMEM supplemented with 10% FBS, Washing, secondary antibody, and developing steps were per- 100 U/mL Penicillin–Streptomycin, 2 mmol/L GlutaMAX, and formed according to the manufacturer's instructions. 100 mmol/L sodium pyruvate. JHOS-2 and JHOS4 cells were maintained in DMEM/F12 supplemented with 10% FBS, Growth assays 100 U/mL Penicillin–Streptomycin, 2 mmol/L GlutaMAX, and For short-term growth assays, 3,000–5,000 cells were plated per nonessential amino acids. HIO-80 ovarian surface epithelial well in 96-well plates and allowed to adhere and equilibrate cells were a generous gift supplied by Dr. Andrew Godwin overnight. Drug was added the following morning and after (The University of Kansas, Lawrence, Kansas) and were main- 120 hours of drug treatment, cell viability was assessed using the tained in MCDB 105/Media 199 þ 5% FBS, 7.5 mg/mL insulin, CellTiter-Glo luminescent cell viability assay according to the 100 U/mL Penicillin–Streptomycin, and 2 mmol/L GlutaMAX. manufacturer's instruction (Promega). Student t tests were per- To generate trametinib-resistant cell lines, A1847 cells were formed for statistical analyses and P < 0.05 was considered chronically exposed to 50 nmol/L trametinib and propagated in significant. For colony formation assays, cells were plated in the continued presence of 50 nmol/L trametinib. For drug 24-well dishes (3,000–5,000 cells per well) and incubated over- washout experiments, trametinib was removed from resistant night before continuous drug treatment for 14 or 28 days, with cells for approximately 1 month prior to retreatment. A1847 drug and media replaced twice weekly. At the end of treatment, doxycycline–inducible shRNA BRD4 cells were generated by cells were rinsed with PBS and fixed with chilled methanol for 10 infecting A1847 cells with lentiviral particles made with the minutes at 20C. Methanol was removed by aspiration, and cells trans-lentiviral shRNA packaging system and a TRIPZ lentiviral were stained with 0.5% crystal violet in 20% methanol for 20 human BRD4 shRNA plasmid. Cells were selected with and minutes at room temperature, washed with distilled water, and maintained in media containing 2 mg/mL puromycin. For scanned. SILAC labeling, cells were grown for seven doublings in arginine- and lysine-depleted media supplemented with MIBs preparation and chromatography either unlabeled L-arginine (84 mg/L) and L-lysine (48 mg/L) Experiments using MIB-MS were performed as described pre- or equimolar amounts of heavy isotope-labeled viously (11, 18). Briefly, endogenous kinases were isolated by 13 15 10 13 6 [ C6, N4]arginine (Arg )and[ C6]lysine (Lys ) (Sigma) as flowing lysates over kinase inhibitor–conjugated sepharose beads described previously (14). (purvalanol B, VI16832, PP58 and CTx-0294885 beads were

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Targeting MEK and BET Proteins in NF1-Altered Ovarian Cancer

used) in 10 mL gravity-flow columns. Kinases were eluted posttransfection. A list of siRNAs used can be found in Supple- from the column by boiling in 2 500 mL of 0.5% SDS, mentary Excel S1. 0.1 mol/L Tris-HCl (pH 6.8), and 1% 2-mercaptoethanol. Eluted peptides were reduced by incubation with 5 mmol/L DTT at 60C RNA-seq for 25 minutes, alkylated with 20 mmol/L iodoacetamide at room The sequencing libraries were constructed from 500 ng of total temperature for 30 minutes in the dark, and alkylation was RNA using the Illumina's TruSeq RNA Sample Kit V2 (Illumina) quenched with DTT. Kinase peptides were cleaned with PepClean following the manufacturer's instruction. The fragment size of C18 Spin Columns (Thermo Fisher Scientific) and ethyl acetate RNAseq libraries was verified using the Agilent 2100 Bioanalyzer extraction and subsequent LC/MS analysis was performed. (Agilent), and the concentrations were determined using Qubit Instrument (Life Technologies). The libraries were loaded Mass spectrometry and spectra analysis onto the Illumina HiSeq 2500 at 8 pmol/L and run the rapid Proteolytic peptides were resuspended in 0.1% formic acid mode for 2 40 bp paired end sequencing. The fastq files were and separated with a Thermo RSLC Ultimate 3000 on a Thermo generated on the Illumina's BaseSpace, and further data analysis Easy-Spray C18 PepMap 2 mm column with a 235-minute was performed using the TopHat platform for read alignment gradient of 4%–25% acetonitrile with 0.1% formic acid at and the Cufflinks Assembly and DE for gene expression 300 nL/minute. Eluted peptides were analyzed by a Thermo assessment (19). Q Exactive plus mass spectrometer utilizing a top 15 methodology, in which the 15 most intense peptide precursor ions were Statistical analysis subjected to fragmentation. The AGC for MS1 was set to 3 106 All P values were two-sided unless otherwise specified. with a maximum injection time of 120 milliseconds and the AGC for MS2 ions was set to 1 105 with a maximum injection time of 150 milliseconds and the dynamic exclusion was set to Results 90 seconds. Protein identification was performed by searching Single-agent MEK inhibition shows minimal efficacy in MS-MS data against the Swiss-Prot human protein database NF1-deficient EOC cell lines downloaded on July 26, 2018, using Andromeda 1.5.6.0 built Mutation or deletion of the RAS suppressor NF1 has been in MaxQuant 1.6.1.0. reported in several EOC cell lines (Fig. 1A; ref. 5, 20). Character- ization of NF1 protein levels by Western blot analysis confirmed Statistical analysis of MIB-MS NF1-altered lines A1847, CAOV3, SNU119, and JHOS-2 lacked For trametinib-induced MIBs signatures, we performed at least NF1 protein, as well as identified NF1–wild-type (wt) lines two biological MIB-MS experiments using reciprocal SILAC COV362, JHOS-4, and OVCAR8 to be deficient in NF1 protein heavy/light and light/heavy. A one-paired t test (Benjamini– (Fig. 1B). SKOV3 cells have a reported mutation in NF1 but Hochberg P < 0.05) of SILAC MIB-binding ratios from biological expressed NF1 protein, whereas other NF1-wt EOC cell lines replicates of L-drug/H-control or H-drug/L-control was per- KURAMOCHI, OVCAR4, OVSAHO, and OVCAR5 have detect- formed to generate MIB-MS response signatures to trametinib able NF1 protein similar to normal ovarian surface epithelial cells using Perseus software v 1.5.2.6. Visualization of MIBs-kinome (HIO80; Supplementary Fig. S1A). Differential activation of RAS signatures using heatmaps were generated using Perseus software effector AKT signaling was detected amidst NF1-deficient cells v 1.5.2.6. with majority of NF1-deficient cells exhibiting activation of RAF- MEK-ERK activity (Fig. 1B). Treatment of EOC cells with trame- qRT-PCR tinib had minimal impact on cell viability across EOC cell lines, The GeneJET RNA Purification Kit (Thermo Fisher Scientific) with the exception of JHOS-2 and the KRAS–mutant OVCAR5 was used to isolate RNA from cells according to the manufacturer's cells. Notably, the majority of NF1-deficient cell lines were resis- instructions. qRT-PCR on diluted cDNA was performed with tant (9) to trametinib therapy with GI50 values > 100 nmol/L inventoried TaqMan gene expression assays on the StepOne (Fig. 1C; Supplementary Fig. S1B). Moreover, trametinib treat- real-time PCR system. A list of TaqMan gene expression assay ment of NF1-deficient A1847 cells only partially reduced colony probes used can be found in Supplementary Excel S1. formation and failed to induce apoptosis as observed with the KRAS–dependent OVCAR5 cells (Fig. 1D and E). Inhibition of RNAi knockdown studies MEK-ERK-RSK1 pathway by trametinib at 4 hours was confirmed siRNA transfections were performed using 25 nmol/L siRNA by Western blot analysis in A1847 cells, however, activation of duplex and the reverse transfection protocol. 3,000–5,000 cells ERK phosphorylation returned by 48 hours, consistent with per well were added to 96-well plates with media containing the kinome reprograming (Fig. 1F). siRNA and transfection reagent (Lipofectamine RNAiMax) according to the manufacturer's instructions. In experiments MEK inhibition dynamically reprograms the kinome in NF1- where inhibitors were used, the inhibitor was added at the time mutant EOC cells of transfection. Cells were allowed to grow for 72–120 hours To explore adaptive kinase resistance mechanisms to MEK posttransfection prior to CellTiter-Glo (Promega) analysis. Two- inhibition in NF1-deficient EOC, we applied MIB-MS in conjunc- to-three independent experiments were performed with each cell tion with RNA-seq to measure MEKi-induced transcriptional and line and siRNA. Student t tests were performed for statistical proteomic reprogramming (Fig. 2A). Using this proteogenomic analyses and P < 0.05 was considered significant. For Western approach, we can identify the fraction of the kinome promoting blot or MIB MS studies, the same procedure was performed with resistance to the MEK inhibitor trametinib in NF1-deficient cells volumes and cell numbers proportionally scaled to a 60 mm, to rationally predict MEKi combination therapies that provide 10 cm, or 15 cm dish, and cells were collected 48–72 hours more durable therapeutic responses (11, 21). Kinome profiling of

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Figure 1. Single-agent MEK inhibitors show limited efﬁcacy across the majority of NF1-deﬁcient EOC cell lines. A, Table of NF1 alterations in EOC cell lines used in study. NF1 mutation status obtained from (5) and # (20). B, Loss of NF1 protein frequently occurs in EOC cell lines with differential impact on RAS effector signaling. NF1 protein levels and RAS downstream effector PI3K and RAF signaling was determined by Western blot analysis. KRAS–mutant OVCAR5 cells represent a

MEK-addicted EOC control. C, Line graph depicts GI50 of trametinib (nmol/L) across EOC cells. NF1-deﬁcient cells (red) lack detectable NF1 protein and NF1 proﬁcient cells (gray) express detectable NF1 protein as determined by Western blot analysis. Cells were treated for 5 days with escalating doses of trametinib or

DMSO and cell viability determined by CellTiter-Glo. Data presented are from triplicate experiments SEM. GI50 were determined using Prism. D, MEK inhibition blocks colony formation in A1847 cells to a lesser extent then KRAS–mutant OVCAR5 cells. Long-term 14-day colony formation assay of A1847 or OVCAR5 cells treated with MEKi trametinib (10 nmol/L) or DMSO. Colony formation was assessed by crystal violet staining. E, MEK inhibition does not induce apoptosis in A1847 cells. A1847 or OVCAR5 cells were treated with escalating doses of trametinib (0.8, 4, 20, 100, and 500 nmol/L) for 48 hours and cleaved PARP protein levels determined by Western blot analysis. F, Transient inhibition of ERK by trametinib therapy in A1847 cells. A1847 cells were treated with 10 nmol/L trametinib for 4 or 48 hours and activation of ERK determined by Western blot analysis. Antibodies recognizing activation-loop phosphorylation of ERK1/2 or ERK-substrate RSK1 were used to determine ERK1/2 activity. Drug was replenished every 24 hours.

NF1-deficient A1847 cells using MIB-MS and RNA-seq revealed of RTKs in response to trametinib was observed in several widespread transcriptional and proteomic rewiring of kinase additional NF1-deficient and NF1-wt EOC cells, demonstrating networks following MEK inhibition. Increased MIB binding of MEKi-induced RTK reprogramming was a common adaptive the RTKs PDGFRB, DDR1, EPHB3, EPHA4, and MST1R, the mechanism in EOC. Trametinib treatment increased expression tyrosine kinase (TK) FRK and PTK2B, as well as MYLK3, ULK1, of PDGFRB in NF1-deficient CAOV3, COV362, OVCAR8, MAP2K6, MAP3K3, MAP2K5, and MAPK7 were observed in SNU119, and JHOS-2 cells, as well as in NF1-wt SKOV3 A1847 cells following 48 hours trametinib treatment (Fig. 2B and OVSAHO cells. Elevated DDR1 RNA levels were detected and C; Supplementary Excel S2A). Reduced MIB binding of in JHOS-2, OVCAR8, OVCAR4, OVSAHO, and KURAMOCHI EPHA2, AURKA, AURKB, and PIK3R4 was also observed follow- cells, whereas ERBB2 expression was induced in JHOS-2, ing trametinib treatment. Trametinib treatment of A1847 cells for OVCAR8, and A1847 cells following MEK treatment (Supple- 48 hours increased RNA levels of several kinases including mentary Fig. S2). PIK3C2A, MYLK3, MYLK, PDGFRB, DDR1, RIPK2, JAK1, MST1R, Consistent with MIB-MS and RNA-seq analysis, increased tyro- and ATM, and decreased RNA levels of MAP2K7, MAP2K3, STK11, sine phosphorylation of PDGFRB and DDR1 in response to MAP2K2, and MAPK12 (Fig. 2D; Supplementary Excel S2B). Many trametinib treatment was observed by RTK arrays in A1847 cells, of the kinases that showed induced MIB binding following as well as the time-dependent upregulation of PDGFRB and trametinib treatment also exhibited increased RNA levels, downstream AKT, and STAT3 survival pathways (Fig. 2F and including PDGFRB, DDR1, MST1R, MAP2K6, MAPK7, and ULK1, G). In addition, an increase in RNA levels of PDGFRB ligands suggesting that a large component of the kinome rewiring is PDGFB and PDGFD were observed in A1847 cells following MEK transcriptional (Fig. 2E). Notably, the transcriptional induction inhibition, demonstrating the MEKi-induced autocrine/paracrine

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Figure 2. Dynamic reprogramming of the kinome in response to MEK inhibition in NF1-deficient A1847 cells. A, Flowchart of experimental design. Combining MIB-MS and RNA-seq to define the proteogenomic response of the kinome to MEKi in NF1-deficient EOC cells. RNAi and small-molecule inhibitors are used to define MIB- nominated kinase survival functions. Kinome tree reproduced courtesy of Cell Signaling Technology. B, Dynamic response of the kinome to 48 hours trametinib

(10 nmol/L) treatment in A1847 cells. Heatmap depicts SILAC-determined log2 fold changes in MIB binding as a ratio of trametinib/DMSO. Statistical changes in SILAC determined MIB binding were determined by one-paired t test (P < 0.05) using Perseus software. Biological duplicates of SILAC H-trametinib/L-DMSO or L-trametinib/H-DMSO are shown in heatmap. C, MIB-MS signature of kinases induced or repressed by trametinib treatment in A1847 cells. Kinases that exhibited a 1.3-fold increase or decrease in MIB binding following 48 hours trametinib treatment are depicted in kinome tree. D, Kinome-wide transcriptome analysis of trametinib-treated A1847 cells as determined by RNA-seq. Volcano plot depicts statistical changes in kinase expression (in blue, FDR < 0.05) induced (1.5-fold) or repressed (1.5-fold) in response to 48 hours trametinib (10 nmol/L) treatment. E, Scatterplot shows overlap of kinases (in red) induced (1.5-fold) or repressed (1.5-fold) at the protein and RNA level in A1847 cells following 48 hours trametinib (10 nmol/L) treatment as determined by MIB-MS and RNA-seq analysis. F, RTK tyrosine phosphorylation induced or repressed by trametinib treatment in A1847 cells. Cells were treated with 10 nmol/L trametinib for 48 hours and tyrosine phosphorylation monitored by RTK array. G, Time-dependent increase in PDGFRB protein levels and downstream kinase signaling following trametinib treatment. A1847 cells were treated with trametinib (10 nmol/L) for 0, 4, 24, or 48 hours and protein levels and phosphorylation determinedby Western blot analysis. H, Induction of PDGFB and PDGFD expression in A1847 cells following exposure to trametinib (10 nmol/L) for 48 hours. Line graph depicts log2 fold changes in growth receptor ligand RNA levels as a ratio of trametinib/DMSO cells, as determined by RNA-seq.

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loop involving upregulation of both the PDGFRB receptor and its provided superior growth inhibition relative to single agents activating ligands (Fig. 2H). across all NF1-deficient and NF1-wt EOC cells, signifying the MEKi-induced RAF dependency was a global adaptive mecha- MEKi-induced kinase signature is chronically maintained in nism to MEK inhibitors in EOC (Supplementary Fig. S3B). NF1-deficient cells and is reversible Treatment of RAS-altered cells with MEK inhibitors has been Chronic exposure of A1847 cells to trametinib abolished the shown to induce PI3K activity through a compensatory feedback growth inhibition observed following short-term exposure to mechanism, supporting the combined blockade of PI3K and trametinib (Fig. 3A). Kinome profiling of A1847 cells chronically MEK (2, 3, 24). Consistent with these findings, knockdown of exposed to 50 nmol/L trametinib (A1847-T) demonstrated PIK3CA or small-molecule inhibition with GDC0941 blocked cell induced MIB binding of the majority of the kinases activated by growth in A1847-T cells to a greater extent than parental cells short-term MEKi exposure, including PDGFRB, DDR1, MST1R, (Fig. 4G and H). Kinome profiling of A1847-T cells treated with BRAF, and MAPK7, signifying trametinib-resistant cells continu- GDC0941 for 48 hours resulted in reduced MIB binding of several ously maintain the kinome signature induced at 48 hours (Fig. 3B cell-cycle protein kinases including PLK1, PLK4, CDK1, BUB1, and C; Supplementary Excel S3A and S3B). Increased MIB binding TTK, AURKA, and AURKB consistent with the growth inhibition of JAK3, RIPK2, IKBKB, CHUK, TAOK1, RPS6KA1, and PRKCQ observed (Fig. 4I; Supplementary Excel S4). Furthermore, the were uniquely observed in A1847-T cells, whereas MIB binding of combination of trametinib and GDC0941 showed significant PDGFRB and JAK1 were further induced relative to short-term synergy in A1847 parental cells, as well as across all EOC cell trametinib exposure (Fig. 3D). Consistent with activation of lines with the exception of OVCAR4 and KURAMOCHI cells, several RTKs in A1847-T cells, elevated downstream survival demonstrating activation of PI3K-AKT axis was an essential com- signaling including STAT3, SRC, AKT, and CRAF, as well as the ponent of the kinome reprogramming response to MEK inhibi- reactivation of ERK, was observed in cells chronically exposed to tion in both NF1-deficient and NF1-wt EOC cells (Fig. 4J; Sup- trametinib (Fig. 3E). Notably, removal of trametinib from A1847- plementary Fig. S3C). T cells nearly completely reversed the MEKi-induced kinome Taken together, targeting the MEKi-induced kinase signature signature, demonstrating the absolute requirement of trametinib with a variety of small-molecule inhibitors acting at the level of to maintain the RTK-driven resistance signature (Fig. 3F). Fur- RTK or their downstream kinase pathways such as RAF, MEK5, or thermore, removal of trametinib resensitized A1847-T cells to PI3K provides more durable responses relative to single agents trametinib (Fig. 3G). alone in NF1-deficient cells. Moreover, these findings demon- Collectively, NF1-deficient cell line A1847 dynamically strate numerous distinct kinase pathways are triggered in response reprogrammed the kinome inducing a number of RTKs, to MEK inhibition that are integral to the MEKi-induced kinome including DDR1 and PDGFRB and their subsequent down- reprogramming response. stream RAF-MEK-ERK, JAK-STAT, and PI3K-AKT signaling that was maintained indefinitely in the presence of trametinib MEKi-mediated degradation of FOSL1 alleviates repression of (Fig. 3H). Following removal of trametinib, A1847-T cells RTK signaling promoting trametinib resistance revert to initial kinome state and regain sensitivity to MEK The RAF-MEK-ERK pathway has been shown to regulate the inhibition. stability of a number of cancer-associated transcription factors including MYC and FOSL1 (25, 26). Phosphorylation of MYC Kinase inhibitor combination therapies overcome adaptive at S62 and/or FOSL1 at S265 by ERK1/2 prevents proteasome- resistance to trametinib in NF1-deficient EOC cells mediated degradation leading to protein stabilization promot- Knockdown of MIB-MS–nominated kinases in A1847 and ing growth and survival. Loss of MYC repressor activity follow- A1847-T cells revealed enhanced dependency of cells chroni- ing MEKi therapies has been shown to result in induced cally exposed to trametinib for RTKs (PDGFRB, EGFR, FGFR2, expression of RTKs in breast and colorectal cancers promoting FGFR4, and DDR1), TKs (JAK1 and JAK3), MAPK pathway drug resistance (11, 13). Herein, reduced phosphorylation of components (BRAF, MEK1/2, ERK1/2, and ERK5), as well as MYC at S62 and total MYC levels were observed at higher RIPK2 (Fig. 4A). Genetic depletion or small-molecule inhibi- concentrations of trametinib (>100 nmol/L) in NF1-deficient tion of PDGFRB using the pan-TK inhibitor sorafenib (22), A1847 and JHOS-2 EOC cells (Fig. 5A; Supplementary Fig. greatly enhanced the efficacy of trametinib in A1847 or A1847- S4A). Notably, reduced levels of phosphorylated FOSL1 at S265 T cells, demonstrating the dependency of these cells for and FOSL1 total protein were observed at picomolar concen- PDGFRB to overcome single-agent MEK inhibition (Fig. 4B– trations of trametinib that coincided with ERK inhibition and D). In addition, cotreatment of NF1-deficient EOC cell lines the induction of RTKs PDGFRB and DDR1. Reduced FOSL1 and JHOS-2, OVCAR8, JHOS-4, SNU119, and CAOV3, as well as increased PDGFRB RNA and protein levels were detected within NF1-wt KURAMOCHI and OVSAHO cells with trametinib 4 hours of trametinib treatment, demonstrating MEK inhibi- and sorafenib enhanced growth inhibition relative to single- tion rapidly mediates transcriptional reprogramming in A1847 agent therapies (Supplementary Fig. S3A). Targeting the MEK5- cells (Supplementary Fig. S4B and S4C). Furthermore, knock- ERK5 pathway using MEK5 inhibitor BIX02189 or treatment down of FOSL1 in A1847 cells induced transcription of several with RAF inhibitor LY3009120 blocked cell growth to a greater RTKs, including DDR1, ERBB2, FGFR3, IGF1R, INSR, MERTK, extent in A1847-T cells than parental, demonstrating the MET,andPDGFRB,whereasMYC knockdown induced ERBB3, dependency of trametinib-resistant cells for MEK5 and ERBB2,andPDGFRB RNA levels (Fig. 5B). Knockdown of MYC CRAF-BRAF signaling (Fig. 4E and F). Activation of RAF fol- uniquely increased protein levels of ERBB3 in both A1847 and lowing MEK-ERK inhibition has been shown to occur in cancer JHOS-2 cell lines, whereas FOSL1 depletion distinctly elevated cells due to alleviation of ERK-dependent negative feedback on DDR1 protein levels. Either MYC or FOSL1 knockdown RAF (23). Here, the combination of LY3009120 and trametinib increased PDGFRB protein levels with FOSL1 depletion

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Figure 3. Trametinib-induced kinase signature is chronically maintained in A1847 cells and is reversible. A, Trametinib treatment has minimal impact on cell viability of A1847-T cells. Parental (A1847) or trametinib-resistant A1847 cells (A1847-T) were treated with escalating doses of trametinib for 72 hours and cell viability assessed by CellTiter-Glo. A1847-T–treated cell viabilities were normalized to DMSO-treated A1847-T cells. B, MIB-MS kinome profiles of A1847 cells following chronic exposure to trametinib (A1847-T) or short-term 48 hours 10 nmol/L trametinib treatment (A1847 þ trametinib). Heatmap depicts SILAC-determined log2 fold changes in MIB binding as a ratio of (A1847-T/parental) or as a ratio of (A1847 þ trametinib/DMSO). C, Scatterplot shows overlap of kinases (in red) induced (1.5-fold) or repressed (1.5-fold) following chronic exposure to trametinib or short-term exposure (48 hours at 10 nmol/L), as determined by MIB-MS profiling. D, Scatterplot shows differences in MIB binding of kinases (in red) induced (1.5-fold) or repressed (1.5-fold) following chronic exposure to trametinib or short- term exposure (48 hours at 10 nmol/L) analyzed by MIB-MS profiling. E, Activation of RTK downstream survival signaling pathways in A1847 cells chronically exposed to trametinib. A1847 or A1847-T cells were treated with escalating doses of trametinib (0.8, 4, 20, 100, and 500 nmol/L) for 48 hours and kinase activity determined by Western blot analysis using activation-loop phospho-antibodies. F, Reversible nature of trametinib-mediated kinome response in A1847 cells.

MIB-MS kinome proﬁles of A1847-T cells or A1847-T cells following trametinib removal. Heatmap depicts SILAC-determined log2 fold changes in MIB binding as a ratio of A1847-T/A1847-parental or A1847-T, drug removed/A1847-T. G, Removal of trametinib from chronically exposed cells resensitizes cells to trametinib. A1847, A1847-T, or A1847-T with trametinib removed (A1847-T, drug removed) were treated with escalating doses of trametinib and cell viability assessed by

CellTiter-Glo. Trametinib-treated cells were normalized to cells treated with DMSO. GI50 were determined using Prism. H, MEKi-induced dynamic and chronic kinome reprogramming signature in A1847 cells determined by MIB-MS, RNA-seq, and phospho-antibodies. Kinases induced by 48 hours trametinib in A1847 cells that remain elevated in A1847-T cells are highlighted in blue. Data presented in A and G are from triplicate experiments SD; , P 0.05 by Student t test. Data presented in B and F were biological duplicates of SILAC H-drug/L-DMSO or L-drug/H-DMSO. Statistical changes in SILAC-determined MIB binding were determined by one-paired t test (P < 0.05) using Perseus software.

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Figure 4. Targeting MEKi-induced kinases overcomes MEKi resistance due to kinome reprogramming in NF1-deficient cells. A, Targeted siRNA screen identifies acquired kinase vulnerabilities in A1847-T cells. A1847 or A1847-T cells were transfected with siRNAs targeting kinases, cultured for 72 hours and cell viability determined. A1847-T–knockdown cells were normalized to A1847-T cells transfected with nontargeting siRNA. B, Knockdown of PDGFRB sensitizes A1847 cells to MEK inhibition. Growth inhibition of parental A1847 cells in response to escalating doses of trametinib with or without PDGFRB siRNA knockdown. A1847 cells were transfected with siRNAs targeting PDGFRB or control siRNAs, cultured for 72 hours, and cell viability determined. C, A1847-T cells show enhanced sensitivity to pan-TK inhibitor sorafenib relative to parental cells. Parental or A1847-T cells were treated with increasing doses of sorafenib for 72 hours and cell viability determined. D, Cotargeting MEK and PDGFRB enhances growth inhibition of parental A1847 cells. Cells were treated with DMSO, sorafenib (500 nmol/L), or sorafenib (500 nmol/L) in combination with increasing concentrations of trametinib (tramet) for 5 days and cell viability determined. Dotted line indicates growth inhibition achieved by single-agent trametinib (10 nmol/L) treatment. E and F, A1847-T cells show enhanced sensitivity to MEK5 or RAF inhibition relative to parental cells. Parental or A1847-T cells were treated with increasing doses of BIX02189 or LY3009120 for 72 hours and cell viability determined. G, A1847-T cells acquire dependency on PIK3CA for cell growth. A1847 or A1847-T cells were transfected with siRNAs targeting PIK3CA or control siRNAs, cultured for 72 hours, and cell viability determined. A1847-T–knockdown cells were normalized to A1847-T cells transfected with nontargeting siRNA. H, A1847-T cells show enhanced sensitivity to PIK3CA inhibitor (GDC0941) relative to parental cells. Parental or A1847-T cells were treated with increasing doses of GDC0941 for 72 hours and cell viability assessed. I, MIB-MS–defined kinome response profiles of A1847-T cell lines following 48 hours GDC-0941 (1 mmol/L) treatment. Volcano

plot depicts SILAC-determined log2 fold changes in MIB binding as a ratio of GDC-0941/DMSO. Statistical changes in SILAC-determined MIB binding were determined by one-paired t test (P < 0.05) using Perseus software. Biological duplicates of SILAC H-GDC0941/L-DMSO or L-GDC0941/H-DMSO were used to generate volcano plot. Cell-cycle kinases reduced by GDC0941 highlighted in blue. J, Cotargeting MEK and PIK3CA enhances growth inhibition of A1847 cells. Cells were treated with DMSO, GDC0941 (500 nmol/L), or GDC0941 (500 nmol/L) in combination with increasing concentrations of trametinib for 5 days andcell viability determined. Data presented in A–J are from triplicate experiments SD; , P 0.05 by Student t test. Cell viability was determined by CellTiter-Glo assays. In data presented in D, E, F, and H, A1847-T kinase inhibitor–treated cells were normalized to DMSO-treated A1847-T cells.

elevating PDGFRB to a greater extent than MYC ablation Bromo and extra terminal bromodomain proteins BRD2 and (Fig. 5C; Supplementary Fig. S4D). Moreover, preventing BRD4 are essential for MEKi-induced kinome adaptations in MEKi-mediated FOSL1 protein degradation using the protea- NF1-deﬁcient EOC cells some inhibitor bortezomib blocked induction of PDGFRB and The plasticity of the MEKi-mediated kinome reprogramming DDR1, demonstrating the RTKs are transcriptionally induced response, as well as the signiﬁcant transcriptional upregulation of by MEKi through destabilization of FOSL1 proteins (Fig. 5D). RTKs, suggests an epigenetic mechanism may be contributing to

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Figure 5. MEKi-mediated destabilization of FOSL1 promotes RTK upregulation in NF1-defecient cells. A, MEK inhibition promotes degradation of FOSL1 coinciding with RTK upregulation. A1847 cells were treated with escalating doses of trametinib (0.8, 4, 20, 100, and 500 nmol/L) for 48 hours, protein phosphorylation and total levels determined by Western blot analysis. B, FOSL1 or MYC knockdown induces RTK expression. RNA levels of RTKs in A1847 following knockdown of MYC or FOSL1 for 72 hours as determined by qRT-PCR. Bar graph depicts RTK RNA levels following knockdown of MYC or FOSL1 relative to cells treated with control siRNAs. C, FOSL1 or MYC knockdown promotes differential RTK protein induction. MYC, FOSL1, and RTK protein levels were determined by Western blot analysis. A1847 cells were transfected with siRNAs targeting MYC, FOSL1, MYC/FOSL1, or control siRNAs and cultured for 72 hours. D, Proteasome inhibition prevents FOSL1 degradation blocking MEKi-induced RTKs. A1847 cells were treated with DMSO, trametinib (10 nmol/L), bortezomib (3, 10, and 20 nmol/L), or the combination of trametinib 10 nmol/L and bortezomib (3, 10, and 20 nmol/L) for 24 hours, protein phosphorylation and total levels determined by Western blot analysis. the MEKi-resistance in NF1-deficient cells. A key group of epige- formation of trametinib-resistant colonies (Fig. 6B and C; netic remodelers referred to as "readers," contain bromodomains Supplementary Fig. S5B and S5C). that recognize histone modifications and facilitate the assembly Recently, the BET (bromo and extra terminal) bromodomain of chromatin-remodeling complexes controlling gene expres- proteins were shown to be essential for kinase expression and sion (27). To interrogate the role of bromodomain function in promoting adaptive kinase transcription responses to inhibi- transcriptional reprogramming of the kinome in response to MEK tors (18, 28–30), supporting BET proteins as attractive targets for inhibition, we performed a MEKi synthetic lethal siRNA screen of blocking kinase transcription. Here, using qRT-PCR in combina- 46 bromodomain-containing proteins in A1847 cells chronically tion with loss-of-function assays, we determined the role of exposed to trametinib (A1847-T) or A1847 parental cells BET proteins in the regulation of the RTK transcription in NF1- (Fig. 6A). Knockdown of ATAD2B, TRIM33, TAF1, SP140L KAT2B, deficient A1847 cells. Differential regulation of kinases at the TAF1L, and BRD4 enhanced growth inhibition of MEKi-resistant transcriptional level was observed among BET proteins, where A1847-T cells relative to parental cells (Supplementary Fig. S5A). BRD2 knockdown uniquely reduced DDR1, DDR2, IGF1R, and Moreover, siRNA-mediated knockdown of BRD4 dramatically INSR RNA levels, whereas BRD4 knockdown solely inhibited improved growth inhibition by trametinib and blocked the PDGFRB transcription (Fig. 6D). Interestingly, knockdown of

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Figure 6. BET proteins required for MEKi-induced kinome reprogramming in NF1-deficient cells. A, siRNA screen targeting bromodomain proteins in parental (A1847) or trametinib-resistant (A1847-T) cells identifies BRD4 as a mediator of trametinib resistance. Cells were transfected with siRNAs targeting 46 bromodomain containing proteins or control siRNAs, cultured for 72 hours, and cell viability assessed by CellTiter-Glo. Cells treated with bromodomain siRNAs were normalized to cells transfected with nontargeting siRNA. B, Knockdown of BRD4 sensitizes A1847 cells to trametinib. A1847 cells were transfected with siRNAs targeting BRD4 or control siRNAs, treated with DMSO or trametinib for 72 hours, and cell viability assessed by CellTiter-Glo. C, Downregulation of BRD4 prevents development of trametinib-resistant colonies in A1847 cells. Cells stably expressing doxycycline-inducible BRD4 shRNA were treated with trametinib (10 nmol/L) or DMSO in the presence or absence of doxycycline for 14 days and colony formation assessed by crystal violet. D, Knockdown of BRD2, BRD4, or both reduce RTK transcription. A1847 cells were transfected with siRNAs targeting BRD2, BRD4, BRD2,andBRD4, or control siRNAs for 72 hours and RTK RNA levels determined by qRT-PCR. E, Knockdown of both BRD2 and BRD4 provide superior repression of trametinib-induced RTK transcription. A1847 cells were transfected with siRNAs targeting BRD2, BRD4, BRD2,andBRD4, or control siRNAs, treated with DMSO or trametinib (10 nmol/L) for 48 hours, and RTK RNA levels determined by qRT-PCR. F, BRD4 knockdown blocks or reduces trametinib-induced kinome reprogramming response. A1847 cells stably expressing doxycycline-inducible BRD4 shRNA were treated with trametinib or DMSO for 48 hours and subjected to MIB-MS kinome profiling. Line graph depicts SILAC-

determined log2 fold changes in MIB binding as a ratio of trametinib/DMSO. Statistical changes in SILAC-determined MIB binding were determined by one-paired t test (P < 0.05) using Perseus software. Biological duplicates of SILAC H-trametinib/L-DMSO or L-trametinib/H-DMSO were used to generate line graph. Data presented in A, B, D, and E are triplicate experiments SD (, P 0.05 by Student t test).

BRD4 reduced protein levels of ERBB3 without suppressing RNA inhibition, and depletion of both BRD2 and BRD4 was levels, suggesting that BRD4 depletion may influence the protein required to prevent MEKi-induced ERBB3 transcription stability of ERBB3 (Supplementary Fig. S5D). Notably, knock- (Fig. 6E). Furthermore, downregulation of BRD4 in A1847 down of both BRD2 and BRD4 resulted in overall greater reduc- cells blocked or reduced activation of the majority of the tion in transcription of RTKs, reducing RNA levels of AXL, DDR1, trametinib-mediated resistance signature as shown by MIB-MS, EGFR, ERBB2, ERBB3, IGF1R, and PDGFRB in A1847 cells, dem- including PDGFRB, BRAF, RAF1, DDR1, MAPK7, JAK1, and onstrating combined blockade of BRD2 and BRD4 provided RSK1 (Fig. 6F; Supplementary Excel S5). superior RTK transcriptional repression than knockdown of indi- Taken together, we identified BRD2 and BRD4 as integral to the vidual BET proteins (Fig. 6D). regulation of kinases induced by MEKi in NF1-deficient cells, as Next, we tested whether downregulation of BRD2, BRD4, or well as demonstrated that blockade of BET protein function both prevented MEKi-induced transcriptional induction of prevented the MEKi-mediated upregulation of RTKs RNA and RTKs in A1847 cells. Knockdown of BRD4 blocked the protein levels. These findings highlight the therapeutic implica- MEKi-mediated induction of PDGFRB, whereas BRD2 deple- tions of targeting BET proteins to block kinome reprogramming to tion prevented increased expression of DDR1 following MEK MEKi in NF1-deficient cells.

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BET bromodomain inhibition blocks MEKi-induced kinome ing (5), consequently, PI3K and MEK inhibitors have emerged adaptations sensitizing NF1-deficient cells to trametinib as a promising therapeutic avenue. Moreover, increased frequency Small-molecule inhibitors of BET proteins have been devel- of NF1 mutations has also been reported in patients with EOC oped that interfere with bromodomain binding blocking tran- who have developed resistance to first-line chemotherapy for scription (31). Combined treatment of the BET bromodomain which there are no current therapies (32). Despite these observa- inhibitors (BETi), JQ1, and trametinib for 48 hours blocked the tions and implications, no comprehensive studies have been induction of the majority of the MEKi-mediated reprogramming carried out testing the efficacy of MEK inhibition in NF1-deficient as determined by MIB-MS kinome profiling (Fig. 7A and B; EOC. Here, we performed a detailed proteomics, genomics, and Supplementary Excel S6). Reduced MIB binding of PDGFRB, functional analysis of MEK inhibition in an established NF1- RAF1, DDR1, RIPK2, JAK1, MAPK7, BRAF, MAP2K6, RPS6AK1, deficient EOC cell line, A1847, and then subsequently validated MET, SRC, and CHUK was observed in response to the combi- our findings in several additional NF1-deficient EOC cells. nation therapy, as well as the induction a number of kinases Our studies showed the MEK inhibitor trametinib was ineffec- involved in growth arrest and apoptosis including TGFBR1 and tive at providing durable growth inhibition or inducing apoptosis ACVR2A. Blockade of trametinib-induced activation of PDGFRB in NF1-deficient cells due to MEKi-mediated kinome reprogram- and RAF-MEK-ERK signaling by JQ1 was confirmed by Western ming. Kinome profiling of MEKi-treated or MEKi-resistant NF1- blot and RTK arrays (Fig. 7C; Supplementary Fig. S6A). The deficient A1847 cells using MIB-MS and RNA-seq revealed cells combined treatment of trametinib and JQ1 induced apoptosis activated a network of kinases including a variety of RTKs and their in A1847 cells, increasing cleaved PARP levels relative to single- downstream PI3K-AKT, and RAF-MEK-ERK signaling overcoming agent trametinib therapy. The trametinib and JQ1 combination MEK inhibition. Here, we showed targeting MEKi-induced RTKs, repressed MEKi-induced transcription of PDGFRB, DDR1, and such as PDGFRB in combination with MEK inhibitor, improved ERBB3 in A1847 cells, and blocked trametinib-induced PDGFRB the efficacy of trametinib in NF1-deficient EOC cells, as well as in and DDR1 expression in CAOV3, COV362, JHOS-2, and some NF1-wt EOC cells. Importantly, recent studies have impli- OVCAR8, as well as prevented PDGFRB reprogramming in SKOV3 cated activation of PDGFRB signaling as a biomarker for poor and SNU119 cell lines (Fig. 7D; Supplementary Fig. S6B and S6C). prognosis in patients with EOC (33). Our studies suggest that In addition, BET inhibition blocked MEKi-mediated induction of combination of trametinib and sorafenib may represent a prom- PDGFRB and DDR1 expression following FOSL1 knockdown, ising therapy for EOC tumors with elevated PDGFRB and/or NF1 demonstrating BET proteins were required to promote transcrip- deficiencies. In addition, we showed cotargeting MEK and PI3K or tion of these RTKs following FOSL1 depletion (Supplementary RAF significantly enhanced growth inhibition relative to single Fig. S6D). Notably, continued treatment of JQ1 was required to agents across all NF1-deficient EOC cell lines tested, demonstrat- maintain PDGFRB repression in presence of trametinib, where ing MEKi-induced PI3K and RAF activation were integral to the JQ1 removal from media abolished repressive effects on RTK kinome reprogramming response promoting trametinib resis- transcription (Supplementary Fig. S6E). The JQ1 and trametinib tance in NF1-deficient EOC cells. Notably, cotargeting MEK and combination therapy enhanced growth inhibition of A1847 cells PI3K or RAF enhanced growth inhibition independent of NF1 relative to single agents alone in 5-day viability assays, as well as status, suggesting these combination therapies may have broad blocked the emergence of MEKi-resistant A1847 colonies in long- activity across EOC. Combination therapies targeting MEK and term colony formation assays (Fig. 7E; Supplementary Fig. S6F). PI3K have been extensively explored in a variety of RAS-altered Moreover, cotargeting MEK and BET proteins provided superior cancer models, as well as in clinical trials for RAS-altered can- growth repression over individual agents in all NF1-deficient cell cers (24). However, significant toxicities have been associated lines (Fig. 7F; Supplementary Fig. S6G). Notably, NF1-proficient with this kinase inhibitor combination with ongoing studies cell lines, KURAMOCHI, OVCAR4, and OVSAHO showed no exploring dose regimens and timing of the drug combinations therapeutic benefit to the trametinib–JQ1 combination, suggest- to deal with off-target side effects (34). ing NF1-deficient EOC cells may display enhanced sensitivity to Transcriptional induction of RTKs following destabilization of combined blockade of BET and MEK signaling. key oncogenic transcription factors, such as MYC, has been In addition, A1847 cells that acquired resistance to trametinib observed in response to blockade of RAF-MEK-ERK signaling (11, therapy showed increased sensitivity to JQ1 treatment, where BET 13, 30). Here, we demonstrated that treatment of NF1-deficient inhibition destabilized the MEKi-mediated reprogramming sig- cells with low nanomolar concentrations of trametinib (<10 nature in MEKi-resistant cells (Fig. 7G). MIB-MS kinome profiling nmol/L) resulted in the rapid degradation of the transcription of trametinib-resistant A1847 cells revealed JQ1 treatment factor FOSL1 that coincided with induced expression of RTKs. reduced MIB binding of several of the MEKi resistance signature FOSL1 has been implicated as a master gatekeeper of the EMT including PDGFRB, BRAF, DDR1, RAF1, and MAPK3, as well as program in TNBC claudin low cells, consistent with a role of cell-cycle–related kinases AURKA, AURKB, and PLK1 consistent FOSL1 in the regulation of expression a diverse array of RTKs, with the growth arrest observed (Fig. 7H). These findings dem- particularly those RTKs involved in epithelial and mesenchymal onstrated the requirement for the presence of both BETi and MEKi signaling (35). In addition, we observed differential degradation to efficiently block reprogramming, as well as show MEKi-resis- of MYC or FOSL1 that was dependent on the dose of MEK tant cells require BET proteins to maintain kinome adaptive inhibitor administered, where low doses of trametinib reduced responses. FOSL1 but not MYC protein levels. Moreover, knockdown of MYC or FOSL1 in NF1-deficient cells resulted in induced expression of distinct RTKs, demonstrating the unique repressive functions of Discussion these transcription factors on RTK expression. Interestingly, these Loss of RAS suppressor protein NF1 frequently occurs in EOC findings suggest that the composition of MEKi-induced RTK leading to activation of PI3K-AKT and RAF-MEK-ERK signal- signature may depend on the concentration of trametinib that

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Figure 7. BET bromodomain inhibition blocks MEKi-induced kinome reprogramming providing more durable responses in NF1-deﬁcient cells.A,BET inhibition blocks trametinib-induced kinome response. MIB-MS kinome proﬁles of A1847 cells treated with trametinib (tramet; 10 nmol/L), JQ1 (500 nmol/L), or the combined

treatment of trametinib and JQ1 for 48 hours. Heatmap depicts SILAC-determined log2 fold changes in MIB binding as a ratio of drug/DMSO. Statistical changes in MIB binding were determined by one-paired t test (P < 0.05) using Perseus software. Biological duplicates of SILAC H-drug/L-DMSO or L-drug/H-DMSO are shown in heatmap B, Line graph depicts kinases induced by trametinib that were inhibited by cotreatment with JQ1 as determined by MIB-MS profiling. C, Cotreatment of A1847 cells with trametinib and JQ1 represses PDGFRB induction and reactivation of the RAF-MEK-ERK pathway. A1847 cells were treated with escalating doses of trametinib (0.8, 4, 20, 100, and 500 nmol/L) or in combination with JQ1 (500 nmol/L) for 48 hours and phosphorylation and total protein levels determined by Western blot. D, Combination of JQ1 and trametinib block MEKi-mediated transcriptional induction of PDGFRB. A1847 cells were treated with DMSO, JQ1 (500 nmol/L), trametinib (10 nmol/L), or the combination of JQ1 and trametinib for 48 hours and PDGFRB RNA determined by qRT-PCR. E, Trametinib and JQ1 combination therapy prevents development of trametinib-resistant colonies. A1847 cells were treated with DMSO, JQ1 (500 nmol/L), trametinib (10 nmol/L), or the combination of JQ1 and trametinib for 4 weeks and colony formation determined by crystal violet assays. F, Cotargeting MEK and BET proteins enhances growth inhibition of NF1-deficient EOC cells. EOC cells were treated for 5 days with trametinib (10 nmol/L), escalating doses of JQ1, trametinib (10 nmol/L), and JQ1, or DMSO, and cell viability determined. G, Trametinib-resistant cells exhibit enhanced sensitivity toward BET protein inhibition. A1847-T cells or A1847 cells were treated with escalating doses of JQ1 for 5 days and viability assessed. H, BET protein inhibition suppresses trametinib-induced kinome reprogramming signature in A1847-T cells. MIB-MS kinome profile of A1847-T cells following 48 hours treatment with JQ1 (500 nmol/L). Volcano plot

depicts SILAC-determined log2 fold changes in MIB binding as a ratio of JQ1/DMSO. Kinases induced by chronic trametinib exposure and repressed by JQ1 treatment are highlighted in blue. Statistical changes in SILAC-determined MIB binding were determined by one-paired t test (P < 0.05) using Perseus software. Biological duplicates of SILAC H-JQ1/L-DMSO or L-JQ1/H-DMSO were used to generate volcano plot. Data presented in F and G are from triplicate experiments SD; , P 0.05 by Student t test. Cell viability was determined by CellTiter-Glo assays.

is administered. Low doses of trametinib may alleviate repression and PDGFRB. The heterogeneity of RTKs induced at higher doses of RTKs regulated by FOSL1 such as DDR1, PDGFRB, and IGF1R, of trametinib, due to destabilization of both MYC and FOSL1, whereas higher doses of trametinib may lead to loss of repression may present a signiﬁcant challenge in the design of combination of both FOSL1- and MYC-regulated RTKs such as EGFR, ERBB3, therapies involving MEK and RTKs, particularly as ERBB and

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PDGFR family of receptors are quite distinct in structure and MEKi-reprogramming in NF1-deficient EOC cells, as well as inhibitor sensitivities. disrupt the adaptive response once it is established in MEKi- Previous studies have shown that combined blockade of BET resistant cells. Moreover, simultaneous administration of the bromodomain "reader" proteins and kinase inhibitors such as drugs was required to prevent MEKi-mediated transcriptional ERBB2/EGFR, PI3K, or MEK inhibitors prevented kinase adaptive induction of RTKs, where removal of JQ1 abolished transcrip- responses providing superior growth inhibition in breast cancer tional repression of MEKi-induced RTKs. Our findings suggest BET models (28–30). Here, we showed that the bromodomain pro- inhibitors should be utilized concurrently with trametinib or teins BRD2 and BRD4 were required for the MEKi-induced RTK administered once tumors have adapted to MEKi therapies for transcription and that combination therapies involving BETi and optimal inhibition of MEKi-induced kinome reprogramming. MEKi blocked RTK reprogramming providing more durable ther- However, further exploration of BET and MEK inhibitor combi- apeutic responses in NF1-deficient EOC cells. Intriguingly, our nations in NF1-deficient EOC preclinical models will be required loss-of-function studies suggest distinct functions for BRD2 and to optimize dosing and timing, as well as define potential toxi- BRD4 in regulation of RTK transcription, where BRD2 and BRD4 cities associated with the dual agent therapy. were required for transcription of different sets of RTKs following MEK inhibition in NF1-deficient EOC cells. The relevant functions Disclosure of Potential Conflicts of Interest of the distinct BET bromodomain proteins in the epigenetic No potential conflicts of interest were disclosed. control of cell growth and survival remains poorly characterized (36), with the majority of studies focusing on defining the Authors' Contributions role of BRD4 in promoting cancer. Here, we showed BRD2 was Conception and design: A.M. Kurimchak, J. Chernoff, J.S. Duncan DDR1 Development of methodology: K.E. Duncan, J.S. Duncan required for transcription, and that combined blockade of Acquisition of data (provided animals, acquired and managed patients, both BRD2 and BRD4 was required to reduce transcription of provided facilities, etc.): A.M. Kurimchak, C. Shelton, C. Herrera-Montavez, several RTKs. However, further studies exploring the role of each K.E. Duncan, J.S. Duncan BET protein in controlling kinase transcription will be required to Analysis and interpretation of data (e.g., statistical analysis, biostatistics, define BET protein–specific functions in EOC. computational analysis): A.M. Kurimchak, J.S. Duncan In previous reports, NF1-altered cancers were shown to exhibit Writing, review, and/or revision of the manuscript: A.M. Kurimchak, J. Chernoff, J.S. Duncan enhanced sensitivity to epigenetic inhibitors such as JQ1 due to Administrative, technical, or material support (i.e., reporting or organizing SUZ12 loss of the polycomb repressive complex 2 gene , which data, constructing databases): J.S. Duncan potentiates RAS-driven transcription (37). Here, we found the Study supervision: J. Chernoff, J.S. Duncan majority of NF1-deficienct EOC cells exhibited enhanced sensitivity to the trametinib–JQ1 combination relative to single agents, Acknowledgments whereas the majority of NF1-proficient EOC cells tested showed This work was funded by NIH CORE Grant CA06927 (Fox Chase Cancer minimal to no therapeutic benefit, suggesting NF1-deficient EOC Center), R01 CA211670 (to J.S. Duncan), R01 CA142928 (to J. Chernoff), and NIH T32 CA009035 (to A.M. Kurimchak). The W.W. Smith Charitable Trust cells may harbor unique transcriptional plasticity and dependen- Research Grant (to J.S. Duncan). Kinome profiling studies were funded by the cy for BRD4 for MEKi-mediated kinome reprogramming. How- Cancer Kinome Initiative at Fox Chase Cancer Center, which was established by ever, further proteogenomic studies exploring the distinct a donation from Don Morel. responses of NF1-wt or NF1-deficient EOC cells to trametinib will be required to decipher the differential impact that RAS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in activation has on transcriptional reprogramming to MEK accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibition. Finally, our evaluation of combination therapies using trame- Received December 16, 2018; revised March 14, 2019; accepted April 29, tinib and JQ1 showed BET inhibition can prevent the induction of 2019; published first May 1, 2019.

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1734 Mol Cancer Res; 17(8) August 2019 Molecular Cancer Research

Intrinsic Resistance to MEK Inhibition through BET Protein− Mediated Kinome Reprogramming in NF1-Deficient Ovarian Cancer

Alison M. Kurimchak, Claude Shelton, Carlos Herrera-Montávez, et al.

Mol Cancer Res 2019;17:1721-1734. Published OnlineFirst May 1, 2019.

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