Eif4b Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation by Abl Oncogenes

Published OnlineFirst June 7, 2013; DOI: 10.1158/0008-5472.CAN-12-4277

Cancer Tumor and Stem Cell Biology Research eIF4B Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation by Abl Oncogenes

Jianling Yang1, Jun Wang1, Ke Chen1, Guijie Guo1, Ruijiao Xi1, Paul B. Rothman4, Douglas Whitten5, Lianfeng Zhang2, Shile Huang6, and Ji-Long Chen1,3

Abstract Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl–expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl- transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers. Cancer Res; 73(15); 1– 11. 2013 AACR.

Introduction extensive studies, the precise mechanisms by which Abl onco- Expression of Abl proteins is associated with a variety of proteins cause cancer are not fully understood. hematopoietic malignancies (1, 2). In mice, the v-Abl oncogene Growing evidence suggests that the full transforming activ- – of Abelson murine leukemia virus (A-MuLV) induces pre-B-cell ity of Abl involves the activation of STAT/Pim pathway (2, 5 7). transformation through constitutively activated JAK–STAT For example, previous studies have shown that v-Abl cannot fi signaling (2, 3). In humans, chromosomal translocations result ef ciently transform bone marrow cells (BMC) derived from fi / / in the formation of Bcr-Abl hybrid gene that mediates the Pim-1/Pim-2 double-de cient mice (Pim-1 /Pim-2 ) (6), – pathogenesis of chronic myelogenous leukemia (4). Despite indicating that Pim-1 and Pim-2 are required for v-Abl mediated tumorigenesis. A report from the other group also showed that the signal transmitted through Pim kinases is important for survival of hematopoietic cells transformed by Bcr-Abl (8). Authors' Affiliations: 1CAS Key Laboratory of Pathogenic Microbiology In addition, it has been revealed that Pim-1 expression was and Immunology, Institute of Microbiology, Chinese Academy of Sciences markedly upregulated following Bcr-Abl–dependent activa- (CAS); 2Key Laboratory of Human Disease Comparative Medicine, Ministry of Health, Institute of Laboratory Animal Science, Chinese Academy of tion of STAT5 (9, 10). Together, these observations show that Medical Sciences & Comparative Medical Center, Peking Union Medical Pim pathway contributes to cellular transformation by v-Abl 3 College, Beijing; College of Animal Sciences, Fujian Agriculture and and Bcr-Abl. However, the precise roles of Pim kinases in Abl- Forestry University, Fuzhou, China; 4Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa mediated transformation remain elusive. City, Iowa; 5Research Technology Support Facility, Michigan State Uni- The Pim kinases are a family of 3 ubiquitously expressed 6 versity, East Lansing, Michigan; and Department of Biochemistry and serine/threonine (Ser/Thr) kinases (Pim-1, Pim-2, and Pim-3). Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana Pim-1 was first identified as a common integration site in Moloney murine leukemia virus-induced T-cell lymphoma (11). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Subsequent studies have shown that Pim kinases contribute to both cell proliferation and survival and have thus been impli- Current Address for P.B. Rothman: School of Medicine, The Johns Hopkins University, 733 N. Broadway/Suite 100 Baltimore, Maryland cated in the control of tumor formation (12). For example, Pim- 1 and Pim-2 have been shown to be transcriptionally upregu- Corresponding Author: Ji-Long Chen, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Acad- lated in various hematopoietic malignancies (13). In addition, a emy of Sciences (CAS), Beijing 100101, China. Phone: 86-10-64807300; high frequency of Pim-1 somatic mutation has been found in Fax: 86-10-64807980; E-mail: [email protected] diffuse large B-cell lymphoma cases (14). Recent studies have doi: 10.1158/0008-5472.CAN-12-4277 also revealed that enhanced Pim expression is associated with 2013 American Association for Cancer Research. other tumors including human prostate, pancreatic, colon, and

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gastric cancers (11, 15, 16). These findings suggest that upre- described previously (6, 34). These cell lines were characterized þ þ gulation of Pim kinases or Pim mutation may provide a in our laboratory as CD10 /CD19 pre-B cells. The 32D selective advantage during cellular transformation. On the myeloid cells stably expressing wild-type (WT) Bcr-Abl or other hand, an increasing number of substrates have been Bcr-Abl-T315I were kind gifts from Dr. Guangbiao Zhou (Insti- identified that are phosphorylated by Pim kinases. These tute of Zoology, Chinese Academy of Science, Beijing, China). substrates include transcriptional regulators, cell-cycle regu- Pim- or eIF4B-overexpressing Abl transformants (K562, NS2, lators, signaling intermediates, and apoptosis mediators (17– and W44) were generated by stably infecting the cells with 23). Interestingly, eukaryotic translation initiation factor 4B retroviruses or lentiviruses encoding Pim or eIF4B using viral (eIF4B) has recently been identified as a novel Pim-2 substrate vectors pMSCV-GFP or pNL-GFP (Addgene) as previously (17). Pim-2 kinase has also been shown to modulate protein described (35). Short hairpin RNA (shRNA)-expressing stable translation regulator eIF4E-binding protein 1 (24–27). Abl transformants were generated by infection of the cells The control of mRNA translation is preferentially exerted at with lentiviruses expressing specific shRNA in pSIH-H1-GFP the initiation phase, which is crucial for the specific expression vector (System Biosciences) as described previously (36). The of genes important for development, cell growth, proliferation, shRNA sequences are described in Supplementary Materials and survival. eIF4B is part of the protein complex involved in and Methods. the ribosome recruitment to the 50 ends of eukaryotic mRNAs and is thought to stimulate eIF4F activity by potentiating the Antibodies and reagents eIF4A RNA helicase activity in unwinding secondary structures The following antibodies were used in this study: anti-FLAG in the 50 untranslated region (50UTR) of the mRNA (28). (Sigma F1804 M2); anti-Pim-1 (Santa Cruz sc-13513 12H8); anti- Therefore, eIF4B is a key component in the regulation of c-Myc (Santa Cruz sc-40 9E10); anti-Bcr (Santa Cruz sc-885 N- eukaryotic translation initiation. eIF4B is a phosphorylated 20); anti-Bcl-xL (Cell Signaling 2764 54H6); anti-eIF4B (Cell protein, and its phosphorylation is responsive to extracellular Signaling 3592); anti-phospho-eIF4B (Ser406; Cell Signaling stimuli including serum, insulin, and phorbol esters (29, 30). In 5399); anti-phospho-eIF4B (Ser422; Cell Signaling 3591); anti- addition, it has been revealed that eIF4B is regulated by several Bcl-2 (Cell Signaling 2870 50E3); anti-c-Abl (Merck Millipore proto-oncogenic signaling pathways including mitogen-acti- OP19 19–110); anti-p70 S6K, and anti-phospho-p70S6K [Thr389] vated protein kinase, PI3K/mTOR, and Akt (30–32). Moreover, (Cell Signaling 9202 and 9205). All other antibodies were obt- eIF4B has been shown to regulate translation of proliferative ained and used as described previously (7). Pim-1/Pim-2 kinase and prosurvival mRNAs with structured 50UTR and thus inhibitor SMI-4a was purchased from Calbiochem. control cell proliferation and apoptosis (24, 33). For example, recent studies have shown that eIF4B is involved in regulating GST pull-down experiment and in vitro kinase assay the expression of Cdc25C, c-Myc, Bcl-2, and XIAP. These Glutathione S-transferase (GST) fusion proteins were exp- observations suggest that eIF4B may contribute to cellular ressed and purified as previously described (6, 35). Pull-down transformation. assays were conducted by incubating aliquots of purified GST In this study, we investigated the role of Pim kinases leading fusion protein beads with cell lysates at 4C overnight. Beads to eIF4B phosphorylation downstream of Abl signaling. Our were washed and examined by Western blotting. For in vitro data revealed that expression and phosphorylation of eIF4B are kinase assay, purified GST fusion proteins were mixed with Abl kinase dependent in Abl transformants. Furthermore, we each other in the kinase buffer incubated at 30C for 30 showed that Pim kinase–phosphorylated eIF4B, especially on minutes (37). Samples were separated by electrophoresis and Ser422, is required for efficient cellular transformation by Abl probed with the indicated antibodies. oncogenes. These results provide novel insights into complex mechanisms by which Abl oncoproteins cause hematopoietic Immunoprecipitation, immunoblotting, and apoptosis malignancies. In addition, our experiments establish a key role assay for eIF4B phosphorylation and thus, eIF4B may be a potential Preparation of cell extracts, immunoprecipitation, and imm- target for treatment of Abl-induced cancers. unoblotting were conducted as previously described (6, 36). Where indicated, immunoblot signals were quantified by densitometry. For apoptosis assay, cells were treated with Materials and Methods 10 mmol/L imatinib or 50 mmol/L SMI-4a. Cells were then Cell lines and cell culture stained with propidium iodide/Annexin V–FITC and analyzed Cancer cell lines K562, 293T, Jurkat, A549, and HeLa were by fluorescence-activated cell sorter (BD Biosciences). purchased from American Type Culture Collection. Hepato- cellular carcinom cell line SMMC-7721 and breast carcinoma Examination of tumorigenicity using xenograft model in cell line MCF-7 were purchased from National Platform of nude mice Experimental Cell Resources for Sci-Tech (http://cellresource. Nude-mouse injection was carried out as described previously cn). Cells were grown in Dulbecco's Modified Eagle Medium or (35). Tumor growth was monitored and measured in volume RPMI-1640 supplemented with 10% FBS (Gibco) and antibio- (length height width) at the indicated time points after tics (penicillin and streptomycin; Invitrogen) as previously inoculation. Bioluminescent imaging was used to detect tumor described (7). The v-Abl–transformed mouse cell lines named growth from GFP-expressing cells. Images were quantified as as NS2 and W44 in this study were generated and cultured as photons/s using the indigo software (Berthold Technologies).

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Generation of eIF4B-knockdown transgenic mice eIF4B-knockdown transgenic mice were generated by the A kDa CBS 32P WB microinjection method as previously described (38). Western 100 blotting using anti-eIF4B antibody was conducted to deter- 75 eIF4B mine the interference efficiency. The transgenic founders with high interference efficiency were selected and maintained on a C57BL/6J genetic background. 50 Pim-1 Pim-1 Primary murine bone marrow transformation assay 37 BMCs were freshly isolated from 5- to 6-week old mice. v- SOCS-1 SOCS-1 Abl- and Bcr-Abl–mediated bone marrow transformation was 25 conducted as previously described (6, 35). Transformation efficiency was scored by counting the number of the wells containing the transformed cell clones showing cytokine-inde- B GST + –––––– pendent growth 3 weeks after infection. GST-eIF4B –+ ––+ –– GST-eIF4BS406A – – + – – + – DNA construction and mass spectrometry GST-eIF4BS422A –––+ ––+ Myc-Pim-1 + + + + – – – DNA construction and mass spectrometry (MS) analysis are – – – – + + + described in the Supplementary Materials and Methods. Myc-Pim-1-KD Myc-Pim1 (bound) Results Myc-Pim1 (input) eIF4B was found to be a potential target of Pim-1 kinase GST-eIF4B in v-Abl transformants GST Substrates of Pim kinases in Abl transformants are poorly characterized. To identify novel Pim targets in Abl transfor- C mants, we used v-Abl–transformed pre-B-cell lines derived GST + –––––– GST-Pim-1 – + + + – – – from mice (6). The v-Abl transformants were metabolically 32 GST-Pim-1-KD – – – – + + + labeled with H3 PO4. Radiolabeled Pim-1 was then isolated by FLAG-eIF4B + + – – + – – immunoprecipitation, and phosphorylation was examined FLAG-eIF4BS406A ––+ ––+ – by autoradiography. Several phosphorylated proteins were FLAG-eIF4BS422A –––+ ––+ detected in the precipitated protein complex (Fig. 1A). The FLAG-eIF4B (bound) identities of Pim-1 and SOCS-1 were confirmed by Western blotting. As expected, when expressed in cells, Pim-1 became FLAG-eIF4B (input) constitutively autophosphorylated. The observed phosphory- GST-Pim-1 lation of SOCS-1 is consistent with the previous studies (6, 39). GST Interestingly, we observed a highly phosphorylated protein at approximately 80 kDa. Subsequent analysis of tryptic frag- fi ments by MS led to the identi cation of this protein as eIF4B Figure 1. Pim-1 interacts with eIF4B in v-Abl–transformed cells and 293T (Fig. 1A and Supplementary data S1). cells. A, phosphoamino acid(s) in v-Abl–transformed cells was 32 To confirm the interaction between Pim-1 and eIF4B in the metabolically labeled by adding 500 mCi (18.5 MBq) of H3 PO4 for 2 hours. 32P-labeled Pim-1 protein was isolated by immunoprecipitation, v-Abl transformants, we conducted the GST pull-down experi- 32 ments. Association of eIF4B with Pim-1 was observed when and phosphorylation was detected by autoradiography ( P). Pim-1 and SOCS-1 were examined by Western blotting (WB) and Commassie Blue either GST-eIF4B was used to pull down Pim-1 (Fig. 1B) or GST- staining (CBS). eIF4B was identified by MS. B, 293T cells were Pim-1 was used to pull down eIF4B (Fig. 1C). Furthermore, we transfected with Myc-Pim-1 or Myc-Pim-1-KD (Pim-1K67M). Lysates found that eIF4B WT and its Ser406Ala (S406A) and Ser422Ala were incubated with purified GST, GST-eIF4B, GST-eIF4BS406A, or fi (S422A) mutants had similar capacity to bind Pim-1. Strikingly, GST-eIF4BS422A coupling beads. The bound proteins were identi ed by WB using anti-Myc antibody. C, 293T cells were transfected with Flag- however, kinase-dead (KD) mutant of Pim-1 (Pim-1 K67M) lost eIF4B, Flag-eIF4BS406A, or Flag-eIF4BS422A. Pull-down assay using the ability to interact with eIF4B. These results suggest that purified GST, GST-Pim-1, or GST-Pim-1-KD coupling beads was interaction between Pim-1 and eIF4B may require Pim kinase conducted as described in B. activity, or the residue lysine 67 on Pim-1 may be critical for the physical interaction between these proteins. activity of eIF4B (17, 30, 31). Also, previous experiments have revealed the substrate consensus sequence of Pim kinases (R- Pim-1 and Pim-2 can directly phosphorylate eIF4B on X-R-R/H-X-S/T; ref. 17). Using an in silico search, we found that Ser406 and Ser422 Ser406 and Ser422 in the arginine-rich motif region of eIF4B Previous studies have shown that eIF4B is serine phosphor- are potential Pim phosphorylation sites. To test this possibil- ylated on Ser406 and Ser422 by several kinases, and phosphor- ity, 293T cells were cotransfected with eIF4B and Pim kinase, ylation of these residues is essential for optimal translational and eIF4B phosphorylation status was then determined by

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A B pcDNA3.1(-) + –––– pcDNA3.1(-) + –––– pcDNA3.1(-)-Pim-1 – + – + – pcDNA3.1(-)-Pim-2 – + – + – pcDNA3.1(-)-Pim-1-KD ––+ –+ pcDNA3.1(-)-Pim-2-KD ––+ –+ FLAG-eIF4B + + + – – FLAG-eIF4B + + + – – FLAG-eIF4BS422A –––+ + FLAG-eIF4BS422A –––+ + Figure 2. Pim-1 and Pim-2 kinases p-eIF4B-S422 p-eIF4B-S422 directly phosphorylate eIF4B. A, FLAG-eIF4B FLAG-eIF4B 293T cells were cotransfected with Pim-1 or Pim-1-KD and eIF4B or Myc-Pim-1 Myc-Pim-2 eIF4BS422A mutant. Cell lysates were examined by Western blotting C D using indicated antibodies to pcDNA3.1(-) + –––– pcDNA3.1(-) + –––– determine the phosphorylation of pcDNA3.1(-)-Pim-1 – + – + – pcDNA3.1(-)-Pim-2 – + – + – eIF4B Ser422 mediated by Pim-1. pcDNA3.1(-)-Pim-1-KD ––+ –+ pcDNA3.1(-)-Pim-2-KD ––+ –+ B, experiments were carried out to FLAG-eIF4B + + + – – FLAG-eIF4B + + + – – test phosphorylation of eIF4B FLAG-eIF4BS406A –––+ + FLAG-eIF4BS406A –––+ + Ser422 mediated by Pim-2 as p-eIF4B-S406 p-eIF4B-S406 described in A. C and D, FLAG-eIF4B FLAG-eIF4B experiments were carried out as described in A to examine the Myc-Pim-2 Myc-Pim-1 effect of Pim-1 (C) and Pim-2 (D) on the phosphorylation of eIF4B E F Ser406. E–H, in vitro kinase assay: GST + –––– GST + –––– E, puriﬁed recombinant GST-eIF4B GST-Pim-1 – + – + – GST-Pim-2 – + – + – or GST-eIF4BS422A protein was GST-Pim-1-KD ––+ –+ GST-Pim-2-KD ––+ –+ ﬁ + + + – – + + + – – incubated with puri ed GST, GST-eIF4B GST-eIF4B GST-Pim-1, or GST-Pim-1-KD. GST-eIF4BS422A –––+ + GST-eIF4BS422A –––+ + Phosphorylation of eIF4B on p-eIF4B-S422 p-eIF4B-S422 Ser422 was examined by GST-eIF4B GST-eIF4B immunoblotting, and GST-fusion proteins were stained with GST-Pim-1 GST-Pim-2 Commassie Blue. F, experiment GST GST was carried out as described in E to test the effect of Pim-2 on the phosphorylation of eIF4B Ser422. G GST + –––– H GST + –––– G and H, experiments were carried GST-Pim-1 – + – + – GST-Pim-2 – + – + – out as described in E to determine GST-Pim-1-KD ––+ –+ GST-Pim-2-KD ––+ –+ whether Pim-1 (G) or Pim-2 (H) GST-eIF4B + + + – – GST-eIF4B + + + – – directly phosphorylates eIF4B GST-eIF4BS406A –––+ + GST-eIF4BS406A –––+ + Ser406. p-eIF4B-S406 p-eIF4B-S406 GST-eIF4B GST-eIF4B GST-Pim-1 GST-Pim-2 GST GST

Western blotting with the speciﬁc antibodies. We found that eIF4B is highly expressed and phosphorylated in v-Abl expression of Pim-1 or Pim-2 resulted in marked increase in and Bcr-Abl transformants in an Abl kinase-dependent Ser422 phosphorylation and slight increase in Ser406 phos- manner phorylation of eIF4B (Fig. 2A–D). Importantly, expression of It is believed that deregulated translational control plays an Pim-1-KD or Pim-2-KD mutant abolished the phosphoryla- important role in oncogenic transformation (40). To explore tion, indicating that phosphorylation of eIF4B is Pim kinase the contribution of eIF4B in Abl-induced cellular transforma- dependent in the cells (Fig. 2A–D). However, expression of tion, we investigated the eIF4B expression and phosphoryla- Pim-3 had little effect on eIF4B phosphorylation (Supplemen- tion status in Abl transformants as compared with other cell tary Fig. S1). We further conducted in vitro kinase assay by lines and normal controls. Interestingly, we observed that incubating the puriﬁed recombinant GST-Pim kinases with eIF4B was overexpressed in all 3 Abl-transformed cell lines eIF4B protein. Indeed, Pim-1 or Pim-2 was able to phosphor- analyzed (v-Abl–transformed cell lines W44 and NS2, and Bcr- ylate eIF4B at Ser406 and Ser422, and mutation of these sites to Abl–expressing K562 leukemic cell; Fig. 3A and Supplementary Ala abolished the phosphorylation (Fig. 2E–H). In addition, Fig. S2A). Strikingly, eIF4B was also highly phosphorylated in kinase-dead mutation of Pim kinases failed to phosphorylate Abl-transformed cells, especially on Ser422 residue (Fig. 3A). eIF4B at Ser406 and Ser422. These experiments show that To examine whether eIF4B is expressed and phosphorylated Pim-1 and Pim-2 directly phosphorylate eIF4B on Ser406 and downstream of Abl signaling, Abl transformants were treated Ser422. in a time course with imatinib (Fig. 3B–D and Supplementary

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A Human Mouse B

Normal v-Abl–transformed cell 1# 2# A549 Hela 293T K562 W44 NS2 Jurkat BMC MCF-7 SMMC-7721 Imatinib (h) 0 5 10 15 20 p-eIF4B-S422 p-eIF4B-S422 p-eIF4B-S406 p-eIF4B-S406 eIF4B eIF4B Bcr-Abl Actin v-Abl Actin

C D Bcr-Abl-WT Bcr-Abl-T315I

120 eIF4B Imatinib (h) 0 5 10 15 20 0 5 10 15 20 p-eIF4B-S406 100 p-eIF4B-S422 p-eIF4B-S422 80 p-eIF4B-S406 60 eIF4B 40 Bcr-Abl 20 Pim-1 0

Change from baseline (%) Change from baseline Pim-2 0 h 5 h 10 h 15 h 20 h Imatinib treatment (v-Abl) Actin

Figure 3. Expression and phosphorylation of eIF4B exhibit an Abl kinase-dependent manner. A, eIF4B expression and phosphorylation were examined by Western blotting in Abl-transformed cells (K562, W44, NS2), indicated human cell lines, human peripheral blood mononuclear cells (normal) and mouse BMCs. B, v-Abl–transformed cells were treated in a time course with imatinib (10 mmol/L). Cell lysates were analyzed by immunoblotting to examine the eIF4B expression and phosphorylation with indicated antibodies. C, eIF4B expression and phosphorylation levels in B were quantitated by densitometry and normalized to actin expression levels. The levels of eIF4B expression and phosphorylation are 100% at 0 hour. Plotted are results from 3 independent experiments. Error bars represent SEM, n ¼ 3. D, Bcr-Abl–transformed cells (32D-Bcr-Abl and 32D-Bcr-Abl -T315I) were treated in a time course with imatinib (20 mmol/L). Cell lysates were analyzed by immunoblotting with indicated antibodies.

Fig. S2B–S2E). The results showed that the endogenous protein time course with SMI-4a (Fig. 4A and B). Interestingly, treat- levels of eIF4B decreased after treatment with imatinib, sug- ment with SMI-4a led to a more rapid reduction in the gesting that expression of eIF4B is Abl kinase dependent. phosphorylation of eIF4B on Ser422 (Fig. 4A and B and Sup- Similar to the situation seen with eIF4B expression, the phos- plementary Fig. S3A and S3B), similar to the pattern observed in phorylation of eIF4B on Ser406 displayed a similar decrease imatinib treatment (Fig. 3B and C). To determine the respon- after imatinib treatment. However, the eIF4B phosphorylation sible kinases that phosphorylate eIF4B in Abl transformants, we on Ser422 was more sensitive to imatinib treatment as indi- generated shRNA-based Pim-1, Pim-2, or S6K knockdown cells cated by rapidly reduced phosphorylation levels after exposure (Fig. 4C–E and Supplementary Fig. S3C–S3E). Phosphorylation to the compound (Fig. 3C and Supplementary Fig. S2B and of eIF4B on Ser422 was greatly suppressed by silencing the Pim- S2C). To conﬁrm that expression and phosphorylation of eIF4B 1 and clearly decreased after Pim-2 depletion (Fig. 4C and data is Abl kinase dependent, we used Bcr-Abl T315I mutant to test not shown). In contrast, eIF4B is still phosphorylated in Abl the effect of imatinib. As expected, imatinib treatment had transformants upon silencing S6K (Fig. 4D and E). Similar little effect on eIF4B expression and phosphorylation in cells results were obtained from experiments using rapamycin expressing the imatinib-resistant mutant (Fig. 3D). These (Supplementary Fig. S3F and S3G). These data suggest that results reveal that eIF4B is expressed and activated by Abl the ability of Abl oncoproteins to regulate eIF4B phosphory- signaling, and Ser422 is a critical residue in eIF4B that can be lation, especially on Ser422 residue, is mainly mediated by their regulated by this signaling. downstream effectors Pim kinases. To further test this possibility, we generated v-Abl- and Bcr- Pim-1 and Pim-2 regulate eIF4B phosphorylation in Abl–transformed cell lines stably expressing either Pim WT, v-Abl and Bcr-Abl transformants Pim KD mutants, or GFP control using bicistronic retroviruses To determine whether Pim kinases are able to regulate eIF4B (Supplementary Fig. S3H). Although the endogenous Pim phosphorylation in Abl-transformed cells, SMI-4a, a novel expression was inhibited by imatinib treatment, the ectopic small-molecule inhibitor of Pim-1 and Pim-2 kinases (41), was expression of Pim-1 and Pim-2 caused a marked increase in used. We found that eIF4B protein level and phosphorylation eIF4B phosphorylation on Ser422 compared with the GFP on Ser406 slightly decreased in Abl transformants treated in a control (Fig. 4F and G and Supplementary Fig. S3I). As expected,

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Figure 4. Pim-1 and Pim-2 kinases A v-Abl– B Bcr-Abl– regulate eIF4B phosphorylation in transformed cell transformed cell Abl transformants. A, v-Abl– SMI-4a (h) 0 6 12 18 24 SMI-4a (h) 0 6 12 18 24 transformed cells were treated in a p-eIF4B-S422 p-eIF4B-S422 time course with SMI-4a, an inhibitor of Pim-1 and Pim-2 p-eIF4B-S406 p-eIF4B-S406 kinases. Cell lysates were analyzed eIF4B eIF4B by immunoblotting to examine Actin the eIF4B expression and Actin phosphorylation levels as indicated. B, K562 cells were C D E treated with SMI-4a and analyzed by immunoblotting as described in A. C, K562 cells expressing luciferase-specific shRNA or Pim- 1–specific shRNAs were analyzed Pim-1 S6K S6K by immunoblotting with indicated p-eIF4B-S422 p-eIF4B-S422 p-eIF4B-S422 antibodies. D and E, v-Abl– transformed cells (D) or K562 cells p-eIF4B-S406 p-eIF4B-S406 p-eIF4B-S406 (E) expressing luciferase-specific eIF4B eIF4B eIF4B shRNA or S6K-specific shRNAs Actin Actin Actin were analyzed by immunoblotting F G with indicated antibodies. F, v-Abl v-Abl–transformed cell Bcr-Abl–transformed cell transformants ectopically expressing either empty vector EV Pim2-WT Pim2-KD EV Pim2-WT Pim2-KD (EV), Pim-2-WT, or Pim-2-KD were Imatinib (h) 0 5 10 0 5 10 0 5 10 Imatinib (h) 0 12 24 0 12 24 0 12 24 treated with imatinib and analyzed by immunoblotting as indicated. G, p-eIF4B-S422 p-eIF4B-S422 experiment using K562 cells eIF4B eIF4B expressing EV, Pim-2-WT, or Pim- Myc-Pim-2 Myc-Pim-2 2-KD was carried out as described in F. Actin Actin

the effect of Pim kinases was dependent on the catalytic activity undergo apoptosis induced by imatinib, although there is because the cells expressing Pim KD mutants recapitulated that no significant difference in the survival of untreated cells of the control. These results confirm that phosphorylation of expressing control or eIF4B shRNA (Supplementary Fig. S4B eIF4B on Ser422 is regulated by Pim-1 and Pim-2 kinases in Abl and S4C). transformants. Furthermore, we asked whether eIF4B functions downstream of Pim signaling in Abl transformants. To address this eIF4B plays a critical role in regulating survival of Abl issue, we generated Abl transformants stably expressing either transformants and functions downstream of Pim GFP control, eIF4B WT, or its phosphomimetic mutants signaling in these cells (Supplementary Fig. S4D). These cells were treated in a time Because eIF4B was found to be regulated by Abl kinase- course with Pim inhibitor SMI-4a and analyzed for cell survival. dependent oncogenic Pim signaling, and eIF4B is involved in Inhibition of Pim kinases with SMI-4a for 20 hours reduced cell control of prosurvival mRNA translation, we hypothesized viability to approximately 25% in the control v-Abl cells (pNL), that eIF4B might play an important role in regulating survival to approximately 40% in the cells expressing eIF4B WT or of Abl transformants. To examine this possibility, we gener- eIF4B S406E, and 52% to 54% in the cells expressing eIF4B ated stable v-Abl- and Bcr-Abl–transformed cell lines expres- S422E or eIF4B S406E/S422E (Fig. 5E). Similar results were sing eIF4B-specific shRNAs (Fig. 5A and B and Supplementary observed in K562 cells (Fig. 5F). These data indicate that Fig. S4A). v-Abl transformants initiated apoptosis following expression of eIF4B WT and its phosphomimetic mutants imatinib treatment, and approximately 51% of WT cells conferred various resistance to apoptosis induced by the Pim remained viable after incubation with this inhibitor for 15 inhibitor. It seemed that the cells expressing S422E or S406E/ hours under our culture condition. In contrast, only approx- S422E were more resistant to SMI-4a–induced apoptosis than imately 28% eIF4B silencing cells were viable under the same those expressing WT or S406E. Because it has been suggested conditions (Fig. 5C). Similarly, approximately 48% of Bcr-Abl– that Janus-activated kinase (Jak)-2 and Stat5 are key upstream positive K562 cells expressing control shRNA were viable after factors that regulate Pim expression, we further tested the treatment with imatinib for 32 hours. Strikingly, only 27% of effects of silencing these factors on eIF4B phosphorylation. K562 cells expressing eIF4B-specific shRNA exhibited viable Indeed, Pim-1 expression and phosphorylation of eIF4B on following the same treatment (Fig. 5D). These experiments Ser422 were markedly reduced upon depletion of Jak2 and show that silencing eIF4B sensitizes Abl transformants to Stat5 (Supplementary Fig. S4E and S4F). Together, these results

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A E 100 v-Abl–transformed cell 80

60 eIF4B pNL Actin 40 pNL-eIF4B pNL-eIF4BS406E Percentage viability Figure 5. eIF4B plays a critical role 20 B pNL-eIF4BS422E in regulating survival of Abl transformants. A and B, eIF4B Bcr-Abl–transformed cell pNL-eIF4BS406E/S422E expression in v-Abl–transformed 0 cells (A) and K562 cells (B) 0 h 5 h 10 h 15 h 20 h expressing luciferase shRNA (sh- SMI-4a treatment (v-Abl) luciferase) or eIF4B-specific shRNAs (sh-eIF4B) targeting F 100 different regions of eIF4B eIF4B sequences were analyzed by immunoblotting. C and D, survival Actin 80 of v-Abl–transformed cells (C) and K562 cells (D) expressing luciferase shRNA or eIF4B-specific shRNAs C 100 sh-luciferase 60 upon treatment of imatinib was sh-eIF4B pNL analyzed by flow cytometry after 80 40 pNL-eIF4B propidium iodide staining. Plotted are the results from 3 independent 60 pNL-eIF4BS406E Percentage viability experiments. Error bars represent 20 pNL-eIF4BS422E SEM. E and F, cell survival analysis 40 of v-Abl–transformed cells (E) and pNL-eIF4BS406E/S422E K562 cells (F) ectopically 20 0 Percentage viability expressing either empty vector 0 h 12 h 24 h 36 h 48 h 0 (pNL), eIF4B-WT, or its SMI-4a treatment (Bcr-Abl) phosphomimetic mutants treated 0 h 5 h 10 h 15 h with SMI-4a. G, immunoblot Imatinib treatment (v-Abl) analysis of K562 cells expressing sh-luciferase or sh-eIF4B probed D G as indicated. 100 sh-luciferase sh-eIF4B 80

60 eIF4B c-Myc 40 Pim-1 20 Percent viability Bcl-2

0 Bcl-XL 16 h8 h0 h h0 16 h8 24 h 32 h Actin Imatinib treatment (Bcr-Abl)

reveal that Pim kinase-dependent eIF4B phosphorylation is Silencing eIF4B signiﬁcantly inhibits tumor formation important for the viability of Abl-transformed cells, and Ser422 induced by K562 leukemic cells in xenograft mouse in eIF4B is a critical phosphorylation site that is regulated by model Abl-Jak-Stat-Pim signaling. In an attempt to gain a better understanding of the role of In an attempt to provide insights into the mechanism by eIF4B in Abl-mediated tumorigensis, we investigated the effect which eIF4B regulates survival of Abl transformants, we eval- of silencing eIF4B on the tumor formation of Abl-transformed uated the expression of Pim kinase target, c-Myc, and several cells using xenograft model in nude mice. Each mouse was Bcl-2 family members that are the critical regulators of apo- inoculated subcutaneously with K562 cells stably expressing ptosis. Our results indicate that silencing eIF4B did not alter shRNA targeting either eIF4B or luciferase control. Remark- protein expression of Bcl-XL, but obviously reduced protein ably, we found that the tumors formed by cells expressing expression of Bcl-2 and c-Myc (Fig. 5G). luciferase-speciﬁc shRNA grew much faster than those formed

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A B 1,000 sh-luciferase sh-eIF4B

) sh-eIF4B 3 800 sh-luciferase

600 Experiments 1 2 3 4 5

400

200 D Tumor volume Tumor volume (mm

0 5 d 10 d 15 d 20 d C

120 * 100 sh-luciferase sh-eIF4B 80 E 60 sh-luciferase + – sh-luciferase + – 40 sh-eIF4B –+ sh-eIF4B –+ 20 eIF4B eIF4B 0 Actin Actin Change from baseline (%) Change from baseline sh-luciferase sh-eIF4B Experiment 1 Experiment 2

Figure 6. eIF4B deﬁciency attenuates tumor formation caused by K562 cells. A, nude mice were subcutaneously injected with K562 cells stably expressing sh-luciferase or sh-eIF4B. The tumor volumes were measured at indicated time points. Plotted are results from 3 independent experiments. Error bars, SEM; n ¼ 9. B, tumors were excised from mice. Shown are representative images from 5 independent experiments with similar results. C, relative volume of tumors excised from nude mice injected with K562 cells expressing sh-luciferase or sh-eIF4B. The average volume of tumors caused by K562 cells expressing sh-luciferase is 100%. Error bars, SEM; n ¼ 9; , P < 0.05. Differences between variables were tested for signiﬁcance using the Student t test. D, over a 14-day period after inoculation, tumors stably expressing sh-luciferase or sh-eIF4B were measured by bioluminescent imaging. Shown are representative images from at least 3 independent experiments with similar results. E, eIF4B expression in representative tumors expressing sh-luciferase or sh-eIF4B was examined by immunoblotting.

by cells expressing eIF4B-specific shRNA (Fig. 6A). These cells infected with the A-MuLV displayed v-Abl transformation experiments were repeated at least 3 times to ensure the with average results of 14 wells showing cytokine-independent reliability of the results and consistency of data. By statistical growth of cell clones per 96-well plate (Fig. 7B). However, analysis, we found that silencing eIF4B expression in K562 cells silencing eIF4B significantly decreased v-Abl transformation significantly inhibited the tumor growth (Fig. 6B and C). These efficiency. This study shows that disrupting the eIF4B expres- observations were further confirmed by bioluminescent imag- sion has profound effects on the v-Abl–mediated bone marrow ing conducted to detect tumors (Fig. 6D). In addition, Western transformation. blot analysis of tumor extracts showed the eIF4B depletion in To confirm this observation, we generated bicistronic retro- the tumor cells expressing eIF4B-specific shRNA (Fig. 6E). viruses encoding the v-Abl or Bcr-Abl and either eIF4B, eIF4B- Together, these results suggest that eIF4B is required for S422A, or GFP control (Supplementary Fig. S5C and S5D). BMCs Abl-dependent tumor growth. derived from eIF4B-knockdown or WT mice were infected with these retroviruses. As expected, GFP and eIF4B-S422A failed to Primary bone marrow transformation assay using complement eIF4B deficiency for v-Abl- or Bcr-Abl–mediated transgenic mice shows a requirement for eIF4B in transformation (Fig. 7C and D). In contrast, significantly efficient cellular transformation by Abl oncogenes increased transformants were found in cells infected with the To further define the role of eIF4B in Abl-mediated tumor- retroviruses encoding Abl and eIF4B WT proteins. To show the igenesis, we wished to establish a more physiologic model involvement of Pim kinase in the cellular transformation, we system for analysis of eIF4B involvement in the Abl transfor- generated bicistronic retroviruses encoding the v-Abl or Bcr- mation. For this, we generated eIF4B-knockdown transgenic Abl and either Pim-1, Pim-1-KD mutant, or GFP control (Sup- mice as previously described (Supplementary Fig. S5A and S5B; plementary Fig. S5E and S5F) and conducted the bone marrow ref. 38). The transgenic founders with high interference effi- transformation. We observed that overexpression of Pim-1 ciency were selected (Fig. 7A). BMCs derived from eIF4B- resulted in significantly increased Abl transformation efficiency knockdown mice or their WT littermates were infected with in WT mice but not in eIF4B-knockdown mice (Fig. 7E and F), equal titer of A-MuLV. The capacity of the virus to transform suggesting that Pim promotes Abl transformation through BMCs was measured as previously described (6, 35). eIF4B WT phosphorylating eIF4B. Together, these results suggest that

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A B ** 20

WT TG 16 78 10 72 84 15 11 86 92 95 Figure 7. eIF4B is required for 89 98 12 12 efﬁcient bone marrow eIF4B transformation by Abl oncogenes. 8 Actin A, eIF4B expression in representative tissues (spleen) 4 from eIF4B-knockdown transgenic 0

mice (TG) and WT littermates was wellsWells of survivors/96 examined by immunoblotting. B, WT TG BMCs from WT and eIF4B- C D knockdown transgenic mice were ** ** * * infected with A-MuLV viruses. 25 25 Transformation efﬁciency was scored as described in Materials 20 20 and Methods. Plotted are results 15 15 from 3 independent experiments. WT WT Error bars, SEM; n ¼ 3. , P < 0.01. TG C and D, experiments were carried 10 10 TG out as described in B. BMCs from WT and eIF4B-knockdown 5 5 transgenic mice were infected with

the bicistronic retroviruses wellsWells of survivors/96 0 wellsWells of survivors/96 0 expressing v-Abl (C) or Bcr-Abl (D) and either GFP, eIF4B-WT, or eIF4B-S422A as indicated. Plotted are results from 3 independent experiments. Error bars, SEM; n ¼ 3. , P < 0.05; , P < 0.01. E and E F F, BMCs from WT and eIF4B- knockdown transgenic mice were 25 * ** 25 * ** infected with the bicistronic retroviruses expressing v-Abl (E) or 20 20 lentiviruses expressing Bcr-Abl (F) 15 15 and either GFP, Pim-1-WT, or Pim- WT WT 1-KD as described in C and D. Plotted are results from 3 10 TG 10 TG independent experiments. Error bars, SEM; n ¼ 3. , P < 0.05; 5 5 , P < 0.01. Wells of survivors/96 wellsWells of survivors/96 0 wellsWells of survivors/96 0

v-Abl- and Bcr-Abl–mediated cellular transformation critically by Abl oncogenes (6, 8). Despite these progresses, the mechan- requires activated eIF4B that is regulated by Pim-dependent isms underlying Pim action in Abl transformation and sub- phosphorylation mainly on Ser422. strates of Pim kinases in Abl transformants are poorly characterized. Here, we, for the ﬁrst time, show that Pim-1 and Pim-2 phosphorylate eIF4B mainly on Ser422 and to a Discussion lesser extent, on Ser406 in Abl-transformed cells, revealing a Abl-induced tumorigenesis is a complicated process, involv- novel Pim kinase-dependent eIF4B activation that plays a ing the activation of signaling pathways that regulate cell crucial role in Abl-mediated cellular transformation (Supple- survival and proliferation (2, 42). It has been shown that mentary Fig. S5G). However, Pim-3 has little effect on eIF4B constitutive activation of STAT signaling endows the Abl- phosphorylation. This is consistent with the previous obser- transformed cells with cytokine-independent growth (43– vation that Pim-3 is not required for v-Abl transformation and 45). Importantly, activation of STAT signaling strongly induces it might have different functions in these cells (6). the expression of Pim kinases in response to Abl oncogenic Pim kinases are able to phosphorylate multiple targets and signaling. Pim-1 and Pim-2 have been shown to be critically have therefore been implicated in diverse biologic processes. required for malignant transformation of hematopoietic cells For example, there is compelling evidence showing that Pim

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kinases are of great importance in cellular transformation due survival signals on eIF4B is an attractive alternative for devel- to their oncogenic activities that exert influence in regulation opment of anticancer therapies. of cell survival signaling by phosphorylating BAD and regula- Our study has also begun to address the mechanism by tion of cell-cycle progression through phosphorylation of which eIF4B may affect Abl transformation. We observed that p21CIP1, p27KIP1, Cdc25A, and Cdc25C, as well as regulation depletion of eIF4B led to a significant reduction in the expres- of c-Myc activity (25). In addition, Pim-1 and Pim-2 kinases are sion of Bcl-2 and c-Myc. This is consistent with the previous involved in regulating phosphorylation of SOCS-1, and thereby experiments showing that eIF4B regulates translation initia- alter the inhibitory effects of SOCS-1 (34, 39). On the basis of tion of mRNAs harboring strong to moderate secondary their oncogenic potential, the Pim kinase family is emerging as structures in their 50UTRs such as Bcl-2 and c-Myc (40). It an important new target for drug discovery (46). However, has long been suggested that resistance to apoptosis in Abl- malignant transformation of lymphoid and myeloid cells transformed cells is associated with increased expression of involves the dysregulation or mutation of genes including antiapoptotic proteins including Bcl-2 (49, 50). Importantly, PI3K/Akt pathway and Pim family (14, 47). The Akt and Pim our observation indicates that Abl kinase-dependent expres- kinases produce parallel oncogenic signals and share many sion and phosphorylation of eIF4B are responsible for control molecular targets normally involved in regulating the cell of the antiapoptotic protein expression. Further research is proliferation and survival. Therefore, resistance to targeted required to address whether this regulation occurs in human Pim or PI3K/Akt therapeutics may become a major clinical leukemia patients and has any prognostic significance in the problem because the redundancy of oncogenic signaling path- leukemia. ways provides back-up mechanisms that allow cancer cells to escape (26, 48). Disclosure of Potential Conflicts of Interest fl In this study, our results represent the first report of Pim No potential con icts of interest were disclosed. kinases-mediated phosphorylation of eIF4B in Abl transfor- Authors' Contributions mants, suggesting that Pim kinases regulate cap-dependant Conception and design: J.-L. Chen protein translation in these cells. We found that expression of Development of methodology: J. Yang, G. Guo eIF4B S422E phosphomimetic mutant was able to complement Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Chen, D. Whitten inhibition of Pim kinases for Abl transformant survival, imply- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ing that phosphorylation of this site plays a key role in eIF4B computational analysis): J. Yang, J. Wang, D. Whitten Writing, review, and/or revision of the manuscript: J. Yang, P.B. Rothman, S. activity. These data indicate that eIF4B Ser422 phosphoryla- Huang, J.-L. Chen tion is critically required for efficient cellular transformation by Administrative, technical, or material support (i.e., reporting or orga- Abl oncogenes. In addition, the results reveal that eIF4B nizing data, constructing databases): R. Xi, L. Zhang mediates some of the effects of the Pim kinases on hemato- Study supervision: J.-L. Chen poietic cell transformation. Previous studies from other group Acknowledgments have shown that activity of eIF4B can be regulated through The authors thank members of the Chen laboratory for helpful discussions. phosphorylation by Akt (30). These experiments reveal that ability of Akt to phosphorylate eIF4B is potentially critical for the transforming capacity of this kinase. Our results presented Grant Support This work was supported by National Basic Research Program (973) of China in this study provide strong evidence that eIF4B can also be (2009CB918902), National Key Technologies Research and Development Pro- phosphorylated by Pim kinases. Together, these data suggest gram of China (2013ZX10004-611), Natural Science Foundation of China that the function of eIF4B in regulating cell survival and (81171943, 30971476), and Hundreds of Talents Program of Chinese Academy of Sciences 2009–2014 to J.-L. Chen. proliferation is mediated by several proto-oncogenic signaling The costs of publication of this article were defrayed in part by the payment of pathways. These findings indicate that eIF4B represents one of page charges. This article must therefore be hereby marked advertisement in the focal points whereby Akt, Pim, and other oncogenic accordance with 18 U.S.C. Section 1734 solely to indicate this fact. signaling converge to exert their effects on malignant trans- Received November 22, 2012; revised May 3, 2013; accepted May 28, 2013; formation. Therefore, targeting the convergence of oncogenic published OnlineFirst June 7, 2013.

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www.aacrjournals.org Cancer Res; 73(15) August 1, 2013 OF11

eIF4B Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation by Abl Oncogenes

Jianling Yang, Jun Wang, Ke Chen, et al.

Cancer Res Published OnlineFirst June 7, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-4277

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2013/06/07/0008-5472.CAN-12-4277.DC1

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