Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Home , Casein kinase 2, Emerin, GSK3B, MAPK1, MTORC2

Published OnlineFirst May 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0938

Molecular Cancer Cancer Biology and Signal Transduction Therapeutics

Robert Parker1, Roderick Clifton-Bligh2, and Mark P. Molloy1

Abstract Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAPK able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized; however, how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this, we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation-selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identiﬁed thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that coinhibition of the pleiotropic acidophilic protein kinase CK2 (CK2) and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These ﬁndings highlight a role for CK2 blockade in potentiating the antiproliferative effects of BRAF and MEK inhibition in BRAF cancers. Mol Cancer Ther; 13(7); 1894–906. 2014 AACR.

Introduction kinase able to hyper-phosphorylate MAPK kinases MEK1/ Aberrant activity of protein kinases drives many of the 2, driving cell transformation through unrestrained ERK1/ hallmarks of cancer and also participates in the develop- 2 activation. The discovery of specific activating mutations ment of resistance to current treatments (1). In many has led to the clinical development of mutation-specific, cancers, dysregulation of the MAPK pathway is associated small-molecule kinase inhibitors. In melanoma, one such with poor prognosis, which results from activating muta- compound, vemurafenib (PLX4032), has shown high effi- tions in genes encoding cytosolic signaling proteins (e.g., cacy in BRAFV600E-positive patients (5), and the logical BRAF and RAS) or receptor tyrosine kinases (e.g., EGFR application of this approach in thyroid cancer is under and RET). The BRAF gene is found mutated in approxi- investigation. However, as with many targeted therapies, mately 1 of 4 of all anaplastic thyroid carcinomas (ATC), acquired resistance to treatment is common, and thus and 40% to 60% of papillary thyroid carcinomas (PTC; successful application of targeted therapies will benefit refs. 2–4). BRAF is commonly mutated by a single transver- from more sophisticated understanding of the events con- sion (T1799A) that codes for a missense protein (V600E). trolled by oncogenic mutations and the molecular Biochemically, BRAFV600E mimics phosphorylation of the responses that result from inhibiting these enzymes. T598 and S601 residues producing a constitutively active The impact of pharmacologic blocking of BRAFV600E by selective inhibition in thyroid cancer has been demonstrated in vivo. Inhibition reduces cell proliferation in Authors' Affiliations: 1Australian Proteome Analysis Facility, Department PTC/ATC mouse tumor xenograft (6) and recently of Chemistry and Biomolecular Sciences, Macquarie University; and 2Kol- ling Institute of Medical Research, University of Sydney, Sydney, New vemurafenib was shown to suppress tumor growth in South Wales, Australia BRAFV600E human ATC (7). At a molecular level, kb Note: Supplementary data for this article are available at Molecular Cancer BRAFV600E mediates activation of the NF- transcrip- Therapeutics Online (http://mct.aacrjournals.org/). tion factor, epigenetic reprogramming through methyla- TIMP3 HMGB2 Corresponding Author: Mark P. Molloy, Macquarie University, Research tion, and/or expression of genes such as , , Park Drive, Sydney, NSW 2109, Australia. Phone: 612-9850-6218; Fax: metalloproteases, and other structural extracellular 612-9850-6200; E-mail: [email protected] matrix genes that can promote proliferation and cell doi: 10.1158/1535-7163.MCT-13-0938 invasion (8–10). NF-kb–driven expression of TIMP1 is 2014 American Association for Cancer Research. also implicated in BRAFV600E thyroid cancers, able to

1894 Mol Cancer Ther; 13(7) July 2014

Antiproliferative Synergism of CK2 and BRAF Inhibitors

activate the PI3K/AKT pathway sustaining cell prolifer- tive inhibitor; 2 mmol/L vemurafenib (PLX4032; Selleck- ation, with one possible consequence being the over chem) or 1 to 2 mmol/L selumetinib (AZD-6244; Sell- activation of the mTOR pathway (8, 11). Gene expression, eckchem) or DMSO (Sigma) for 30 minutes before cell methylation, and molecular studies have revealed several lysis. Three independent cell cultures were used per cell key processes regulated by BRAF in thyroid cancer, but line and condition. Cells were lysed as described in (12) currently little is known about dysregulated posttransla- with minor modifications (see Supplementary Methods) tional control of protein signaling. In particular, little is and protein amounts determined. A total of 1 to 25 pmol of known about how selective inhibition of BRAFV600E siRNA-targeting transcripts of the human CK2a (Cell alters the output of cell signaling processes. Data outlining Signaling) and nontarget control siRNA were used to the signaling events regulated by BRAFV600E, and how knock down CK2 expression in melanoma cells. siRNA cells respond to small-molecule–based inhibition, are was delivered using Lipofectamine RNAiMAX for 72 vital to designing combination therapy programs that hours, protein levels were examined by Western blot are effective and combat the development of acquired analysis. resistance. Lack of knowledge of phosphorylated sites within Protein digestion and phosphopeptide enrichment proteins and identification of upstream regulating kinases A total of 500 mg of proteins were reduced, alkylated, are major limiting factors in understanding what path- and digested with trypsin (Promega) overnight at 37C. ways are activated in cancer. Currently, several reliable Samples were acidified, adjusted to 80 mg/mL glycolic techniques for phosphopeptide enrichment and charac- acid, and phosphopeptides purified by the addition of m terization using mass spectrometry have been developed 5 mg of TiO2 beads (Titansphere, 10 m) for 1 hour. Beads and promise to add significantly to this knowledge base were extensively washed and phosphopeptides were (12, 13). Temporal characterization of the phosphopro- eluted with consecutive 100 mL additions of 1% (v/v) teome cellular landscape provides a direct readout ammonia (Sigma) with 0%, 30%, and 50% (v/v) acetoni- of kinase substrates and can be used to develop hypoth- trile. Samples were immediately dried and resuspended eses about likely active kinases under those conditions. in 1% (v/v) trifluoroacetic acid, 5% (v/v) acetonitrile for Here, we used established techniques to profile the LC/MS-MS. phosphoproteome of a drug-sensitive BRAFV600E-positive ATC cell line (SW-1736), and monitor the quanti- Mass spectrometry (LC-MS/MS) tative response of approximately 2,000 phosphosites Samples were analyzed by LC-MS/MS using a Triple- after selective blocking of BRAFV600E and MEK1/2 TOF 5600 mass spectrometer (AB Sciex) and peptides with the clinically tested small-molecule inhibitors identified using ProteinPilot v4.5 and Mascot v2.2 as vemurafenib (PLX4032) and selumetinib (AZD6244). described in Supplementary Methods. Label-free quanti- We used thyroid cancer cell lines with defined muta- tation (LFQ) using extracted ion chromatograms was con- tions in BRAF and RET to demonstrate mutation-specific ducted using Skyline (14) and statistical analysis carried response in cell growth, cell cycle, and phosphorylation of out using the DanteR scripts. Parallel reaction monitoring known and novel effectors. Motif analysis of the flanking was conducted on the TripleTOF 5600 and analyzed using linear amino acid sequences of regulated phosphosites Skyline as described in Supplementary Methods. revealed several regulatory kinases that function in BRAFV600E signaling and in response to inhibition. Phosphosite localization and kinase assignment Although ERK1/2 and cyclin dependent kinase (CDK1/ To localize modifications, search results were processed 2) substrates were expected in downregulated sites after using Scaffold PTM (ProteomeSoftware), which uses A- BRAF inhibition, we also observed increases in some score (15) and a probability function to assign confidence phosphosubstrates consistent with the activity of acido- to amino acid modification location based on available philic kinase(s). We demonstrated that combinatorial inhi- peak depth present in MS/MS spectra. Upstream kinases bition of BRAF and the acidophilic kinase CK2 in thyroid were putatively assigned using the NetworKIN algorithm cancer and patient-derived melanoma cells is synergisti- (16), and the output was analyzed in excel and using the cally lethal and associated with decreased AKT signaling. program phosphosite analyzer (17), which performs statistical enrichment analysis. Materials and Methods Cell culture, siRNA, and protein preparation Viability assays Thyroid cell lines were tested for authenticity by short Cells were seeded in 96-well plates at 5,000 to 10,000 cells tandem repeat profiling according to the ANSI/ATCC per well in triplicate for each drug treatment and time ASN-0002-2011 standards. Melanomas established from point. After 2 hours, cells were treated with dilutions 0.02, primary tissue were genotyped previously (14). All cell 0.2,1,2,10mmol/L for vemurafenib (PLX4032), and 0.002, lines were cultured in RPMI 1640 medium supplemented 0.02, 0.1, 0.2, 1 mmol/L of selumetinib (AZD6244) and 0.8, 4, with 10% (v/v) bovine serum (Life Technologies) at 37C 8 mmol/L of CK2 inhibitor (CX-4945; Selleckchem) in m in a humidified atmosphere of 5% CO2, and grown to 80% thyroid cells. For melanomas, 0.5, 1, 2, 4, 8 mol/L of all to 90% confluence. Cells were incubated with the respec- compounds were used either singularly or in combination.

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After 72 hours, cell viability was assessed by Presto blue ulated via this kinase. We used both vemurafenib and assay (Life Technologies). selumetinib to find common and distinguishable changes to phosphorylation regulated by either BRAF or MEK1/2, Western blot respectively. Following short-term drug exposure (15 m A total of 25 g of protein was mixed with NuPAGE and 30 minutes), proteins were extracted, digested, and loading buffer (Life Technologies). Membranes were phosphopeptides were enriched using TiO2 before being blocked with TBS/Tween-20 supplemented with 5% analyzed by LC-MS (Fig. 1D). In total, 2,329 unique peptide (w/v) skimmed milk, incubated with primary antibody sequences, of which approximately 90% were phosphory- O/N at 4 C, and with secondary antibody conjugated to lated (STY), were identified at an FDR of approximately 1%. fluorescent tag (LiCor) for 1 hour at room temperature and From these, 2,457 p[S], p[T], p[Y] sites could be localized imaged using an Odyssey system (LiCor). with high confidence (>95%) using MS/MS spectra (Sup- plementary Table S1). These class-one sites mapped to Results approximately 1,200 phosphoproteins. For the class-one Mutations in the MAPK pathway predict sensitivity sites that represent the highest confidence phospho-site to MAPK inhibitors assignments in the dataset, the NetworKIN algorithm was To measure the sensitivity of thyroid cell lines to MAPK used to map the most likely kinase responsible for the inhibitors, we measured the relative effect on cell prolif- phosphorylation of each site (Supplementary Table S2). eration using the oncogenic BRAF inhibitor vemurafenib NetworKIN uses motif analysis and context information to (PLX4032; ref. 18) and the MEK1/2 inhibitor selumetinib generate a score for each kinase–substrate relationship (16). (AZD6244; ref. 19; Fig. 1A). The cell lines SW-1736 (BRAFV600E; ref. 3), TPC-1 (RET/PTC1 translocation; Phosphoproteins regulated by BRAF and MEK ref. 20), and the BRAF wild-type thyroid cell, Nthy-ori inhibition 3.1 (derived from normal tissue and SV40 immortalized; We next quantified the difference in relative abundance ref. 21), were used as a comparative system for MAPK of phosphopeptides in SW-1736 cells treated with vemur- activation in thyroid cancer, and treated for 48 to 72 hours afenib or selumetinib (Supplementary Table S3). General- with 0.2 mmol/L selumetinib and 2 mmol/L vemurafenib ly, data were well matched across treatments and biologic (Fig. 1B and C). The BRAFV600E-mutant cell line SW-1736 replicates; however, each peak was manually validated to exhibited sensitivity (>40% growth inhibition at 1–2 ensure accurate peak integration, resulting in approxi- mmol/L) to both BRAFV600E and MEK1/2 inhibition, mately 1,800 phosphopeptides reliably quantified after whereas both TPC-1 and Nthy-ori 3.1 were insensitive to drug inhibition (Supplementary Table S3). To assign high vemurafenib as expected (10% growth inhibition at 1–2 confidence changes in altered phosphopeptides, a robust mmol/L; Fig. 1B). Both cell lines with activating MAPK ANOVA (P < 0.05) with a fold change of 1.5 was used as a pathway mutations (SW-1736 and TPC-1) showed similar cutoff; FDR was controlled by P value adjustment using sensitivity to selumetinib (30%–40% growth inhibition the Benjamini and Hochberg method (Q < 0.2; Supplemen- at 1 mmol/L), demonstrating the convergence of pathway tary Table S3). At this level of stringency, 23 phosphopep- activation through the downstream component MEK1/2 tides (33 phosphorylation sites) showed significant (Fig. 1B). ERK1/2 output was detected by Western blot changes in abundance in all treatment groups and most analysis of phospho-retinoblastoma binding protein (RB), were coregulated by BRAFV600E and MEK inhibition total cyclin-D1, p27KIP, phospho-ERK and total-ERK in (Table 1). Supplementary Table S4 describes the key the presence or absence of 2 mmol/L vemurafenib (Fig. function (derived from UniProt and PubMed) of each 1C). At 48 hours, clear reduction in phospho-ERK1/2 was protein and the most likely (NetworKIN; ref. 16) or verified evident in the BRAFV600E cells after treatment with kinase(s) for these sites and also compares with expression vemurafenib. A concurrent reduction in phosphorylation observed previously for these phosphorylation sites in of RB, cyclin-D1 and a moderate increase in p27KIP cell-cycle phases G1–M (22). In general, the direction in protein are consistent with reduced ERK1/2 output. which sites were regulated by 30 minutes of MAPK inhi- Both TPC1 and Nthy-ori 3.1 exhibited no change in phos- bition showed limited overlap with G1 (5/14) or M (2/9) pho-ERK, phospho-RB, and cyclin-D1, confirming that phase arrest observed in a previous study in HeLa cells ERK1/2 output is not affected by vemurafenib in these (22). Several phosphoproteins with sites regulated in our cells. TPC1 cells exhibited loss of p27KIP expression upon study were annotated with roles in the regulation of exposure to vemurafenib; however, no significant change chromatin structure/organization, control of transcrip- in proliferation was observed. tion, and nuclear envelope organization. Gene ontology analysis using Ingenuity pathway analysis indicated that Phosphoproteomic analysis of MAPK pathway processes DNA methylation and transcriptional repres- inhibition sion (P ¼ 1.29 10 9; >50% pathway coverage) and ERK/ In SW-1736 cells, signaling through the MAPK pathway MAPK signaling (P ¼ 8.11 10 6) were highly represented is dominated by the oncogenic BRAFV600E mutation, so (Supplementary Table S5). The relative change in abun- that screening for changes to protein phosphorylation after dance of these core MAPK components correlated with BRAFV600E inhibition will identify proteins that are reg- both treatments (30 minutes; R2 ¼ 0.62) indicating a high

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A B C μ Nthy-ori TPC1 SW1736 2 mol/L vemurafenib 48 h 120% +–+–+– WT TPC WT RET RET RET 100% p-RB

80% p-ERK BRAF WT BRAF WT BRAF V600E 60% t-ERK Vemurafenib 40% t-p27KIP MEK MEK MEK % Viable 3 days 20% Selumenitib t-Cyclin D1 0% ERK1/2 ERK1/2 ERK1/2 Nthy-ori TPC1 SW1736 2 μmol/L vemurafenib 0.2 μmol/L selumetinib

D E 2 DMSO 2 mmol/L Vem 1 mmol/L Sel 1 15 min 30 min 15 min 30 min 30 min vemurafenib PAK4 MAPKi SW1736 0 p-ERK (BRAFV600E) –5 –4 –3 –2 –1 RRAS2 210 PPP1R12A –1 Phosphopeptide –2 enrichment –3 Label-free quant R 2 = 0.6231 –4 by LC-MS MAPK1

–5 30 min selumetinib

Figure 1. Experimental design and phosphoproteomics. A, three thyroid cell lines were chosen for their known mutations in the ERK1/2 MAPK pathway. Nthy- ori provided a wild-type MAPK pathway response. TPC1 is activated by RET/TPC translocation, and SW-1736 is activated by BRAFV600E, representing two major mutations found in the MAPK pathway in thyroid cancer. The points of inhibition by vemurafenib and selumetinib in MAPK are indicated (light gray). B, the viability of each cell line was measured after exposure to vemurafenib and selumetinib for 3 days; error bars, SD. C, Western blot of ERK1/2 signaling output after treatment for 2 days with 2 mmol/L vemurafenib. D, phosphoproteomic workflow showing SW-1736 cells treated with MAPK inhibitors and MAPK activity assessed by Western blot for p-ERK; samples were digested with trypsin and phosphopeptides enriched by TiO2 and analyzed by LC-MS. E, scatterplot of the log2 relative effect measured for phosphopeptides after 30-minute exposure to vemurafenib and selumetinib. Phosphopeptides labeled (solid boxes) are from proteins involved in the canonical MAPK pathway. degree of similarity in response to MAPK inhibition at the proportion in the entire dataset. Figure 2 summarizes blockade through BRAF or MEK (Fig. 1E). Among these the results of this analysis; we observed that approximately peptides, S695 in SMARCA4 and S297 in SGTB exhibited 50% (RRP1B S245, TMF1 S344, MAX S2 and S11 and OSTF1 an early 15-minute response to MAPK inhibition with S213) of the upregulated sites with NetworKIN kinase MEK and were nearer to control levels after 30-minute predictions could be attributed to the CK2 family (Fig. inhibition. Two other peptides, S331 of BIN1 and S389/393 2A–C). This pattern was confirmed using the phosphosite in SRRM1, were regulated specifically by BRAF inhibition. analyzer program, which performs a statistical test where the frequency of regulated clusters of phosphosites is Explorative kinase enrichment compared with the background frequency of all phospho- To investigate if any kinase(s) could account for a sig- sites (17; Supplementary Fig. S1). CK2 is an acidophilic nificant proportion of the regulation observed in the phos- kinase that requires an acidic or phosphorylated residue at phopeptide dataset, we explored the NetworKIN results position þ3 in their substrate (SXXE/D). In our data, one for phosphorylation sites changing in abundance versus site OSTF1 S213 is the penultimate amino acid at the

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Table 1. Phosphoproteins regulated by BRAF and MEK inhibition in SW1736 thyroid carcinoma cells

PLXa PLXa AZDa AZDa Siteb P mct.aacrjournals.org

Gene name UniProt Protein name Peptide sequence P-site (15 min) (30 min) (15 min) (30 min) value Published OnlineFirstMay13,2014;DOI:10.1158/1535-7163.MCT-13-0938 BIN1 O00499 Myc box-dependent–interacting protein 1 VNHEPEPAGGATPGATLPK[pS]PSQLR S331 0.1 0.9 0.3 0.0 0.97 PPP1R12A O14974 Protein phosphatase 1 regulatory KTG[pS]YGALAEITASK S445 0.9 1.4 1.3 0.8 1 subunit 12A TPR P12270 Nucleoprotein TPR TDGFAEAIH[pS]PQVAGVPR S2155 1.4 2.2 3.3 2.6 1 MAPK1 P28482 MAPK 1 VADPDHDHTGFL[pT]E[pY]VATR T185, Y187 0.8 1.4 2.7 3.3 1,1 EMD P50402 Emerin DSAYQSITHYRPV[pS]ASR S171 0.3 0.8 0.7 0.6 0.97 on September 24, 2021. © 2014American Association for Cancer Research. SMARCA4 P51532 Transcription activator BRG1 KIPDPD[pS]DDVSEVDAR S695 0.4 0.3 3.6 0.2 1 MAX P61244 Protein MAX [pS]DNDDIEVE[pS]DADKR S2, S11 0.1 0.7 0.7 1.2 1 TMF1 P82094 TATA element modulatory factor SVSEIN[pS]DDELSGK S344 0.4 0.5 0.7 1.0 1 AHNAK Q09666 Neuroblast differentiation-associated FKAEAPLP[pS]PK S5110 0.3 1 0.8 2.2 1 protein LK[pS]EDGVEGDLGETQSR S135 0.4 1.2 1.6 2.9 1 ISMPDLDLHLK[pS]PK S2397 0.3 1.1 0.8 1.7 1 VDIN[pT]PDVDVHGPDWHLK T4766 0.1 0.6 0.1 1.0 1 RRP1B Q14684 Ribosomal RNA processing protein VGDGDL[pS]AEEIPENEVSLR S227 (S245) 3.1 2.8 3.7 3.5 1 1 homolog B EZH2 Q15910 Histone-lysine N-methyltransferase EZH2 VKESSIIAPAPAEDVD[pT]PPR T448 0.3 0.8 0.1 0.7 1 RBBP6 Q7Z6E9 Retinoblastoma-binding protein 6 ENF[pS]PER S861 0.9 1.2 1 1.6 1 SRRM1 Q8IYB3 Serine/arginine repetitive matrix protein 1 KPPAPP[pS]PVQSQ[pS]PSTNWSPAVPVK S781, S787 0.7 0.4 1 0.3 1,1 RL[pS]PSA[pS]PPR S389, S393 0.1 1.7 0.2 0.3 1,0.98 OSTF1 Q92882 Osteoclast-stimulating factor 1 TLSNAEDYLDDED[pS]D S213 0.5 1.2 1.4 1.9 1 SGTB Q96EQ0 Small glutamine-rich tetratricopeptide SF[pS]SSAEEHS S297 0.5 0.7 2.6 0.9 0.99 repeat-containing protein beta FAM129B Q96TA1 Niban-like protein 1 AAPEAS[pS]PPA[pS]PLQHLLPGK S679, S683 0 0.2 0.2 0.8 1,1 oeua acrTherapeutics Cancer Molecular PDS5B Q9NTI5 Sister chromatid cohesion protein PDS5 GHTA[pS]ESDEQQWPEEK S1257 0.1 0.8 0.4 0.6 0.84 homolog B NOP16 Q9Y3C1 Nucleolar protein 16 FGY[pS]VNR S16 1.3 1.9 1.6 2.1 1 PLEC Q15149 Plectin TQLA[pS]WSDPTEETGPVAGILDTETLEK S4406 0.24 0.39 0.06 1.39 0.73

NOTE: Q < 0.2 shown in bold. a Log2 fold change. bP-site localization probability. Published OnlineFirst May 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0938

Antiproliferative Synergism of CK2 and BRAF Inhibitors

A B C

min min min min Upregulated CDK2/CDK3 10% CDK2/CDK3, p38,JNK 30% Figure 2. Kinase enrichment CK2 analysis. A, clustered heatmap of 50% RCK,CLK the log2 relative effect of 10% phosphosites regulated by MAPK Others inhibition (Q < 0.2; red > 0; green < 0). B, web logo display of stacked CDK2/CDK3 Background histograms generated from the 11 amino acid linear motifs p38 surrounding upregulated and 20% downregulated sites, with 42% CK2 background (all) sites shown for 18% comparison. C, pie charts for the JNK top 5 (occurrence) kinase groups found to match (using NetworKIN) 7% 13% Others the substrates of sites upregulated and downregulated Downregulated CDK2/CDK3 phosphoproteins. CDK2/CDK3, 19% p38, JNK 38% CK2 19% Unassigned

12% 12% Others

C-terminus, and thus no þ3 position is possible; however, validation of 8 of the 23 phosphopeptides (Supplementary the phosphoacceptor site is surrounded by acidic residues Table S6). Four of these, MAPK1, translocated promoter in positions 4toþ1 and NetworKIN predicted it as a region (TPR), niban-like protein 1 (FAM129B), and oste- CK2 family substrate. Searching the literature revealed the oclast-stimulating factor 1 (OSTF1), were plotted along- sites regulated in protein MAX and RRP1B as verified CK2 side a phosphopeptide from chaperone DnaJ (DNAJ) substrates, whereas others have no bona fide kinase rela- measured as a loading control. These data were consistent tionships assigned (23, 24). Other upregulated phospho- with the original screen and confirmed that these phos- sites were associated with CDK2/3, RCK, CLK family phorylation sites are regulated by BRAFV600E inhibition kinases. Among the downregulated sites, the majority with vemurafenib in the BRAFV600E cell line SW-1736. were predicted substrates of the p38 family and CDK2/ All but one (S213 in OSTF1) of the phosphopeptides 3 family. CDK2/3 and MAPK are highly related protein responded to MEK inhibition in BRAFV600E, RET/TPC, kinases and share similar substrate specificity (PXSP and and the BRAF wild-type normal thyroid cell line, indicat- SPXK/R). ing that these substrates were normally regulated by the MAPK pathway requiring activated MEK1/2. The RET/ MAPK blockade with vemurafenib is mutation TPC mutant exhibited small increases in phosphorylation specific of MAPK1, AHNAK, TPR and a decrease in OSTF1 phos- Next, we investigated the regulation of several phosphorylation after treatment with vemurafenib. This is phosites by MAPK blockade in the background of the two consistent with the paradoxical activation of the MAPK most common thyroid cancer mutations (BRAF and RET). pathway observed in a BRAF wild-type background, with To perform this analysis, we prepared fresh cell lysates an activated Ras signal (26), and is corroborated by the loss and used a relatively novel mass spectrometric technique of p27KIP determined by Western blot (Fig. 1C). called parallel-reaction monitoring (PRM) that quantitates with high-specificity, predetermined peptides present in Inhibition of CK2 is synergistically lethal with the samples (25). We selected a subset of the phosphopep- vemurafenib or selumetinib in BRAF-mutated tides whose levels are altered upon MAPK inhibition and thyroid carcinoma and melanoma cell lines applied this method to quantitate the specific phospho- Kinase enrichment analysis indicated that blocking peptide and a detectable counterpart unmodified peptide BRAFV600E signaling with vemurafenib or selumetinib from the same protein (Fig. 3A–D). This approach was less was associated with an increase phosphorylation of sub- sensitive than the LFQ experiment but still enabled cross strates of acidophilic kinase(s). Of the acidophilic kinases,

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A B

3 MAPK1 4 TPR 2 3 1 2 1 0 ratio ratio 2 2 0 –1 –1 Log Log –2 –2 –3 –3 BRAFi MEKi BRAFi MEKi BRAFi MEKi –4 SW1736 TPC1 Nthy-ori BRAFi MEKi BRAFi MEKi BRAFi MEKi Figure 3. Inhibitor-based phosphopeptide regulation in - SW1736 TPC1 Nthyori BRAFV600E, RET/TPC1, and wild-type genetic backgrounds GQVFDVGPR (unmodified) FLADQQSEIDGLK (unmodified) quantiﬁed by mass spectrometry. VADPDHDHTGFL[pT]E[pY]VATR (T185, Y187) TDGFAEAIH[pS]PQVAGVPR (S2155) A–D, bar plot for log2 ratio of 4 phosphopeptides, counterpart SL[pS]TSGESLYHVLGLDK (DNAJ loading control) SL[pS]TSGESLYHVLGLDK (DNAJ loading control) nonphosphopeptides, and phospho-DNAJ (loading control) relative to control after 30-minute 3 C FAM129B D 3 treatment with vemurafenib OSTF1 2 and selumetinib in SW-1736 2 1 (BRAFV600E), TPC1 (RET/TPC), 1 and Nthy-ori (wild-type) thyroid ratio 0 ratio 2 2 0 cells. –1 Log Log –1 –2 –2 –3 –3 BRAFi MEKi BRAFi MEKi BRAFi MEKi BRAFi MEKi BRAFi MEKi BRAFi MEKi

SW1736 TPC1 Nthy-ori SW1736 TPC1 Nthy-ori

TLMNTIMQLR (unmodified) GYADIVQLLLAK (unmodified)

AAPEAS[pS]PPASPLQHLLPGK (S679) TLSNAEDYLDDED[pS]D (S213)

SL[pS]TSGESLYHVLGLDK (DNAJ loading control) SL[pS]TSGESLYHVLGLDK (DNAJ loading control)

CK2 was predicted by NetworKIN to regulate several of 4A). In cells with wild-type BRAF protein (TPC1 and these sites, and two, S2/11 in MAX and S245 in RRP1B, Nthy-ori 3.1 cells), the combination of CX-4945 with have been reported previously (23, 24). As CK2 can vemurafenib showed only a minor effect on cell prolifer- activate AKT signaling, a known mechanism for drug ation. In TPC1 cells treated with selumetinib, the addition resistance to MAPK inhibitors, we hypothesized that CK2 of the CK2 inhibitor acted antagonistically and positively activity might interact with the inhibition of BRAFV600E regulated cell proliferation (19%, Bliss additivity; Fig. as a positive regulator of cell proliferation. To investigate 4A). To determine if CK2 inhibition in the RET-mutant cell this, the thyroid cell lines were treated with vemurafenib line TPC1 would be more effective in combination with a (2 mmol/L) or selumetinib (1 mmol/L) in combination MAPK inhibitor tailored to this mutation, we assessed with increasing concentrations of the speciﬁc CK2 inhib- possible synergistic effects of vandetanib and CK2. TPC1 m m itor CX-4945, (0.8–8 mol/L; ref. 27) and cell viability cells were sensitive to vandetanib (GI50 1 mol/L); how- determined after 3 days (Supplementary Fig. S2). In the ever, TPC1 was relatively insensitive to CK2 inhibition BRAFV600E-mutant SW-1736 cells, CK2 inhibition alone with CX-4945, and no synergistic effect of coinhibition was reduced cell growth by approximately 13%, vemurafenib observed (Supplementary Fig. S3), conﬁrming the require- reducedgrowthbyapproximately36%consistentwith ment of the BRAF lesion for CK2 synergism to be effective. our earlier observations. Coadministration of CX-4945 To test the effect of combined treatment in melanoma, and vemurafenib was synergistic, reducing growth by four patient-derived cell lines were evaluated. Genotyp- approximately 60% compared with controls (Fig. 4A). ing of C0045 and C0088 showed they carry the The antiproliferative activity was greater than (6%–15%) BRAFV600E/K mutation, whereas C0037 and C0084 are that of Bliss additivity at all concentrations used (Sup- BRAF wild-type (14). For both oncogenic BRAF melanoma plementary Fig. S2 and Supplementary Table S7). The cell lines, the effects of combining CK2 inhibition with level of synergism observed between selumetinib and vemurafenib were additive (Supplementary Fig. S4). For CX-4945 was considerably lower (5% at 4 mmol/L; Fig. these lines, we also calculated the combination index

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Antiproliferative Synergism of CK2 and BRAF Inhibitors

Figure 4. Response to combinatorial MAPK/CK2 inhibition. A, bar plot of growth inhibition in SW-1736, TPC-1, and Nthy-ori cells after 3-day exposure to 4 mmol/L CX-4945, 2 mmol/L vemurafenib (PLX-4032), 1 mmol/L selumetinib (AZD6244), 2 mmol/L vemurafenib in combination with 4 mmol/L CX-4945, 1 mmol/L selumetinib in combination with 4 mmol/L CX-4945. Additivity >5% is marked with a dotted line and shaded, and was calculated as the theoretical combined fractional response effect [(FaþFb)(100-Fa)/100], where F is the percentage of cells killed at given concentration, Fa is vemurafenib or selumetinib, and Fb is CX-4945. B, bar plot for the CI based on the fractional responses measured for four patient-derived melanoma cell lines after treatment with a 1:1 mixture of CX-4945 and vemurafenib 2-fold serially diluted (1.0–16 mmol/L). C, Western blot for CK2a after 72 hours of transfection with CK2a (siCK2a)or control (siCON) siRNA in melanoma cells C0037 (BRAF wild-type) and C0088 (BRAFV600K); b-tubulin is shown to represent total protein loading. D, bar plot of viability of melanoma cells C0037 (BRAF wild-type) and C0088 (BRAFV600K) after siRNA interference with 1 pmol of siCON, siCK2a,4mmol/L vemurafenib (PLX-4032), 4 mmol/L CX-4945, and combinations with siRNA treatments.

(CI) for serially diluted 1:1 mixtures of vemurafenib and cells. However, when CK2 siRNA was combined with CX-4945 (0.5–8 mmol/L) using the Chou and Talalay 4 mmol/L vemurafenib, an additive response was method (28). Synergism was observed for all combina- observed in the BRAFV600K-mutant C0088, whereas tions in both oncogenic BRAF melanoma cell lines, with no significant change in viability was detected in BRAF strong synergism (CI ¼ 0.21 at 4 mmol/L total dose) wild-type C0037 cells, consistent with the observations observed for V600E-mutant C0045, and synergism we reported for CK2 inhibition with CX-4945. observed for V600K-mutant C0088 (Fig. 4B). Both BRAF wild-type melanoma lines were unaffected by vemur- CK2 affects AKT signaling in oncogenic BRAF afenib alone (<10%), and no advantage was observed thyroid cells with coadministration of CX-4945, confirming the Several possible mechanisms may underlay the syner- requirement of the BRAF lesion for drug synergism as gism observed between vemurafenib and CK2 inhibition observed above with thyroid cells. Interestingly, all in BRAF-mutant cells. To explore one avenue, we per- melanoma cell lines were more sensitive to CK2 inhi- formed Western blot analysis of signaling through the bition alone (20%–35% at 4 mmol/L) when compared PI3K–AKT pathway in the BRAFV600E thyroid cells. SW- with thyroid carcinoma cell lines (2%–13% at 4 mmol/L). 1736 cells were treated with vemurafenib (2 mmol/L), CX- To confirm specificity of the observed BRAF/CK2 drug 4945 (4 mmol/L), AKT inhibitor perfosine (4 mmol/L), and synergism, we knocked down the expression of CK2a with binary combinations for 30 minutes (Fig. 5B). Phos- using siRNA in two of the melanoma cell lines C0088 phospecific antibodies for two well-known AKT phos- (BRAF) and C0037 (wild-type). We repeated the viabil- pho-activation sites (T308 and S473) were downregulated ity assay using 4 mmol/Lvemurafenibandcomparedit by CK2 treatment alone, and when CK2 inhibition was with effects of dual inhibition using CX-4945/vemur- combined with vemurafenib (Fig. 5B). Phosphorylation of afenib (Fig. 4C–D). The cell viability response to CK2 the CK2-regulated AKT priming site, pSer129, was siRNA alone was minimal in both C0037 and C0088 reduced upon exposure to CX-4945, providing a clear

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A Inhibition of V600E BRAF Vemurafenib cell invasion

FAM129B PPP1R12A Selumenitib MEK1/2

NUAK1 Cytosol PTEN/AKT?

ERK1/2 OSTF1 CK2 Cell motility CDK1/2 Figure 5. Summary of ? phosphoproteomic pathways. A, network diagram consisting of several proteins with sites regulated (red, up; green, down) in TPR Nucleus MAX Myc? this study; connections place regulatory signiﬁcance in context of knowledge about speciﬁc EZH2 PRCR – ? Myc genes substrate kinase relationships. Several regulatory kinases are Regulation of gene transcription implicated, and the potential Polycomb genes mechanistic sites of regulation (gray dashed boxes) and biologic B C processes are hypothesized. B, Western blot analysis of the PI3K/ RTK AKT pathway after CK2, BRAF, and AKT inhibition for 30 minutes in AKT pSer129 SW-1736 thyroid cancer cells. C, PIP3 PIP2 network interpretation of Western AKT pThr308 PI3K blot of the PI3K/AKT pathway. NC, PTEN AKT pSer473 no change. NC P NC P AKT PDK1

GAPDH CK2

P PDK1 P AKT GSK-3β pSer9 P

PTEN pSer380 P Cell cycle mTORC2 GSK3 C-RAF pSer258 Apoptosis Metabolsim GAPDH

link between CK2 and the AKT prosurvival pathway. tified approximately 2,300 phosphopeptides and quanti- BRAF inhibition alone was unable to illicit changes to tated approximately 1,800, demonstrating comprehensive AKT phosphorylation, indicating the response to be depth of analysis. Gene enrichment analysis of protein dependent on CK2 activity. Interestingly, PTEN phos- function indicated that a high abundance of components phorylation was not affected, nor was CRAF, a target of involved in the regulation of gene transcription and sig- AKT1. Little effect was observed for PDK1, the kinase naling through MAPK are constitutively phosphorylated. responsible for AKT1 phosphorylation at T308 after RTK Using a PTM site localization algorithm, linear motif activation. The inhibitory site Ser9 in the N-terminal of the analysis, contextual and literature searches, phosphopep- AKT target GSK3b was downregulated, indicating CK2 tides could be confidently assigned as putative and bona activity is required for AKT signaling to GSK3b in SW- fide substrates to several upstream regulatory kinases. The 1736 cells. LFQ mass spectrometry approach was able to quantify the relative effect of shutting down MAPK signaling by blocking BRAFV600E. We observed highly significant Discussion reduction in the MEK1/2 substrate of the MAPK In this work, we measured the response of the phos- ERK1/2, and several other MAPK signaling molecules phoproteome of BRAFV600E-activated thyroid cancer showed high correlation between treatments, indicating cells to pharmacologic blockade with the clinically tested highly overlapping responses to inhibiting at BRAF and inhibitors vemurafenib and selumetinib. Our work iden- MEK in thyroid carcinoma cells (Fig. 1E).

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Antiproliferative Synergism of CK2 and BRAF Inhibitors

It was important to see if the phosphorylation sites proteins in a melanoma cell line. In that report, physio- identified to be inhibited by the compounds used in logic characterization demonstrated that phosphorylation this study were also reported to be inhibited by other MEK drives cell invasion by an unknown mechanism (33). In inhibitors (29, 30). Site S135/136 in neuroblast differenti- our dataset, gene enrichment analysis demonstrated the ation-associated protein (AHNAK) was significantly large part played by phosphoproteins in the regulation of downregulated after both BRAF and MEK inhibition and gene transcription. EZH2, BIN1, and TPR all regulate has previously been shown to be regulated in response to chromatin structure and organization and were regulated the MEK inhibitor I (CAS 297744-42-4) in the MEK-sensi- by MAPK inhibition at sites corresponding to p38/ tive acute myelogenous leukemia (AML) cell line, MV4-11. CDK1/2 phosphorylation. PRM mass spectrometry con- In their study, phosphorylation of S135/136 was nonre- firmed the phosphorylation of S2155 in TPR, a nuclear sponsive to MEK inhibition in the resistant AML cell line, pore complex protein important for nuclear transport and HEL. In a second study, melanoma cells were treated with mitosis (34). S2155 is a confirmed substrate of ERK1/2, MEKi GDC-0973 and PI3Ki GDC-0941, and immunopre- flanked by ERK1/2 docking domains; phosphorylation cipitation of phosphopeptides was used to profile DNA enhances ERK1/2 binding and is thought to sequester damage response signaling (29). The protein SMARC4 ERK1/2 to nuclear targets enabling gene transcription showed initial decrease (not significant) in phosphoryla- (35). These data indicate several processes in which tion after MEKi and increased after long-term treatment BRAFV600E drives through promoting ERK1/2 or with PI3Ki singularly and in combination with MEKi (29). CDK1/2 activity in the nucleus. The phosphorylation of Consistent with data presented here, S695 in SMARC4 is EZH2, TPR, and BIN1 may be key in modifying chroma- affected at 15 minutes with the MEKi AZD-6244 and tin-interacting proteins to regulate gene expression nec- returns to control levels by 30 minutes. These data confirm essary for proliferation (Fig. 5A). that several phosphosites are consistently regulated in The enrichment of putative phosphopeptide substrates response to MEK inhibition across several different cell of CK2 following BRAFV600E inhibition indicates that types in agreement with our observations. blocking MAPK has consequences for CK2 activity. CK2 is We investigated the kinases that are involved in driving constitutively active with >300 known substrates (36). The the BRAFV600E phenotype and the response to kinase specificity of CK2 is achieved by regulatory b-subunits inhibition. The results were striking; the majority of phos- and compartmentalization (36, 37). The N-terminal phosphorylation sites downregulated were flanked by the phopeptide from protein MAX, a nuclear component of highly conserved p38/CDK2/3 linear motif, indicating the c-MYC transcription factor complex identified in our prominent activity of ERK1/2 and/or CDK2/3. However, screen, was upregulated after vemurafenib blocking in surprisingly, many sites (40%) were upregulated and BRAFV600E thyroid cancer cells (38). The peptide was N- were highly enriched (4-fold over background) for sub- terminally acetylated and phosphorylated at positions S2 strates of the acidophilic kinases. Several of the phosphor- and S11. S11 is flanked by a CK2 consensus motif and ylation sites proposed to be regulated by these kinases are phosphorylated by CK2 to prevent cleavage by caspases from proteins with characterized functions in tumor biol- to inhibit caspase-5 Fas-mediated apoptosis (23, 39). A ogy; most function in chromatin regulation and, for a few, second protein that was predicted to be a CK2 substrate the function of the phosphorylation site has been reported was OSTF1; phosphorylation of S213 increased after (Supplementary Table S4). Figure 5A is a summary of BRAF or MEK inhibition, and this was confirmed by PRM these data and attempts to place the protein phosphory- mass spectrometry. S213 is at the C-terminal of the OSTF1 lation events detected in networks with respect to their protein, a human ortholog of SH3P2 in mice, and both are upstream regulatory kinases and the downstream pro- indirectly involved in osteoclast formation (40). Recently, cesses they affect. phosphorylation of SH3P2 at S202 was shown to be ERK Of those phosphoproteins affected by MAPK inhibi- dependent and catalyzed by ribosome S6 kinase (RSK; tion, the cytosolic protein, PPP1R12A, is a key regulatory ref. 41). RSK phosphorylation promoted cell motility by component of protein phosphatase 1 (PP1); the site S445 inhibiting the activity of SH3P2 (41). Our data indicate a was assigned by NetworKIN as a substrate of the AMPK role for regulation of the closely situated S213 by a CK2 group of kinases and is a bona fide substrate of the AMPK- family kinase. It would be interesting to investigate the related kinase NUAK1 (31). Phosphorylation of S445 by role S213 may play in cell motility and investigate how the NUAK1 inhibits targeting of PP1 to myosin, suppressing plasticity of phosphosite localization in SH3P2 relates to phosphatase activity and maintaining phospho-myosin function and cell phenotype. essential for the actin-cytoskeletal rearrangements The increase in levels of phosphoprotein substrates required for cell migration (32). Another protein involved predicted to be regulated by acidophilic kinases following in cell migration/invasion was niban-like protein MAPK pathway inhibition was intriguing. As the activity (FAM129B). Kinase-mediated regulation of the sites of the archetypal acidophilic kinase CK2 is usually asso- S692 and S696 was confirmed by PRM mass spectrometry, ciated with the maintenance of cell proliferation in cancer with no detectable change in the unmodified protein (42), this led us to evaluate the therapeutic potential of amount. These sites were also found phosphorylated in controlling this signaling axis in BRAF cancers. We treated an inhibitor screen for BRAFV600E-regulated phospho- cells with a relatively new and specific inhibitor of CK2

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Parker et al.

(CX-4945; ref. 43) alone and in combination with vemur- The development of many new anticancer drugs is based afenib or selumetinib. Growth inhibition was additive on the knowledge that cancers arise from specific genetic when combined with vemurafenib, increasing cell death aberrations in genes controlling regulatory pathways (27). by 15% to 25% in both thyroid and melanoma cell lines Logically targeting these pathways with high specificity harboring the BRAFV600E mutation. This suggests that should provide a personalized antitumor effect while the observed increase in abundance of CK2 phospho- reducing side effects of more generalized cytotoxic chemo- substrates was potentially a survival adaptation to MAPK therapy. Here, we reveal several novel sites of regulation pathway blockade. that occur in the phosphoproteome of BRAFV600E-mutat- CK2 promotes antiapoptotic, prosurvival mechanisms ed thyroid cancer cells treated with therapeutic molecules through potentiation of NF-kb, Wnt/b-catenin, PI3K/ designed to target specific components of MAPK signaling. AKT pathways, and inhibition of caspase-mediated apo- CK2 inhibition has already been shown to be effective in ptosis (37, 42, 44). CK2 regulation is key to the develop- combination with EGFR inhibition by erlotinib in lung ment of multidrug resistance (MDR) phenotypes and carcinoma, and to prevent DNA repair after treatment with plays a role in "nononcogene addiction" in several cancers, the chemotoxic agents gemcitabine, cisplatin, and carbo- including T-cell leukemia, prostate, colon, and breast platin (27, 55), and our data suggest that cotargeting cancer (42). In patients with melanoma with the BRAFV600E and CK2 is a valid path for further investiga- BRAFV600 mutation, the long-term effectiveness of tion. This could be especially useful in some BRAF-mutant vemurafenib is limited by the development of acquired colon cancers that seem unresponsive to direct BRAF drug resistance. In humans and cell models, the develop- inhibition due to feedback mechanisms that reactivate RTK ment of resistance is often associated with the activation of signaling (56). This feedback results in AKT activation, and the PI3K/AKT pathway usually suppressed by MAPK our data suggest that the novel combination of BRAF and activation (45, 46). So far, cotargeting with AKT, mTOR, CK2 inhibition would suppress proliferation in tumors that and MEK can reduce resistance in melanoma cell lines (46, are addicted to AKT-driven survival. Indeed, there is 47). The phosphoproteomic data presented here highlight substantial interest in direct targeting of PI3K and AKT in CK2 activity as a mechanism by which BRAFV600E cells combination with MAPK pathway inhibitors in BRAF- may adapt to vemurafenib treatment. Our data are con- mutant tumors (57, 58). As CK2 regulates a myriad of sistent with a role for CK2 in activating prosurvival pro-growth substrates, it would be of interest to determine mechanisms through pathways including AKT signaling whether controlling CK2 activity is advantageous com- (48), potentiating the ERK nuclear localization (49) and pared with current approaches trialing PI3K/AKT target- regulation of c-MYC transcription (23). ing in combination with MAPK inhibition. Western analysis of several components from the PI3K/ AKT pathway indicated that CK2 regulates AKT phos- Disclosure of Potential Conflicts of Interest phorylation in BRAF-mutated thyroid cancer cells. We No potential conflicts of interest were disclosed. have shown that AKT pSer129, a site directly regulated by CK2 (50), is indeed greatly reduced by treatment with CX- Authors' Contributions 4945 (Fig. 5B). S129 phosphorylation has been shown to Conception and design: R. Parker, R. Clifton-Bligh, M.P. Molloy hyperactivate AKT through a priming step that recruits Development of methodology: R. Parker Acquisition of data (provided animals, acquired and managed patients, HSP90 and inhibits phosphatase activity, increasing occu- provided facilities, etc.): R. Parker, M.P. Molloy pancy at the activation site T308 and most likely S473 (50). Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): R. Parker, R. Clifton-Bligh, M.P. Molloy Consistent with this, we observed reduced phosphoryla- Writing, review, and/or revision of the manuscript: R. Parker, R. Clifton- tion of both the PDK1 and mTORC2 phosphorylation Bligh, M.P. Molloy sites in AKT following CK2 inhibition. No change in Study supervision: M.P. Molloy PDK1 phosphorylation or PTEN phosphorylation was observed, indicating that PI3K was still active and CK2 Acknowledgments did not act upstream of PDK1 as reported previously for The authors thank Chris Schmidt, Cathy Lanagan, Linda O’Connor, Ken Dutton-Regester, and Nick K Hayward at QIMR Berghofer Medical PTEN (51, 52). CK2 inhibition demonstrated reduction of Research Institute for establishing and genotyping the melanoma cell lines. AKT kinase activity evidenced by reduced phosphoryla- They also thank Jimmy Parker for providing scripts facilitating the com- tion of GSK3b Ser9 (Fig. 5B). GSK3b is a multifunctional piling of datasets. protein kinase that is constitutively active until phosphorylated by AKT at position Ser9 (53). Reduced GSK3b Grant Support M.P. Molloy was financially supported by Macquarie University and phosphorylation observed here would enhance phos- the Cancer Institute NSW. Aspects of this research were supported by phorylation of the G1–S phase cyclins D, E, and transcrip- facilities funded by the Australian Government’s National Collaborative tion factors c-JUN (AP-1) and c-MYC, targeting them for Research Infrastructure Scheme. The costs of publication of this article were defrayed in part by the proteolysis and enhancing cell-cycle arrest (53). The rel- payment of page charges. This article must therefore be hereby marked evance of CK2 regulation of AKT activity is highlighted in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate a recent report showing that CX-4945 prevents AKT this fact. activation and promotes survival in mice with intracranial Received November 5, 2013; revised May 8, 2014; accepted May 8, 2014; glioblastoma xenografts (54). published OnlineFirst May 13, 2014.

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1906 Mol Cancer Ther; 13(7) July 2014 Molecular Cancer Therapeutics

Phosphoproteomics of MAPK Inhibition in BRAF-Mutated Cells and a Role for the Lethal Synergism of Dual BRAF and CK2 Inhibition

Robert Parker, Roderick Clifton-Bligh and Mark P. Molloy

Mol Cancer Ther 2014;13:1894-1906. Published OnlineFirst May 13, 2014.

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