Targeting Phospho-MARCKS Overcomes Drug-Resistance and Induces Antitumor Activity in Preclinical Models of Multiple Myeloma

ORIGINAL ARTICLE Targeting phospho-MARCKS overcomes drug-resistance and induces antitumor activity in preclinical models of multiple myeloma

Y Yang1,2,7, Y Chen1,2,3, MN Saha1,2, J Chen4, K Evans4, L Qiu5, D Reece6, GA Chen3 and H Chang1,2,3,6

Multiple myeloma (MM) is incurable in virtually all patients due to the presence of innate and emergent drug-resistance. To identify potential drug resistance mechanisms in MM we used iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry to compare protein expression profiles of drug-resistant (RPMI 8226-R5) and sensitive (RPMI 8226-S) isogenic cell lines. We identified selective overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in drug-resistant R5 cells. MARCKS overexpression was also observed in several drug-resistant human myeloma cell lines (HMCLs) and in drug-resistant primary MM samples. Functionally, inhibition of MARCKS phosphorylation by enzastaurin or knockdown of the gene by RNAi significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and to other anti-myeloma drugs, providing evidence that MARCKS can modulate drug response. Mechanistically, pMARCKS (phosphorylated form of MARCKS) was found to function as an E2F-1 cofactor to regulate SKP2 transcription. pMARCKS promoted cell-cycle progression by facilitating SKP2 expression, suppressing p27Kip1 and potentially counteracting drug-induced cell-cycle arrest by promoting Cyclin E/CDK2 activity. Importantly, MARCKS knockdown in combination with bortezomib treatment overcame bortezomib resistance, significantly inhibited tumor growth and prolonged host survival in a MM xenograft model. These data provide a rationale for therapeutic targeting of pMARCKS to improve the outcome of patients with refractory/relapsed MM.

Leukemia (2015) 29, 715–726; doi:10.1038/leu.2014.255

INTRODUCTION and the control of interleukin-6 receptor shedding in MM.19,24 Multiple myeloma (MM) is an incurable plasma cell malignancy Thus, inhibitors of PKCs have been considered as strong molecular characterized by intrinsic and acquired drug-resistance.1,2 The candidates for therapeutic targeting due to their increased 25 19,20 mechanisms underlying this therapeutic resistance are complex. activation in many cancers including MM. However, diverse Activation of NFκB has been postulated to contribute to both signaling effects of PKCs along with their large number of isoforms chemoresistance and disease pathogenesis through canonical and have made the development of drugs targeting these enzymes 26 noncanonical signaling pathways,3–8 whereas several recent studies difﬁcult, whereas inhibition of pMARCKS (phosphorylated form have suggested that targeting insulin-like growth factor-1 receptor, of MARCKS), a downstream mediator of PKC, may be an alternative phospho-mesenchymal epithelial transition factor or NIMA-related and more direct approach in targeted therapy of MM. 9–11 ﬁ kinase 2 may overcome multiple drug resistance in MM. In this study we identi ed overexpression of MARCKS protein in Myristoylated alanine-rich C-kinase substrate (MARCKS) is a drug-resistant MM cells using global proteomic assessment, and protein kinase C (PKC) substrate that has previously been reported demonstrated a novel role of MARCKS/pMARCKS in modulating to play a role in cell adhesion, spreading and mitogenesis.12–14 the SKP2/p27 signaling axis and in promoting cell proliferation. When phosphorylated by PKCs, MARCKS is shuttled from the Our in vitro and in vivo results provide preclinical evidence that plasma membrane to the cytosol and is implicated in modulation targeting MARCKS is an attractive therapeutic strategy to over- of several critical events including cell growth, cell-cycle progres- come drug resistance in MM. sion and apoptosis.15–17 MARCKS has also been found in direct regulation of invasion and metastasis in several types of solid tumors.11,18 Association of MARCKS upstream molecule PKC with MATERIALS AND METHODS drug resistance has been reported in different cancers including Myeloma cell lines and primary MM samples 19,20 21–23 MM. MM samples display upregulation of PKCβ and PKC RPMI8226-R5, a multidrug-resistant MM cell line that is cross-resistant to itself has been shown to be involved in anti-apoptosis, vascular bortezomib (BTZ), was kindly provided by Dr R Buzzeo.27 The MM.1R cell endothelial growth factor- and wingless-type-induced migration line, which is resistant to dexamethasone, was obtained from ATCC

1Division of Molecular and Cellular Biology, Toronto General Research Institute, Toronto, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; 3Department of Hematology/Oncology, First Affiliated Hospital, Nanchang University, Nanchang, China; 4Ontario Cancer Biomarker Network, Toronto, Canada; 5Department of Lymphoma & Myeloma, Institute of Hematology & Blood Diseases Hospital, State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences & Peking Union of Medical College, Tianjin, People's Republic of China and 6Department of Laboratory Hematology and Medical Oncology, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada. Correspondence: Professor H Chang, Department of Laboratory Hematology and Medical Oncology, Toronto General Hospital, University Health Network, 200 Elizabeth Street, Toronto, 11E-413, Ontario, Canada M5G 2C4. E-mail: [email protected] 7Current address: Novade Pharmaceutical Co., Ltd., Tianjin 300308, PR China. Received 30 April 2014; revised 8 August 2014; accepted 14 August 2014; accepted article preview online 2 September 2014; advance online publication, 14 October 2014 Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 716 (Manassas, VA, USA). Resistance of 8226-R5 and MM.1R to the proteasome OPM-2BR (Supplementary Figure S2), suggesting that MARCKS inhibitors BTZ and MG132 is shown in Supplementary Figure S1. Three protein levels are correlated with a phenotype of drug resistance, at pairs of BTZ-resistant and sensitive cell lines namely 8226-BR, 8226-S; least in above HMCLs. Notably, the MARCKS protein that was OPM-2BR, OPM-2S; and ANBL-6BR, ANBL-6S were kindly provided by Dr 9 expressed in 8226-R5 and MM.1R cells appeared to be phosphory- Orlowski. These cell lines developed by exposing parental cells to serially lated (Figure 1b). increased concentrations of BTZ were cultured as previously described and fi showed no PSMB5 mutations.9 Methods for isolation and culture of To determine if similar ndings could be observed in primary primary MM samples, peripheral blood mononuclear cells and bone MM tumors, we next sought to determine if MARCKS protein was marrow mononuclear cells are described in Supplementary Materials and expressed in primary MM and whether this was correlated with + Methods. This study was approved by the Research Ethics Committee of treatment response. CD138 MM cells were isolated from patients’ University Health Network, Toronto, Canada, in accordance with the bone marrow samples at diagnosis or at relapse and evaluated for Declaration of Helsinki. phosphorylated MARCKS protein by immunoblotting (Figure 1d). Conspicuously, MM patient samples collected at relapse (n = 4), Isobaric tags for relative and absolute quantitation and selected following prior treatment with BTZ, showed significantly higher reaction monitoring/mass spectrometry proteomic analysis pMARCKS expression than MM samples collected at diagnosis Quantitative proteomic analyses of 8226-R5, grown in the presence of (n = 5; Figure 1d), consistent with our initial observations 10 nM BTZ, and of wild type (Wt) 8226 were performed in duplicate using in HMCLs. an iTRAQ (isobaric tags for relative and absolute quantitation) mass spectrometry (MS)-based platform. Briefly, extracted proteins from cell Inhibition of phosphorylation of MARCKS suppresses the growth lysates were digested with trypsin and labeled with iTRAQ tags according of drug-resistant MM cells to the manufacturer's instruction (AB Sciex, Framingham, MA, USA), and then fractionated by off-line strong cation exchange using a Thermo As MARCKS is a downstream target of PKC and reportedly Biobasic strong cation exchange column (AB Sciex). Each collected fraction promotes cell migration, invasion and metastasis in several types was analyzed on a nanoflow liquid chromatography–MS/MS platform of solid tumors,24,28 we next sought to determine whether (QStar Elite, AB Sciex). The data were analyzed using Protein Pilot software inhibition of MARCKS influenced the viability of drug-resistant (AB Sciex). A 1.5-fold difference in expression in both platforms was used MM cells. As no direct inhibitors of MARCKS are known, we used as an initial threshold in screening for differentially expressed proteins. enzastaurin (LY317615.HCl, Enza, Selleck Chemicals, Houston, TX, Targets of interest were selected for SRM (selected reaction monitoring) fi USA), an adenosine triphosphate-competitive inhibitor of PKC, to con rmation. Transitions were generated using MRMPilot (AB Sciex). block the phospho-activation of MARCKS by PKC. Protein extracts from cell lines were digested using trypsin for SRM analysis fi on a nanoflow HPLC–MS/MS (4000 QTrap, AB Sciex) platform. One We rst examined whether Enza had direct inhibitory effects on microgram of protein equivalent was injected. MM cells. Although the viability of both parental 8226-S and drug- resistant 8226-R5 cells was reduced by Enza (Figure 2a), drug- resistant 8226-R5 cells conspicuously showed a flatter dose Cell culture, immunoblotting, cell viability, apoptosis assays, fl response and 10-fold higher IC50 than parental 8226 cells, immuno uorescence microscopy, immunoprecipitation, reflecting their higher basal levels of pMARCKS. Notably, siRNA- chromatin immunoprecipitation, and immunohistochemistry mediated silencing of MARCKS produced effects that paralleled assays those produced by Enza, including decreased 8226-R5 colony These assays are detailed in Supplementary Methods. formation and migration (Figures 2b and c), suggesting that effects of Enza on MM cells might be largely mediated by loss of MARCKS gene silencing, gene qPCR arrays, RT–PCR and animal pMARCKS activity. xenograft model studies As MARCKS is of interest because of its higher expression in These methods are detailed in the Supplementary Methods. drug-resistant MM cells, we next sought to determine whether Enza-induced suppression of MARCKS phosphorylation might Statistical analysis re-sensitize resistant MM cells to BTZ treatment. Both 8226-R5 and fi MM.1R cells were examined. Drug combination studies revealed Statistical signi cance levels were determined by two-tailed t-test or one- μ way analysis of variance. Survival data were analyzed using log-rank test that combination of low doses of BTZ (5 nM) and Enza (2.5 M) and the data were plotted using the Kaplan–Meier method. P-values of displayed synergistic cytotoxic response in 8226-R5 (Figure 2d and o0.05 were considered significant. Supplementary Figure S3). Like Enza, inhibition of MARCKS by siMARCKS also impaired 8226-R5 viability and further potentiated the cytotoxic response of BTZ in this cell line (Figure 2e). RESULTS We next examined the effect of MARCKS knockdown on the Identification of MARCKS overexpression in drug-resistant activity of other anti-MM agents including dexamethasone (Dex), myeloma cells doxorubicin (Dox) and lenalidomide (Len). Silencing of MARCKS by fi To identify possible mechanisms of drug resistance in MM, we siRNA signi cantly enhanced the cytotoxic response of these examined drug-sensitive (8226-S) and drug-resistant (8226-R5) drugs in 8226-R5 and MM.1R cells (Figures 2f and g). These results subclones of RPMI8226 cell line using iTRAQ MS for differentially indicate that inhibition of MARCKS activity can produce MM expressed proteins that correlated with the resistance phenotype. cytotoxicity that circumvents resistance mechanisms to BTZ, Len, Independent MS-based SRM studies were then used to verify and Dex and Dox. Supporting the results obtained in cell lines, cytotoxic response further quantitate differences in protein expression levels. From + initial screening a number of proteins appeared to show differential of Enza was observed in CD138 plasma cells isolated from MM patients in relapse and previously exposed to BTZ. Importantly, expression in these isogenic cell lines (Supplementary Table 1). Enza potentiated the cytotoxic response of BTZ in these samples, Perhaps most strikingly, the PKC substrate, MARCKS, showed a 24.9- confirming that Enza can sensitize MM cells to BTZ treatment fold average increase in expression in 8226-R5 cells compared with (Figure 2h). 8226-S cells, and this markedly differential expression was readily verified by SRM (Figure 1a) and could be detected by both immunobloting and immunostaining (Figures 1b and c). Impor- Inhibition of pMARCKS triggers cell-cycle arrest and apoptosis tantly, relative overexpression of MARCKS was observed in drug- Having demonstrated the role of pMARCKS as a key factor resistant cells, MM.1R and 8226-R5 (Figure 1b), and was also found mediating survival of drug-resistant MM cells, we next sought to in three other BTZ-resistant cell lines, 8226-BR, ANBL-6BR and determine whether the decreased phosphorylation of MARCKS

Figure 1. Detection of MARCKS overexpression in drug-resistant HMCLs and patient samples. (a) Validation and quantification of MARCKS protein expression in crude cytoplasmic extract prepared from wild type and resistant 8226 cells by SRM analysis. Extract ion chromatogram of MARCKS native peptide was quantified by the peak areas for each of the SRM/MS signature ions. (b) MARCKS expression was determined by western blotting in 8226 and MM.1 cells. MM cells resistant or sensitive to BTZ were exposed to 5.0 nM BTZ, respectively (S, sensitive; R, resistant). (c) Immunostaining analysis of MARCKS in 8226 wt and R5 cells, the cells' nuclei are stained blue, but MARCKS stained brown on the cell surface. (d) CD138+ cells obtained from primary diagnosis and relapsed MM patients were freshly isolated, and immunoblotting analysis was performed with indicated antibodies. Quantitative analysis of pMARCKS expression normalized to glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) is shown by bar graph. Statistical analysis was performed by Wilcoxon matched pairs test. was involved in the regulation of cell-cycle arrest and apoptosis. compared to the control (Figure 3c). The degree of apoptosis upon First, 8226-R5 and MM.1R cells were treated with 10 nM BTZ, 5 μM MARCKS knockdown alone, BTZ treatment alone or their Enza or combination with low doses of BTZ (5 nM) and Enza combination was determined through annexin-V staining FACS (2.5 μM) for 48 h. Cell-cycle analysis of 8226-R5 cells revealed an analysis (Figure 3d). The results for the quantitative analysis of accumulation of cells, treated with the indicated drugs, in G1/S apoptosis data are depicted in Figures 3e and f. Similar results phase with a relative paucity of cells traversing through G2/M were observed in MM.1R cells (Supplementary Figure S4). phases (Figure 3a). We further examined whether genetic knock- Induction of apoptosis upon inhibition of pMARCKS by either down of MARCKS by siRNA affected the cell-cycle progression of Enza or siMARCKS was further confirmed by western drug-resistant MM cells. 8226-R5 cells were transiently transfected blotting, which showed cleavage of caspase-8, caspase-3, as well with either siMARCKS or scrambled siRNA, and the transfected as poly(ADP-ribose) polymerase (PARP) but not caspase-9 cells were exposed to BTZ or vehicle for 48 h. Fluorescence- (Figures 3g and h). To identify if apoptosis of BTZ-resistant MM activated cell sorting (FACS) analysis showed that silencing of cells, mediated by inhibition of pMARCKS, was caspase-depen- MARCKS resulted in a prolongation of G1/S of 8226-R5 cells, which dent, 8226-R5 cells were pre-incubated with 2.0 μM pan-caspase was further enhanced by combination of BTZ and siMARCKS inhibitor, Z-VAD-FMK. The cells were subsequently treated with (Figure 3b). Induction of cell-cycle arrest upon reduction of 5.0 μM Enza or transiently transfected with siMARCKS. FACS pMARCKS was associated with apoptosis of the cells as indicated analysis demonstrated that Z-VAD-FMK reduced annexin-V- by an increase in the percentage of annexin-V positive cells positive cells by 54.3 and 63.5% in the presence of either Enza

© 2015 Macmillan Publishers Limited Leukemia (2015) 715 – 726 Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 718 abc 8226 R5 8226 R5 *** *** 120 120 8226 S IC50=0.54 M 125 *** 8226 R5 IC50=3.43 M Resistance Degree=6.43 100 *** 80 80 75

50 40 40 Cell viability

relative to DMSO 25 Number of colonies

0 0 Relative ratio of migration 0 0.0 0.01 0.1 1 10 100 M Enza Enzastaurin Enza Control Control Control Control siMARCKS desiMARCKS 8226 R5 8226 R5 *** 120 *** 120 * ** *** 90 ** 90 * 60 60

30 30 Cell viability Cell viability relative to control relative to control 0 0 – +–+ BTZ (5 nM) – + – + BTZ (5 nM) ––+ + Enza (2.5 M) – – + + siMARCKS

P#1 Relapse fgh100 P#2 Relapse 8226 R5 P#3 Relapse * MM. 1R P#4 Relapse 100 ** 100 * 75 * ** ** *** 75 ** 75 50 ** * 50 50 ** *

Cell viability 25

25 25 relative to DMSO Cell viability (%) Cell viability (%) 0 0 0 –+ –+ –+ –+siMARCKS –+ –+ –+ –+siMARCKS Enza (0.0 M) Enza (2.5 M) Enza (0.0 M) Enza (2.5 M) –– ++ –– ––Dex (5 M) –– ++ –– ––Dex (5 M) BTZ (0.0 nM) BTZ (5.0 nM) –– –– ++ ––Dox (5 M) –– –– ++ ––Dox (5 M) –– –– –– ++Len (10 nM) –– –– –– ++Len (10 nM) Figure 2. Effect of inhibition of pMARCKS by pharmacological inhibitor or knockdown of MARCKS on viability, colony formation and migration in drug-resistant MM cells. (a) 8226-S and 8226-R5 cells were exposed to Enza with indicated concentrations for 48 h, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (b)2×103/well of 8226-R5 cells were seeded in a 12-well plate with methylcellulose RPMI 1640 medium containing 5 μM Enza to form the cell layer; after 8 days colonies were stained with MTT and counted. Control cells were treated with DMSO. For knockdown of MARCKS, 2 × 103/well of 8226-R5 cells transiently transfected with either siMARCKS or scrambled siRNA for 72 h were used in forming the cell layer; 8 days later colonies were stained with MTT and counted. (c) 8226-R5 cells were treated as described in colony formation assay (b), and analyzed for effects on serum-induced migration ability. The migration assay was performed using a 24-well transwell plate. The migrated cells were quantiﬁed by measuring the number of cells in the bottom chamber. The ratio of migrated cells was relative to seeded cells. (d) Combination of BTZ with Enza enhanced suppression of BTZ- resistant MM cell growth. 8226-R5 cells were treated with 5 nM BTZ, 2.5 μM Enza or combination of both for 48 h, and then MTT assay was carried out for measurement of cell viability. (e) Knockdown of MARCKS potentiated the inhibition of cell growth induced by BTZ in BTZ- resistant cells. 8226-R5 cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit (Qiagen) for 48 h, and then the transfected cells were treated with 5.0 nM BTZ or drug vehicle for 24 h. The cell viability was measured by the MTT assay. (f and g) MARCKS silencing potentiated the inhibitory effect of current anti-myeloma agents on the viability of drug-resistant MM cells. 8226-R5 (f) and MM.1 R (g) cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit for 48 h, and then transfected cells were exposed to dexamethasone (Dex, 5 μM), doxorubicin (Dox, 5 μM), lenalidomide (Len 10 nM), or vehicle (DMSO or phosphate buffered saline) for 24 h. The cell viability was measured by the above method. (h) Plasma cells from four relapsed MM patients were treated with indicated concentrations of Enza without (left) or with (right) simultaneous addition of 10 nM BTZ for 24 h, and then cell viability was evaluated as above. All results are presented as means ± s.d. for at least three separate experiments. *Po0.05, **Po0.01, ***Po0.001. DMSO, dimethyl sulfoxide.

or siMARCKS, respectively (Figures 3i and j). Western blotting apoptosis (PAHS-012Z) genes. The qPCR results were analyzed results showing inhibition of caspase-3 and PARP upon Enza with Partek Genomics Suite (Partek Incorporated, St Louis, MO, or siMARCKS treatment in the presence of Z-VAD-FMK USA). A total of 22 genes were upregulated and 16 genes were further confirmed the caspase-dependent apoptotic mechanism downregulated. Interestingly, two-dimensional hierarchical cluster mediated by pMARCKS (Figures 3k and l). Similar results were analysis indicated that 8226-R5 and MM.1R cells transiently observed in MM.1R cells (Supplementary Figure S5). transfected with siMARCKS were clustered to the adjacency of each other with the most similar patterns of expression, and that pMARCKS mediates cell-cycle arrest and apoptosis through both cell lines treated with scrambled siRNA were clustered induction of SKP2/p27 signaling pathway via E2F-1 together (Figure 4a). To further validate qPCR array data for cell- To gain a comprehensive understanding of the effect of MARCKS cycle and apoptosis genes, we examined the expression of inhibition on the cell-cycle regulatory mechanisms in drug- selected genes using qRT–PCR in both 8226-R5 and MM.1R cells resistant MM cells, we assessed gene expression profile with treated as described above, and confirmed fold changes in the customized qPCR array by using Qiagen (Valencia, CA, USA) expression of nine upregulated (for example, PARP, p53 upregu- RT2-Profiler PCR Array for human cell cycle (PAHS-020Z) and lated modulator of apoptosis (PUMA), and CDKN1B/p27) and seven

Leukemia (2015) 715 – 726 © 2015 Macmillan Publishers Limited Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 719 downregulated (for example, SKP2, E2F-1 and CDK2) genes (Figures 4c and d). Time course studies showed a signiﬁcant (Figure 4b). We hypothesized that SKP2/p27 axis was involved in difference in the expression of the indicated proteins observed at the regulation of cell-cycle progression conferring drug-resistance 16 h after treatment with 5 μM Enza. These protein expression to MM cells. To test this hypothesis we explored changes in the patterns were consistent with their mRNA expression proﬁling in expression of SKP2, p27, p21 and other relevant proteins by 8226-R5 cells exposed to Enza. Furthermore, we treated 8226-R5 western blotting in response to pMARKCS inhibition. Enza cells with BTZ (10 nM), Enza (5.0 μM) or the combination downregulated expression of SKP2 as well as pPKCβ and of half doses of each drug. The immunoblotting experiments pMARCKS in a dose- and time-dependent manner. Simulta- showed that combination of low doses of BTZ (5.0 nM) and neously, the expression of p27 and p21 proteins was increased Enza (2.5 μM) resulted in reduction in expression of SKP2,

Figure 3. Inhibition of pMARCKS by pharmacological inhibitor or gene silencing induces cell-cycle arrest and caspase-dependent apoptosis in drug-resistant MM cells. (a) 8226-R5 cells were treated with 10 nM BTZ, 5.0 μM Enza, the combination of 2.5 μM Enza and 5 nM BTZ, or vehicle for 24 h. (b) 8226-R5 cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit for 48 h, and then treated with 5.0 nM BTZ or vehicle for 24 h. The BTZ dose in unsilenced cells was 10 nM. Then the cells were stained with propidium iodide and analyzed by flow cytometry to determine the cell-cycle distribution (a and b). Apoptosis was assessed by flow cytometry with annexin-V/ propidium iodide staining for 8226-R5 cells treated similarly as described in a and b. However, the cells were treated for 48 h with drugs (c and d). (e and f) Quantitative representation of apoptosis in 8226-R5 cells treated as described in c and d, respectively. The results shown are the mean ± s.d. from triplicate experiments; *Po0.05, **Po0.01, ***Po0.001. (g and h) Inhibition of pMARCKS by Enza (g) or siMARCKS (h) induced apoptosis of 8226-R5 cells. The presence of full-length and cleaved caspase-8, caspase-3 and PARP were detected by western blotting with indicated antibodies. (i–l) Inhibition of pMARCKS by Enza (g) or siMARCKS (h) induced caspase-dependent apoptosis of 8226-R5 cells. 8226-R5 cells were exposed to indicated concentration of Enza (i) or transiently transfected with either siMARCKS or scrambled RNA (j), and then treated with 2.0 μM Z-VAD-FMK for 24 h. Cells were then stained with annexin-V/propidium iodide before analysis by flow cytometry. Data presented are representatives of three independent experiments. Statistical analysis of the data was performed with one-way analysis of variance. *Po0.05, **Po0.01, ***Po0.001. (k and l) Cells were processed as described in I and J. Immunoblots of whole-cell lyses were performed with indicated antibodies.

8226 R5 8226 R5 *** 80 *** 60 * ** *** *** *** 60 * *** 40 40

20 20 Apopotosis cells (%) Apopotosis cells (%) 0 0 - + -+BTZ - + -+BTZ - - ++Enza - - ++siMARCKS

8226 R5 8226 R5 *** *** *** 60 * 45 *** *

36 40 27

18 20 9 Apopotosis cells (%) 0 Apopotosis cells (%) 0 _ _ _ _ + + Z-VAD-FMK (2 μM) + + Z-VAD-FMK (2 μM) _ _ _ _ + + Enza (5.0 μM) + + siMARCKS

Figure 3. (Continued).

Leukemia (2015) 715 – 726 © 2015 Macmillan Publishers Limited Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 721 phospho-cyclin E and -CDK2, and increase in expression of p27KIP1 MARCKS alone reduced the expression of SKP2 and cyclin E, (Figure 4e). To determine whether knockdown of MARCKS gene and elevated the expression of p27KIP1 and p21 (Figures 4f and g). affects the expression of SKP2 and SKP2-related proteins, 8226-R5 Combination of MARCKS silencing and BTZ signiﬁcantly poten- and MM.1R cells were transiently transfected with siMARCKS, and tiated the reduction of SKP2 expression and enhancement of the cells were treated with 5.0 nM BTZ. We found that silencing of expression of p27 and p21 proteins compared to BTZ or siMARCKS

1R_siMARCKS R5_siMARCKS 1R_siScramble R5_siScramble

) 15.0 Ct ΔΔ

7.5 5.0

2.5

1.0 Fold change of genes expression

normalized to scramble shRNA(2 0.0

MA P73 IGF RP E2F1 MYC ND1 TNF SKP2CDK2 spas3PU PA CCNE1FOXN3 CC MARCKS CDKN1BCDKN1A Ca

Figure 4. For ﬁgure caption see next page.

** ** *** * 3 *** *** 4 * *** *** *** 3 2

1 1

0 0 Fold change of SKP2 promotor of change Fold Fold change of SKP2 promotor of change Fold

normalized to control ChIP DNA Control TPA siMARCKS Enza Anti-pMARCKS Control TPA siMARCKS Enza Anti-E2F1 to normalized control DNA ChIP Figure 4. Inhibition of pMARCKS induced downregualtion of SKP2 mediated by E2F-1, leading to cell-cycle arrest and apoptosis in drug- resistant MM cells. (a) 8226-R5 cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit for 48 h. Total RNAs were isolated for gene expression profiling with qPCR arrays. The data were analyzed with Partek Genomics Suite (Partek GS) with minimum-fold changes of 41.5 for a gene between control and treated samples. Green represents downregulated and red represents upregulated genes. (b) Validation of gene expression by qRT–PCR. 8226-R5 cells were treated as described in a, total RNA was isolated and applied to qRT–PCR using designed primers. Fold change was expressed as log2-fold induction over scrambled siRNA-treated cells (mean ± s.d.). (c and d) Dose-and time-dependent effect of Enza. on the expression of signaling proteins involved in drug-resistance in MM cells. 8226-R5 cells were exposed to different doses of Enza. (c) for different times (d) at indicated concentration of Enza. Immunoblot analysis of the lysates was performed with indicated antibodies. (e–g) 8226-R5 cells were exposed to either a combination of 2.5 μM Enza and 5.0 nM BTZ or to single agents (5.0 μM Enza or 10 nM BTZ) for 48 h (e). 8226-R5 and MM.1 R cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit for 48 h, and then treated with 5 nM BTZ or vehicle for 24 h (f and g). Western blot analysis was used to examine the expression of MARCKS and cell-cycle-associated proteins. (h) Subcellular localization of the indicated MARCKS and SKP2 proteins. 8226-R5 cells were either treated with 5.0 μM Enza or transiently transfected with siMARCKS for 48 h, and then localization of the indicated proteins was performed by confocal microscopy. Control treatment was performed by scrambled siRNA or vehicle. MARCKS was visualized with Alexa Fluor 488 dye staining (Molecular Probes, Grand Island, NY, USA), SKP2 by Alexa Fluor 568 dye staining and nuclei by 4′6-diamidino-2-phenylindole (DAPI) staining. (i) Whole-cell lysates of 8226-R5 cells were immunoprecipitated with anti-pMARCKS or E2F-1 antibodies, and then immunoblotted with the indicated antibodies. GAPDH was used as a loading control. (j) ChIP analysis of the SKP2 promoter in 8226-R5 cells. Chromatin DNA was immunoprecipitated with indicated antibodies. A DNA fragment corresponding to the SKP2 promoter region was amplified by conventional PCR. GAPDH in the supernatant was used as loading control. (k and l) ChIP analysis of the SKP2 promoter in 8226-R5 cells after treatment with either 200 nM TPA or 5.0 μM Enza or siMARCKS; chromatin DNA was immunoprecipitated with anti-E2F-1 (k) and anti- pMARCKS (l) in equal amounts of whole-cell lysates. A DNA fragment corresponding to the SKP2 promoter region was amplified by quantitative PCR. Data represent mean ± s.d. of three independent experiments. *Po0.05, **Po0.01, ***Po0.001.

treatment alone (Figures 4f and g). As expected, immunostaining formed a cofactor complex in 8226-R5 cells (Figure 4i). In addition, assay (Figure 4h) supported the western blot analysis results. MARCKS was also detected in the IP product by anti-pMARCKS These findings indicated that pMARCKS as an essential factor and anti-E2F-1 antibodies. To further verify the role of pMARCKS / targeted SKP2/p27 axis and promoted cyclin E/CDK2 activity, thus E2F-1 complex in regulating the SKP2 promoter, ChIP (chromatin mediating cell-cycle progression and cell proliferation in the drug- immunoprecipitation) genomic DNA was pulled down by anti- resistant MM cells. pMARCKS and anti-E2F-1, respectively, and the precipitated DNA We next sought to answer the question whether pMARCKS fragments were detected by PCR amplification with specific directly upregulated SKP2 expression. For this, we investigated primers of SKP2 promoter regions. As shown in Figure 4j, promoter activity of the 5′ flanking region of human Skp2 by a amplified fragments of SKP2 promoter were present in both ChIP promoter-luciferase reporter assay in HeLa and 293T cells. The DNA extractions immunoprecipitated by anti-pMARCKS and -E2F-1 cells were transiently cotransfected with the luciferase reporter (Figure 4j). Furthermore, the real-time qPCR analysis was carried plasmid containing Skp2 promoter and pReceiver-M02-MARCKS out for ChIP DNA isolated from 8226-R5 cells that were exposed to fi expression plasmid. There were no signi cant changes of Enza or transiently transfected with siMARCKS. There was an luciferase activity compared to control plasmid (data not shown). apparent decrease of SKP2 promoter DNA in 8226-R5 cells treated This finding suggests that pMARCKS does not directly activate with 5.0 μM Enza or siMARCKS compared to the control (Figures 4k transcription of the SKP2 gene in drug-resistant MM cells. Since and l). E2F-1 has been well-known to be required for activation of SKP2 promoter at the response element (hSRE2),29,30 we hypothesized that pMARCKS may act as a cofactor for E2F-1 and thereby TPA reversed the effects induced by downregulation of Marcks promote SKP2 expression. To test this we performed immunopre- 12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, cipitation (IP) assays to determine whether pMARCKS or MARCKS has been shown to be involved in the regulation of cell growth, binds E2F-1 protein. The results showed that pMARCKS and E2F-1 differentiation, apoptosis, cytokine secretion, migration and

Leukemia (2015) 715 – 726 © 2015 Macmillan Publishers Limited Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 723 membrane permeability during tumorigenesis by homologous proteins such as caspase-8 and PARP (Figures 5a and b). Moreover, PKC activation loop phosphorylation, as well as downstream FACS analysis showed that TPA-induced PKC/MARCKS activation phosphorylation of MARCKS in cancer cells including MM cells.19,20 reduced, by nearly 50%, annexin-V staining induced by MARCKS To verify that pMARCKS signaling was associated with SKP2/p27 silencing in 8226-R5 or MM.1R cells (Figures 5c and d). In addition, axis involvement in cell-cycle arrest and apoptosis in resistant MM treatment of R5 cells with 200 nM TPA signiﬁcantly yielded SKP2 cells, we examined whether abrogation of pMARCKS could be promoter DNA fragments using either anti-E2F-1 or anti-pMARCKS reversed by TPA-triggered activation. 8226-R5 and MM.1R cells (Figures 4k and l). These ﬁndings indicate that SKP2 promoter can were transiently transfected with siMARCKS or scrambled siRNA be activated by pMARCKS and E2F-1 upon TPA stimulation. for 48 h, and then these cells were treated with 200 nM TPA for 12 h. Immunoblotting analysis showed that TPA not only induced Targeting MARCKS retards tumor growth and prolongs survival in phosphorylation of downstream PKCβ/MARCKS, but also reversed drug-resistant MM xenograft model inhibition of pMARKCS by MARCKS silencing, leading to an To assess in vivo activity of MARCKS inhibition, we established a increase in expression of SKP2, cyclin E and CDK2, a reduction in mouse xenograft model with drug-resistant MM cells to evaluate expression of p27, and also the upregulagation of apoptosis marker the effect of abrogation of MARCKS by MARCKS knockdown alone

8226 R5 MM 1R * 40 * *** * *** 32 *** *** 30 24 * 20 16

10 8 Apopotosis cells (%) Apopotosis cells (%) 0 0 - + -+siMARCKS -+-+siMARCKS - - ++TPA (200 nM) --++TPA (200 nM) Figure 5. TPA-triggered phosphorylation of MARCKS reversed cell-cycle arrest and apoptosis in resistant MM cells. (a and b) 8226-R5 (a) and MM.1R (b) cells were transiently transfected with either siMARCKS or scrambled siRNA using the HiPerFect kit for 48 h, and cells were harvested after stimulating with 200 nM TPA for 12 h. Western blot analysis was performed to analyze the expression of pPKC and pMARCKS, as well as cell-cycle and apoptosis regulatory proteins including SKP2/p27. (c and d) Drug-resistant MM cells were treated as described in a and b, and annexin-V/propidium iodide staining was used to evaluate TPA-induced antiapoptotic activity. Data presented are representatives of three independent experiments. Statistical analysis was done with one-way analysis of variance. *Po0.05, **Po0.01, ***Po0.001.

Control 1800 40 BTZ )

3 shMarcks 1350 BTZ/shMar 35

900 30

Weight (g) Weight Control 450 25 BTZ shMarcks

Tumor volums (mm volums Tumor BTZ/shMar 20 21 24 27 30 33 36 39 42 21 24 27 30 33 36 49 42 45 49 Days Days

100 p=0.004 75

Control 25 BTZ(p=0.4219)

Percent survival(%) Percent shMarcks(p=0.0473) BTZ/shMar(p=.0058) 0 0 20 40 60 Days

Figure 6. Inhibition of pMARCKS retards tumor growth and prolongs survival in human plasmacytoma mouse xenograft models. 5 × 106 of GIPZ-shMARCKS-transducted or scrambled vector-transducted 8226-R5 cells were subcutaneously injected into (Severe combined immunodeficient) mice (5 mice per group). Intratumoral injection with GIPZ-scramble or GIPZ-shMARCKS was carried out at an interval of 2 days, and simultaneously administration with or without 0.5 mg/kg BTZ was also performed. Tumor volume was measured with caliper, and then calculated using the following formula: V = 0.5a x b2, where ‘a’ and ‘b’ is the long and short diameters of the tumor, respectively. Survival was evaluated from the first day of tumor injection until death. (a) Knockdown of MARCKS enhanced bortezomib-induced retardation of tumor growth in vivo. Representative image of mice with subcutaneous tumors is shown with the graph. (b) Body weight was measured every third day till day 49 and presented as means ± s.d. (c) Survival was evaluated using Kaplan–Meier curves and log-rank analysis from the first day of tumor cells injection until death or occurrence of an event. (d) Immunoblotting analysis of expression of MARCKS, pMARCKS, SKP2 and E2F-1 proteins in mice tumor sections. (e) Representative microscopic images of tumor sections stained with anti-MARCKS-Alexa Fluor 488 dye and 4′6-diamidino-2-phenylindole (DAPI). (f) Immunohistochemical analysis of tumor sections showed that mice bearing 8226-R5 tumors treated with control, shMARCKS, BTZ or both shMARCKS and BTZ resulted in decrease in the proliferation index (Ki-67) and increase in the apoptotic index (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling), further verifying that targeting pMARCKS induces apoptosis in drug-resistant MM cells in vivo.

Leukemia (2015) 715 – 726 © 2015 Macmillan Publishers Limited Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 725 and in combination with BTZ on tumorigenesis and survival in mice. validation by qPCR and western blot analysis confirmed down- 5×106 Cells from 8226-R5 cell line transduced with GIPZ-lentiviral- regulation of proliferation/antiapoptotic genes (SKP2, CDK2, CCNE1 shMARCKS or control vector were subcutaneously injected into and FOXN3) and upregulation of antiproliferative/proapoptotic the flank of severe combined immunodeficient mice. After the genes (CDKN1B, CDKN1A, CCND1 and PUMA) in 8226-R5 cells appearance of measurable tumors (2–3 mm, day 27), mice were silenced for MARCKS expression. Furthermore, inhibition of treated by intratumoral injection with control, 0.5 mg/kg BTZ, pMARCKS potentiated BTZ-induced upregulation of p27 and p21 25 mM/kg GIPZ-lentiviral-shMARCKS or 0.5 mg/kg BTZ plus 25 mM/ and downregulation of SKP2 suggesting an involvement of SKP2- kg lentiviral-shMARCKS in one-day intervals. Tumor size and mouse p27 axis in regulating MARCKS activity in MM cells. Previous studies body weight were monitored every 3 days. Tumor growth curve have shown that while proteasome inhibitors stabilize p27Kip1, showed that treatment with BTZ or shMARCKS alone had modest agents inhibiting SCFSkp2 (Skp1-Cull-F-box) may represent drugs effects, whereas cotreatment with both agents significantly with more direct targets and with promising enhanced efficacy and suppressed tumor growth (Figure 6a, Po0.05, compared with reduced toxicity in MM.33–35 Incorporation of SKP2 into an SCF BTZ alone, n = 5 per group). Weight loss for all conditions was complex confers the ability to induce ubiquitination of several minimal (for example, o0.64%; Figure 6b). shMARCKS or BTZ alone targets, including p27Kip1 and p21Cip1. Initially we thought that modestly prolonged survival compared to control treatment. Mice regulation of SKP2-p27 might be mediated by direct binding of treated with both BTZ and shMARCAKS had a significantly longer MARCKS to SKP2. However, we did not observe the interaction life span compared to all other three treatments (Figure 6c). (data not shown). We then examined the association with other To assess in vivo MARCKS involvement in the SKP2/p27 transcriptional regulators of SKP2 and found that pMARCKS bound pathway, we investigated targeted protein expression in mice to E2F-1 instead of SKP2 to regulate the SKP2 promoter activity. tumors using immunoblotting and also immunostaining. Consis- Since BTZ and Enza or siMARCKS are synergistic, it is most likely that tent with our in vitro data, the expression of MARCKS, pMARCKS inhibition of E2F-1 would also sensitize myeloma cells to BTZ. and SKP2 proteins was decreased in shMARCKS silencing groups However, other E2F-1 family members such as E2F-2 and E2F-3 compared with control (Figures 6d and e), whereas the expression have similar functions and could compensate for each other.36 of E2F-1 protein did not change. The results of TUNEL assay and Additional experimentation with combination knockdown may proliferation index (Ki-67 staining) of the tumor cells are depicted clarify the effect of specific E2F members in combination with fi in Figure 6f. These ndings indicate that targeting MARCKS MARCKS in MM drug resistance. Moreover, a recent study indicated sensitizes myeloma cells to BTZ treatment and contributes to the that MARCKS regulates proliferation and radiation sensitivity of marked induction of apoptosis of MM cells, as well as suppression human glioma cells through modulation of phosphatidylinositol of MM tumor growth in vivo. 4,5-bisphosphate-dependent signaling pathways such as cell division, PI3K/Akt pathway and cellular senescence.37 Although fi DISCUSSION we did not identify signi cant modulation of any of the important genes associated with PI3/Akt pathway (data not shown), In this study we used MS-based iTRAQ and SRM initially to identify alternative cell signaling pathways regulated by MARCKS cannot fi and con rm the protein(s) associated with MM drug resistance. be excluded and warrant further investigation. fi The proteomic analysis conducted here provides the rst Finally, we evaluated the effect of inhibition of MARCKS in an 8226- fi experimental evidence that MARCKS is signi cantly overexpressed R5 xenografted mouse model. Simultaneous inhibition of MARCKS in human MM cells and is associated with drug resistance. and treatment with BTZ significantly decreased tumor burden and MARCKS and its phosphorylated form (pMARCKS) were highly prolonged survival without showing any toxicity sign via inhibition of expressed in drug-resistant HMCLs compared to their sensitive cell proliferation and induction of apoptosis. In keeping with our counterparts. Importantly, primary MM samples obtained from in vitro observation, the apoptotic effect of reduction of pMARCKS relapsed MM patients showed considerably higher expression of was related to SKP2/p27 activation. From a therapeutic perspective, it pMARCKS compared to the samples obtained from newly is interesting to note that targeting MARCKS, which induces cell-cycle diagnosed patients. This is consistent with the view that small arrest and enhances apoptosis via E2F-1/Skp2/P27 axis in resistant subclones of MM with MARCKS expression (at diagnosis) may have MMcellsisachievablein vivo (model depicted in Figure 7). As the growth advantage and therefore expand to become the existence of several isoforms of PKC has made it a difficult druggable predominant clone at the time of relapsed disease. However, it target in MM, specific blocking MARCKS downstream to PKC would is not clear what causes MARCKS overexpression in drug-resistant MM cells. This is unlikely to be due to protein stabilization as we found that MARCKS transcripts were significantly upregulated in drug-resistant MM cell lines and relapsed primary MM samples (data not shown). Gene amplification may play a role since MARCKS is amplified in several cancer types.31,32 Further studies are required to elucidate the mechanisms by which MARCKS is overexpressed in drug-resistant MM cells. To examine whether attenuationofpMARCKScanaffectdrug response in MM cells, we evaluated MM cell sensitivity to drug treatment by inhibition of MARCKS phosphorylation or knockdown of the gene expression. Reduction of pMARCKS sensitized MM cells to current anti-myeloma drugs such as BTZ, Dex, Dox and Len. In addition, cotreatment of BTZ-resistant MM cells with BTZ and Enza or siMRACKS resulted in a significant inhibition of colony formation and migration. Although previous studies demonstrated anti-myeloma activity of Enza,19 our research further unravels that upregulated MARCKS in drug-resistant MM cells may control drug response, and that inhibition of MARCKS may overcome drug resistance in MM. Figure 7. Schematic model. pMARCKS mediates SKP2/p27 axis via To mechanistically explore the association of pMARCKS with drug E2F-1 transcriptional binding element. Targeting pMARCKS by Enza resistance in MM cells, we performed gene expression profiling by or siMARCKS decreases SKP2 and upregulates p27 activity, leading qPCR array. Our gene expression profiling data and subsequent to cell-cycle arrest and apoptosis.

© 2015 Macmillan Publishers Limited Leukemia (2015) 715 – 726 Targeting MARCKS to overcome drug resistance in myeloma Y Yang et al 726 be a novel and promising therapeutic strategy. Our findings herein motility in activated human hepatic stellate cells. Exp Cell Res 2008; 314: provide a rationale to design new drugs to target MARCKS signaling 1444–1454. in combination with current therapeutic agents to overcome drug 15 Hartwig JH, Thelen M, Rosen A, Janmey PA, Nairn AC, Aderem A. MARCKS is an resistance in MM patients. actin filament crosslinking protein regulated by protein kinase C and calcium- calmodulin. Nature 1992; 356: 618–622. 16 Blackshear PJ. The MARCKS family of cellular protein kinase C substrates. J Biol CONFLICT OF INTEREST Chem 1993; 268: 1501–1504. 17 Aderem A. The MARCKS brothers: a family of protein kinase C substrates. The authors declare no conflict of interest. Cell 1992; 71: 713–716. 18 Estrada-Bernal A, Gatlin JC, Sunpaweravong S, Pfenninger KH. 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