Azab, A K

Azab, F

Ghobrial, I M

Glavey, S V

Maiso, P

Manier, S

Mishima, Y

Moschetta, M

Roccaro, A M

Sacco, A

Sahin, I

Tsang, B

Zhang, Y

Blood Cancer Journal

Harvard University - DASH

 Distinct roles of class I PI3K isoforms in multiple myeloma cellsurvival and dissemination  (Article begins on next page)The Harvard community has made this article openly available.Please share how this access benefits you. Your story matters.Citation Sahin, I., M. Moschetta, Y. Mishima, S. V. Glavey, B. Tsang, F.Azab, S. Manier, et al. 2014. “Distinct roles of class I PI3Kisoforms in multiple myeloma cell survival and dissemination.”Blood Cancer Journal 4 (4): e204. doi:10.1038/bcj.2014.24.http://dx.doi.org/10.1038/bcj.2014.24.Published Version doi:10.1038/bcj.2014.24Accessed February 19, 2015 4:01:15 PM ESTCitable Link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12153007Terms of Use This article was downloaded from Harvard University's DASHrepository, and is made available under the terms and conditionsapplicable to Other Posted Material, as set forth athttp://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAAOPENLETTER TO THE EDITORDistinct roles of class I PI3K isoforms in multiple myeloma cellsurvival and disseminationBlood Cancer Journal (2014) 4, e204; doi:10.1038/bcj.2014.24;published online 25 April 2014The phosphoinositide 3-kinase (PI3K) pathway has a crucial role intumor progression and drug resistance, including both conven-tional chemotherapeutics as well as novel agents.1 Although nomutations have been described in the PI3K/Akt genes in multiplemyeloma (MM), it was shown that this pathway is constitutivelyactivated in MM cells and has pleiotropic effects inﬂuencingproliferation, drug resistance, angiogenesis and cell adhesion.2PI3Ks are divided into three subclasses, and of these, class IPI3Ks—p110a (also known as PIK3CA), p110b (also known asPIK3CB), p110g (also known as PIK3CG) and p110d (also known asPIK3CD)—are well described in terms of their role in cancerdevelopment and progression.1,3 PIK3CA is frequently mutated insolid tumors including carcinoma of the prostate, breast colon andendometrium.4,5 However, there have been no reports of cancer-speciﬁc mutations in MM.6Recently, a number of potential therapeutics targeting speciﬁcPI3K groups or isoforms were developed.3,4 Previous studies haveindicated that p110a, p110b and p110d might be potential targetsfor MM.7–9 Although the basic framework of PI3K signaling hasbeen uncovered, the contribution of the different PI3K isoforms isnot well understood.4 In the current study, we investigated thefunctional role of class I PI3K isoforms in modulating MM celltrafﬁcking in vivo and in vitro.To examine activation of the PI3K/Akt pathway in MM, we ﬁrstperformed gene set enrichment analysis10 on the gene-expressiondata set (Shaughnessy et al. ref. GSE24080) of patients in differentInternational Staging System stages of MM compared with normaldonors;11 and found enrichment of genes related to class I PI3K-activated AKT signaling events. These ﬁndings were observed instage I, II and III MM patients compared with healthy individuals(Figure 1a).To study the role of each isoform (p110a, b, g, and d)i nregulating MM cell survival and trafﬁcking in vivo and in vitro, theexpression of PI3K isoforms was examined in a panel of eight MMcell lines showing different levels of expression of PI3K isoformswith only MM.1S expressing all isoforms (Figure 1b). Thus, MM.1S-GFPþ/lucþ was infected with lentivirus-mediated small hairpinRNAs targeting the different PI3K isoforms. Stable cell lines weregenerated, and efﬁciency of knockdown for each isoform wasconﬁrmed by reverse transcription quantitative PCR (Figure 1c).Speciﬁcity of knockdown was demonstrated by immunoblottingin cell lines using speciﬁc antibodies against each isoform(Figure 1d). Then, we evaluated the effect of each isoform onPI3K–Akt signaling in MM cells in the context of primary MM bonemarrow mesenchymal stromal cells (BM-MSCs) and found inhibi-tion of BM-MSC-dependent induction of phospho(p)-Akt in MMcells with all PI3K isoforms silenced in the tumor clone (Figure 1e).Although p110a, b, and d showed a modest reduction in cellsurvival in vitro (Figure 1f), cell cycle analysis revealed nosigniﬁcant difference on cell cycle distribution patterns(Supplementary Figure 1). We next performed adhesion assay ofMM cells to primary MM-derived BM-MSCs; and found that bysilencing each of class I PI3K isoforms, MM cells inhibited theiradhesion properties, with the p110b and p110d knockdown beingthe most effective (53% reduction and 47% reduction, respec-tively; Po0.001, Po0.01; Figure 1g).To test the effect of the different p110 isoforms on MM tumorprogression in vivo, SCID-Bg mice were injected with MMcells silenced for p110a, b, g and d, and tumor development wasmonitored by bioluminescence imaging. Scramble-infected cellswere used as control. In consistent with in vitro data demonstratingthat the most signiﬁcant changes were observed for adhesion ofMM cells to BM-MSCs in p110b and p110d knockdown cells, tumorprogression was signiﬁcantly lower in p110b- and p110d-knock-down cell-injected mice compared with scramble cell-injectedmice (Po0.05); whereas tumor growth observed in p110a- andp110g-knockdown cell-injected mice was similar to control mice(Figures 2a and b). We speculate that this might be due tomarkedly decreased tumor cell growth triggered by MM celladhesion to BM-MSCs, as the adhesion of MM cells to BM-MSCsactivates many pathways and has a vital role in MM pathogenesisand disease progression.12 We further conﬁrmed that tumorcells showed knockdown for each p110 isoform, as demonstratedex vivo on tumor cells harvested from each cohort of mice(Figure 2c). Mice were followed until the development of hindlimb paralysis or death, and Kaplan–Meier analysis was performedshowing prolonged survival in all groups except p110a mice(p110b and p110g, Po0.05; p110d, Po0.001; Figure 2d). Despitesimilar tumor burden observed between p110g mice andscramble control-injected mice, mice injected with p110g knock-down cells had improved survival compared with control mice.This might be due to the different extent of tumor involvement ofvarious organs13 between the two groups, thus explaining thedifferences in survival.Interestingly, our data indicate that p110a is not critical for thesurvival of MM cells in vivo. Unlike most solid tumor malignancies,where PI3KCA (p110a) mutation is the leading cause of activationof this pathway and is the target of many therapeutic agents indevelopment,3 there have been no reports of this speciﬁcmutations in MM.6 Moreover, it was shown that unlike wild-typep110a, overexpression of the wild-type p110b, p110g and p110d issufﬁcient to induce an oncogenic transformation of ﬁbroblasts incell culture.14In this study, p110b was highly expressed in all MM cell lines,whereas only a minor subset expressed p110d at the protein level(Figure 1b), which is consistent with a recent report9 showingexpression of p110b in 38 MM cell lines in comparison to thedetectable expression of p110d in only 4 cell lines. In addition,another study8 reported similar ﬁndings in cell lines showing lackof p110d expression in most MM cell lines. Of note, we founddiscrepancies in p110d expression in cell lines between our studyand prior published studies but our data was conﬁrmed in theCancer Cell Line Encyclopedia data at the mRNA level (data notshown).15 Importantly, Ikeda et al.8 evaluated p110d levels inpatient samples and detected its expression in all 24 MM patients.This may provide a clinical rationale for targeting p110d despitethe lack of expression of p110d in MM cell lines.Overall, our data suggest that, in contrast with solid tumors, MMmay be more dependent on PI3K p110b and p110d and lessdependent on PI3Ka, and these may be the focus of drugdevelopment in this hematological malignancy.Citation: Blood Cancer Journal (2014) 4, e204; doi:10.1038/bcj.2014.24& 2014 Macmillan Publishers Limited All rights reserved 2044-5385/14www.nature.com/bcj0204060801001200201030507090110406080100120Scramble p110 p110 p110 p110Survival (% of control cells) P<0.05 P<0.01 P<0.05 Scramble  p110 p110 p110 p110Adhesion to BMSCs(% of control cells)P<0.05 P<0.01 P<0.001 P<0.01 Scramble shRNAMM1.S OPM1 OPM2 H929 RPMI LR7 INA6 U266 p110Tubulinp110Tubulinp110Tubulinp110TubulinGene Expression(% of Scramble)p11000.20.40.60.811.21.4Scramble shRNAp110 shRNA 00.20.40.60.811.2p110shRNA Scramblep110  shRNA 00.20.40.60.811.21.4p110Scramble shRNAp110 shRNA 00.20.40.60.811.21.4p110Scrp110p110p110p110shRNAsp110TubulinTubulinTubulinTubulinp110p110p110ScrP-AKT(Thr308)P-AKT(Ser473)AKT+BMSCs Scrp110p110p110p110Enrichment plot: PID_PI3KCIAKTPATHWAY Enrichment plot: PID_PI3KCIAKTPATHWAY Enrichment plot: PID_PI3KCIAKTPATHWAYEnrichment score (ES)Ranked list metric (Signal2Noise)Ranked list metric (Signal2Noise)0.350.300.250.200.150.100.050.00–0.05–0.10–0.15–0.20“Stage 1” (positively correlated) “Stage II” (positively correlated)Zero cross at 8697 Zero cross at 8868 Zero cross at 9894“Healthy” (negatively correlated) “Healthy” (negatively correlated) “Healthy” (negatively correlated)Enrichment score (ES)0.350.300.250.200.150.100.050.00–0.05–0.10–0.15Enrichment score (ES)0.350.300.250.200.150.100.050.00–0.05–0.10–0.150.50.0–0.5–1.0–1.50.51.00.0–0.5–1.0–1.5Ranked list metric (Signal2Noise)0.51.00.0–0.5–1.0Enrichment profile Hits Ranking metric scoresRank in Ordered Dataset0 2,500 5,000 7,500 10,000 12,500 15,000 17,500 20,000Enrichment profile Hits Ranking metric scoresRank in Ordered Dataset0 2,500 5,000 7,500 10,000 12,500 15,000 17,500 20,000Enrichment profile Hits Ranking metric scoresRank in Ordered Dataset0 2,500 5,000 7,500 10,000 12,500 15,000 17,500 20,000“Stage 3” (positively correlated)ISS Stage I vs Healthy ISS Stage II vs Healthy ISS Stage III vs Healthyp110 shRNALetter to the Editor2Blood Cancer Journal & 2014 Macmillan Publishers Limitedp110 p110 p110 p110 ScrambleDay 7 Day 21 Day 35 Scramble shRNA p110 shRNAScramble shRNAp110 shRNAScramble shRNAp110 shRNAp110Scramble shRNAp110 shRNAp110P<0.05Luminescence0.80.60.4x1070.2Radiance(p/sec/cm /sr)Color ScaleMin = 5.98e5Max = 9.23e62.0×108Scramblep110αp110βp110γp110δ1.5×1081.0×1085.0×107Tumor Growth (BLI)9.0×1066.0×1063.0×1066.0×1053.0×10507 21 35Days00.20.40.60.811.200.20.40.60.811.2p110p110Gene Expression(% of Scramble)00.20.40.60.811.200.20.40.60.811.2Figure 2. (Continued)Figure 1. The role of class I PI3K-mediated Akt signaling in MM. (a) Gene set enrichment analysis software analyzed functionally related genesin class I-mediated Akt activation with statistically signiﬁcant enrichment (false-discovery rate q-values o0.25; o0.25 is considered signiﬁcant),using gene-expression data set (GSE24080). Plots show enrichment results for the gene set (left, stage I MM vs normal subjects; middle, stage IIMM vs normal subject; right, stage III MM vs normal subjects). (b) Baseline expression of the different PI3K isoforms (p110a, b, g and d)i nM Mcell lines was detected by immunoblotting using isoform-speciﬁc antibodies. MM tumor cells (MM.1S-GFPþ/lucþ) were infected withlentivirus-mediated small hairpin (sh)RNA. Reverse transcription quantitative PCR (c) and immunoblotting (d) were performed to showinfection efﬁciency and isoform speciﬁcity, respectively. Scramble and knockdown tumor cells (p110a, b, g and d) were cocultured with BMSCsovernight, and MM cells were then separated from the BMSCs, lysed and whole-cell lysates were subjected to immunoblotting (e) with Aktand P-Akt (Thr308 and Ser473), which shows decreased phosphorylation of Akt in knockdown cells. The effects of inhibition of PI3K isoformsby shRNAs on cell survival were assessed by 3-(4,5-dimethylthiazol-2-yl)2-2-diphenyltetrazolium bromide (MTT) assay (f). Adhesion assay (g)was performed to show the ability of knockdown cells to adhere to BMSCs after 2h of incubation.Letter to the Editor3& 2014 Macmillan Publishers Limited Blood Cancer JournalCONFLICT OF INTERESTIMG is on the advisory board for Onyx, BMS and Celgene, and receives research labsupport from Genzyme and BMS. The remaining authors declare no conﬂict ofinterest.ACKNOWLEDGEMENTSThis work was supported by R01CA154648.AUTHOR CONTRIBUTIONSIS: designed and performed the research, analyzed the data and wrote themanuscript; MM, YM, SVG, BT, FA, YZ, PM, AS, AKA and AMR: performed the researchand analyzed the data; IMG: supervised the study and wrote the manuscript.I Sahin1, M Moschetta1, Y Mishima1, SV Glavey1, B Tsang1,F Azab1,2, S Manier1, Y Zhang1, P Maiso1, A Sacco1, AK Azab1,2,AM Roccaro1 and IM Ghobrial11Department of Medical Oncology, Dana-Farber Cancer Institute,Harvard Medical School, Boston, MA, USA and2Cancer Biology Division, Department of Radiation Oncology, Schoolof Medicine, Washington University in St Louis, St Louis, MO, USAE-mail: irene_ghobrial@dfci.harvard.eduREFERENCES1 Rodon J, Dienstmann R, Serra V, Tabernero J. Development of PI3Kinhibitors: lessons learned from early clinical trials. Nat Rev Clin Oncol 2013; 10:143–153.2 Harvey RD, Lonial S. PI3 kinase/AKT pathway as a therapeutic target in multiplemyeloma. Future Oncol 2007; 3: 639–647.3 Courtney KD, Corcoran RB, Engelman JA. The PI3K pathway as drug target inhuman cancer. J Clin Oncol 2010; 28: 1075–1083.4 Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emergingmechanisms of isoform-speciﬁc PI3K signalling. Nat Rev Mol Cell Biol 2010; 11:329–341.5 Zhao L, Vogt PK, Class I. PI3K in oncogenic cellular transformation. Oncogene 2008;27: 5486–5496.6 Ismail SI, Mahmoud IS, Msallam MM, Sughayer MA. Hotspot mutations ofPIK3CA and AKT1 genes are absent in multiple myeloma. Leuk Res 2010; 34:824–826.7 Glauer J, Pletz N, Schon M, Schneider P, Liu N, Ziegelbauer K et al. A novelselective small-molecule PI3K inhibitor is effective against human multiplemyeloma in vitro and in vivo. Blood Cancer J 2013; 3: e141.8 Ikeda H, Hideshima T, Fulciniti M, Perrone G, Miura N, Yasui H et al.PI3K/p110{delta} is a novel therapeutic target in multiple myeloma. Blood 2010;116: 1460–1468.9 Munugalavadla V, Mariathasan S, Slaga D, Du C, Berry L, Del Rosario G et al.The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superioractivity in multiple myeloma. Oncogene 2013; 33: 316–325.P<0.001P<0.05P<0.05Scramblep110 alphaScramblep110 alphaScramblep110 betap110 betaScramblep110 gamap110 gamaScramblep110 deltap110 delta10050035 45 55 65DaysDays35 45 55 65Days35 45 55 50 40 60 70 65Days40 50 60 70Percent survival100500Percent survival10050035 45 55 50 40 60 70 65DaysPercent survival100500Percent survival100500Percent survivalFigure 2. Knockdown of PI3K isoforms regulates tumor progression and survival in vivo. MM.1S-GFPþ/lucþ tumor cell lines (Scr, p110a, b, gand d) were injected intravenously into SCID-Bg mice and tumor growth was assessed by in vivo bioluminescence imaging (BLI).(a) Representative BLI of each group in different time points is shown. (b) Quantiﬁcation of BLI signals demonstrated that p110b and d miceshowed signiﬁcant reduction in tumor growth (Po0.05) compared with scramble mice. (c) Reverse transcription quantitative PCR wasperformed on tumor cells that were harvested from hind leg bones of animals by bone marrow ﬂushing. (d) Survival of mice was evaluateduntil complete hind limb paralysis or death using Kaplan–Meier curves. Compared with scramble mice, all groups except p110a showedprolonged survival (p110b and p110g, Po0.05; p110d, Po0.001).Letter to the Editor4Blood Cancer Journal & 2014 Macmillan Publishers Limited10 Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA et al.Gene set enrichment analysis: a knowledge-based approach for interpretinggenome-wide expression proﬁles. Proc Natl Acad Sci USA 2005; 102:15545–15550.11 Schaefer CF, Anthony K, Krupa S, Buchoff J, Day M, Hannay T et al. PID:the Pathway Interaction Database. Nucleic Acids Res 2009; 37(Database issue):D674–D679.12 Mahindra A, Hideshima T, Anderson KC. Multiple myeloma: biology of the disease.Blood Rev 2010; 24(Suppl 1): S5–S11.13 Huang YW, Richardson JA, Tong AW, Zhang BQ, Stone MJ, Vitetta ES.Disseminated growth of a human multiple myeloma cell line in micewith severe combined immunodeﬁciency disease. Cancer Res 1993; 53:1392–1396.14 Kang S, Denley A, Vanhaesebroeck B, Vogt PK. Oncogenic transformation inducedby the p110beta, -gamma, and -delta isoforms of class I phosphoinositide3-kinase. Proc Natl Acad Sci USA 2006; 103: 1289–1294.15 Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al. TheCancer Cell Line Encyclopedia enables predictive modelling of anticancer drugsensitivity. Nature 2012; 483: 603–607.This work is licensed under a Creative Commons Attribution 3.0Unported License. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicatedotherwise inthecreditline;ifthematerialisnot includedundertheCreativeCommonslicense, users will need to obtain permission from the license holder to reproduce thematerial. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/Supplementary Information accompanies this paper on Blood Cancer Journal website (http://www.nature.com/bcj)Letter to the Editor5& 2014 Macmillan Publishers Limited Blood Cancer Journal

Distinct roles of class I PI3K isoforms in multiple myeloma cell survival and dissemination

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Distinct roles of class I PI3K isoforms in multiple myeloma cell survival and dissemination

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