PIM Kinases Promote Survival and Immune Escape in Primary Mediastinal Large B-Cell Lymphoma through Modulation of JAK-STAT and NF-κB Activity

Primary mediastinal large B-cell lymphoma (PMBL) cells depend on the constitutive activity of NF-κB and STAT transcription factors, which drive expression multiple molecules essential for their survival. In a molecularly related malignant tumor (classic Hodgkin lymphoma), Reed-Sternberg overexpress oncogenic (proviral integration site Moloney murine leukemia virus (PIM) 1, 2, 3 kinases in NF-κB– STAT-dependent manner PIMs enhance survival immunomodulatory molecules. Given overlapping characteristics PMBL cells, we hypothesized that PIM may be overexpressed involved pathogenesis. The diagnostic biopsy specimens was assessed role immune escape determined. were abundantly expressed primary tumors cell lines. Inhibition toxic to attenuated protein translation, down-regulated prosurvival factors BCL2A1, BCL2L1, FCER2. Furthermore, inhibition decreased engaged shaping immunosuppressive microenvironment, including programmed death ligand 1/2 chemokine (C-C motif) 17. Taken together, our data indicate support identify as promising therapeutic targets PMBL. is an aggressive lymphoma, accounting approximately 2% 4% all mature neoplasms, affecting predominantly young girls, manifesting with mass arises from B thymic origin.1Savage K.J. Rare lymphomas: mediastinal, intravascular, effusion lymphoma.Cancer Treat Res. 2008; 142: 243-264PubMed Google Scholar Although cure rates standard immunochemotherapy, such R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) or DA-EPOCH-R (etoposide, prednisone, rituximab), reach 90%, treatment patients relapsed/refractory remains largely ineffective. those patients, new approaches are required improve prognosis. Historically, designated subtype diffuse (DLBCL). However, subsequent molecular studies revealed differences DLBCL highlighted common features shared by nodular sclerosis classic (cHL), transcriptional profile, genomic aberrations leading overexpression (PD-L) 1 -2, microenvironmental interactions, addiction JAK-STAT signaling pathways.2Savage Monti S. Kutok J.L. Cattoretti G. Neuberg D. De Leval L. Kurtin P. Dal Cin Ladd C. Feuerhake F. Aguiar R.C. Li Salles Berger Jing W. Pinkus G.S. Habermann T. Dalla-Favera R. Harris N.L. Aster J.C. Golub T.R. Shipp M.A. signature differs other lymphomas shares classical lymphoma.Blood. 2003; 102: 3871-3879Crossref PubMed Scopus (695) Scholar, 3Rosenwald A. Wright Leroy K. Yu X. Gaulard Gascoyne R.D. et al.Molecular diagnosis identifies clinically favorable subgroup lymphoma.J Exp Med. 198: 851-862Crossref (875) 4Green M.R. Rodig S.J. Juszczynski Currie O'Donnell E. Chapuy B. Takeyama Integrative analysis reveals selective 9p24.1 amplification, increased PD-1 expression, further induction via JAK2 sclerosing 2010; 116: 3268-3277Crossref (843) proviral cHL (RS) boost antiapoptotic proteins, cytokines, chemokines, molecules, PD-L1 PD-L2, thus fostering RS escape.5Szydlowski M. Prochorec-Sobieszek Szumera-Cieckiewicz Derezinska Hoser Wasilewska Szymanska-Giemza O. Jablonska Bialopiotrowicz Sewastianik Polak Czardybon Galezowski Windak Zaucha J.M. Warzocha Brzozka Expression fosters privilege 2017; 130: 1418-1429Crossref (26) might contribute pathogenesis disease. PIM1, -3 decreases proliferation, induces apoptosis, down-regulates formation niche Human Karpas1106P U2940 L-428 maintained RPMI 1640 medium supplemented 20% fetal bovine serum (Lonza Group AG, Basel, Switzerland). SUP-HD1 grown McCoy's 5A AG). acute lymphoblastic SEM line cultured Iscove's modified Dulbecco's 10% dual pan-PIM- FMS-like tyrosine kinase (FLT3) inhibitor MEN1703 provided Menarini Ricerche (Pomezia, Italy), pan-PIM PIM447 purchased Selleckchem (Houston, TX). All chemicals dissolved dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). vehicle-control experiments, final DMSO concentrations 0.5% (control 5-μmol/L dose). Protein lysates resolved SDS-PAGE, transferred polyvinylidene difluoride membranes (Millipore, Burlington, MA), immunoblotted appropriate horseradish peroxidase–labeled secondary antibodies. following antibodies used: anti-PIM1 (catalog number 54523; Cell Signaling Technology, Danvers, anti-PIM2 4730; Technology), anti-PIM3 4165; anti-ph-STAT5-T694 9359; anti-STAT5 9363; anti-ph-STAT3-T705 9145; anti-STAT3 21045; Signalway Antibody LLC, College Park, MD), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MAB374; Millipore), anti-ph-S6-S235/6 4857; anti-S6 2217; anti-ph-4EBP1-S65 9456; anti-4EBP1 9644; anti-ph-RelA-S276 11011-1; SAB Biotherapeutics), anti-RelA SC-8008; Santa Cruz Biotechnology, Dallas, TX), anti-FLT3 3462; Technology). Chemiluminescent signals detected using ECL reagent (PerkinElmer, Waltham, MA) G:Box image acquisition system (Syngene International Ltd., Bangalore, India). To reprobe blots another antibody, incubated stripping buffer 30 minutes at 50°C followed incubation antibody. Band intensities quantified densitometry ImageJ software version 1.52a (NIH, Bethesda, MD; http://imagej.nih.gov/ij). A retrospective group 15 diagnosed according 2017 World Health Organization classification6Swerdlow S.H. Campo Jaffe E.S. Pileri S.A. Stein H. Thiele J. WHO Classification Tumours Haematopoietic Lymphoid Tissues. ed 4 Revised. Vol 2. IARC, Lyon2017Google studied. consent workup tissue specimens. Per local institutional bioethical policies, research use archival, anonymously does not require special review board approval beyond perform workup. Immunohistochemistry formalin-fixed, paraffin-embedded sections performed automated stainer (Dako Denmark A/S, Glostrup, Denmark) indicated antibodies: (ST0513) (1:100, pH 9.0; catalog NBP2-67528; Novus Biologicals, Littleton, CO), (D1D2) (D17C9) (1:50, EnVision Detection System A/S) used signal detection. prostate adenocarcinoma, testis, hepatocellular carcinoma positive staining controls PIM2 PIM3, respectively. Negative (isotype) control stainings ready-to-use FLEX Mouse Control (a cocktail mouse IgG1, IgG2a, IgG2b, IgG3, IgM; code IR750; Dako A/S). Staining scored semiquantitative method (0, no staining; weak, intermediate, 3, strong staining). Samples considered PIM2, PIM3 only if ≥10% exhibited and/or intermediate reactions when ≥20% weakly positive. Analyses independently two hematopathologists (M.P.S. A.S.C.) blinded fashion. microphotographs taken microscope DP72 Olympus BX63 camera (Olympus, Tokyo, Japan). RNA extracted GeneMATRIX Universal purification kit (EURx, Gdańsk, Poland) reverse transcribed Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA), manufacturer's instructions. Gene levels measured CFX Realtime (Bio-Rad Laboratories, Hercules, CA) SYBR Green PCR Master Mix (Life Technologies, gene-specific primers (Table 1). Obtained CT values target genes housekeeping (RPL29) calculate relative transcript abundance 2−ΔΔCt method.5Szydlowski Experiments conducted triplicate, gene compared Gosset's two-sided t-test, GraphPad QuickCalcs/Prism 6 (GraphPad Software, San Diego, CA).Table 1Primer Used Real-Time Quantitative AnalysisPrimerSequenceRPL29-F5′-CAGCTCAGGCTCCCAAAC-3′RPL29-R5′-GCACCAGTCCTTCTGTCCTC-3′NFKBIA-F5′-CCAACTACAATGGCCACACGT-3′NFKBIA-R5′-TCCGGCCATTACAGGGCT-3′TNF-F5′-AGGACGAACATCCAACCTTCCCAA-3′TNF-R5′-TTTGAGCCAGAAGAGGTTGAGGGT-3′CD40-F5′-AACAGGCAGGCACAAACAAGACTG-3′CD40-R5′-TGGCAAACAGGATCCCGAAGATGA-3′BCL2A1-F5′-CAGAAGATGACAGACTGTGAA-3′BCL2A1-R5′-TCCAAGCATGACTTCAGATTC-3′BCL2L1-F5′-GGATTTGAATCTCTTTCTCTCC-3′BCL2L1-R5′-TCAACCACCAGCTCCCGGT-3′CCL5-F5′-ACCATGAAGGTCTCCGCGG-3′CCL5-R5′-GTGACAAAGACGACTGCTGG-3′FCER2-F5′-AATCTCAGGACTTGGAGCTG-3′FCER2-R5′-CCGCTGGACACCTGCAAC-3′CCL17-F5′-CATCCACGCAGCTCGAGG-3′CCL17-R5′-TCTTCACTCTCTTGTTGTTGG-3′CD274-F5′-CATACAACAAAATCAACCAAAGA-3′CD274-R5′-TGGTCTTACCACTCAGGACT-3′F, forward; R, reverse. Open table tab F, Proliferation CellTiter96 AQueous nonradioactive proliferation assay (Promega, Madison, WI). IC50 calculated Prism software. experiments triplicate. For washed phosphate-buffered saline, suspended 1× AnnexinV binding (Becton, Dickinson Co., Franklin Lakes, NJ), stained AnnexinV–fluorescein isothiocyanate propidium iodide Co.), analyzed flow cytometry. DNA-binding activities complexes colorimetric (TransAM kit, 43296; Active Mofif Inc., CA). total 20 μg extracts added microwells. Signals developed instructions Multiskan GO plate reader (Thermo Fisher Scientific, MA). duplicates. PD-L2 cytometry described previously,5Szydlowski phosphatidylethanolamine-conjugated (BioLegend, Cells Cytoflex cytometer (Beckman Coulter, Brea, Differences among continuous variables Gosset’s Software). determine PMBL, PIM-1/2/3 lines first assessed. Twelve PIM1 (80%) [4 biopsies (26%) 12 PIM3] (Figure 1A, Supplemental Table S1). patterns similar observed previously cells.5Szydlowski Scholar,7Brault Menter Obermann E.C. Knapp Thommen Schwaller Tzankov progression markers emerging lymphoma.Br J Cancer. 2012; 107: 491-500Crossref (49) nuclear cytoplasmatic staining, whereas appeared cytosol. investigated cases least one isoform. three higher RS-derived lines, 1B). regulated canonical (RelA), noncanonical (RelB), factors.5Szydlowski We aberrant these induce Therefore, STAT3, STAT5, RelA, RelB Similar (L428 SUP-HD1), phosphorylated (T705) STAT3 but phosphor-STAT5 negative 1C). RelA comparable between highest 1D). factors. viability certain lymphomas, cHL.5Szydlowski 8Yang Q. Chen L.S. Neelapu S.S. Miranda R.N. Medeiros L.J. Gandhi V. Transcription translation Pim SGI-1776 mantle 120: 3491-3500Crossref (53) 9Jablonska Szydłowski Białopiotrowicz Kiliszek Golas Brzózka K.D. novel inhibitor, SEL24-B489, apoptosis inhibits through attenuation Myc NFkB activity. American Society Hematology Annual Meeting. 126. Blood, Orlando, CA2015: 706Google 10Bialopiotrowicz Gorniak Noyszewska-Kania Pula Makuch-Lasica Nowak Bluszcz Szydlowski Piechna Lech-Maranda Budziszewska B.K. Wasylecka-Juszczynska Borg Microenvironment-induced promote CXCR4-triggered mTOR pathway chronic lymphocytic leukaemia migration.J Mol 2018; 22: 3548-3559Crossref (10) 11Paino Garcia-Gomez Gonzalez-Mendez San-Segundo Hernandez-Garcia Lopez-Iglesias A.A. Algarin E.M. Martin-Sanchez Corbacho Ortiz-de-Solorzano Corchete L.A. Gutierrez N.C. Maetos M.V. Garayoa Ocio PIM447, displays antimyeloma bone-protective effects, potently synergizes current standards care.Clin Cancer 23: 225-238Crossref (36) address pan-PIM/FLT3 (MEN1703) Because do express FLT3, this cannot FLT3 2A). markedly dose-dependent manner, 0.97 μmol/L 1.75 2B). whether affects survival, (1.5 5 μmol/L) vehicle alone 96 hours evaluated AnnexinV/propidium staining. fraction apoptotic 7.8% 53% 42.09% 73.92% 2C). Effects confirmed additional, structurally unrelated, (PIM447) (Supplemental Figure S1, B). cytotoxic blockade encouraged us investigate its underlying mechanisms. characterized functions kinases, cap-dependent (phosphorylation 4EBP1 ribosomal S6) NF-κB–RelA evaluated.5Szydlowski Scholar,12Lu Zavorotinskaya Dai Y. Niu X.H. Castillo Sim Wang Langowski Holash Shannon Garcia P.D. Pim2 maintaining myeloma growth modulating TSC2 phosphorylation.Blood. 2013; 122: 1610-1620Crossref (127) Scholar,13Chen Redkar Taverna Cortes J.E. Mechanisms cytotoxicity SGI-1776, myeloid leukemia.Blood. 2011; 118: 693-702Crossref (134) After either S6 phosphorylations Karpas11106P 2D S1C). also (Tyr705) PIM-specific (S276) modulate cytokines/chemokines, proteins.5Szydlowski elucidate regulation direct NF-κB/STAT (NFKBIA, TNF, CD40, CCL5, FCER2, CCL17, CD274) treated inhibitors. NF-κB–RelA, significantly mRNA most 3A S2A). At level, surface 32.89% 48.4% 26.15% (PD-L1) 26.88% (PD-L2) 3B). These observations PIM477 S2B). Collectively, highlight communication cells. -cHL represent distinct entities different strategies features, proteins. Our additional feature tumors. cHL, PMBLs exhibits role, modulates augments signaling, facilitating NF-κB/STAT–driven have reported important inducers Consistent similarity ubiquitously PMBL-derived Aberrant results genetic lesions, REL, BCL10, MALT1 amplifications,14Weniger Gesk Ehrlich Martin-Subero J.I. Dyer M.J. Siebert Moller Barth T.F. Gains REL coincide accumulation protein.Genes Chromosomes 2007; 46: 406-415Crossref (62) Scholar,15Wessendorf Viardot Mueller Kestler H.A. Kohlhammer Lichter Bentz Dohner Schwaenen Further delineation chromosomal consensus regions 37 samples high-resolution profiling (array-CGH).Leukemia. 21: 2463-2469Crossref (59) loss-of-function mutations TNFAIP316Schmitz Hansmann M.L. Bohle Hartmann Mechtersheimer Klapper Vater I. Giefing Stanelle Kuppers TNFAIP3 (A20) suppressor 2009; 206: 981-989Crossref (356) increases genes, BCL2L1 (BCLXL) BCL2A1 (BFL1).17Lam L.T. Davis R.E. Pierce Hepperle Xu Hottelet Nong Wen Adams Dang Staudt L.M. Small molecule inhibitors IkappaB selectively subgroups defined profiling.Clin 2005; 11: 28-40Crossref (104) Scholar,18Hinz Loser Mathas Krappmann Dorken Scheidereit Constitutive NF-kappaB maintains high characteristic network, CD86, set Hodgkin/Reed-Sternberg cells.Blood. 2001; 97: 2798-2807Crossref (221) Similarly, activation JAK downstream caused gain-of-function STAT6 IL4R,19Ritz Guiter Castellano Dorsch Melzner Jais J.P. Dubois Recurrent DNA domain 114: 1236-1242Crossref (76) Scholar,20Vigano Gunawardana Mottok Van Tol Mak Chan F.C. Chong Chavez Woolcock Takata Twa Shulha H.P. Telenius Kutovaya Hung Healy Ben-Neriah Diepstra Kridel Savage Rimsza Steidl Somatic IL4R lead activation.Blood. 131: 2036-2046Crossref (24) amplification region harboring locus,4Green inactivating SOCS1 mutations.21Melzner Bucur A.J. Bruderlein Hasel Leithauser Biallelic mutation SOCS-1 impairs degradation sustains phospho-JAK2 action MedB-1 line.Blood. 105: 2535-2542Crossref (176) Scholar,22Melzner Weniger Ritz deletion within 16p13.13 associated delayed protein.Int 2006; 1941-1944Crossref (55) As consequence, deregulated leads up-regulation BCLXL, CD23 (FCER2),19Ritz evasion, (CCL) 17, PD-L1, PD-L2.4Green Despite central PMBL,17Lam Scholar,23Hao Sun H.H. Selective specifically vitro vivo.Clin 2014; 20: 2674-2683Crossref (94) agents targeting pathways been subject few pilot clinical far. phase study, combination ibrutinib BTK activity) R-ICE (rituximab plus ifosfamide, carboplatin, etoposide) immunochemotherapy substantial response rate relapsing/remitting lymphoma.24Sauter C.S. Matasar Schoder Devlin S.M. Drullinsky Gerecitano Kumar Noy Palomba Portlock Straus D.J. Zelenetz A.D. McCall Miller S.T. Courtien A.I. Younes Moskowitz C.H. study relapsed refractory DLBCL.Blood. 1805-1808Crossref (37) contrast, ruxolitinib progressed rapidly after second cycle treatment.25Kim Kang H.J. Dong-Yeop Lee H.S. Yong Shin Yoon D.H. Hong J.Y. Kong J.H. Sakamoto Ko Y.H. Takeuchi efficacy heavily pretreated hodgkin lymphoma: prospective 2016; 128: 1820Crossref partial overlap controlled NFκB STATs (eg, CCL17), simultaneous both appears strategy could more effectively block because directly phosphorylation subunit STAT3.5Szydlowski pathogenetic dependencies, NF-κB–and STAT-driven CCL17 ligands, affect composition function microenvironment. pharmacologic antitumor setting. show extend list biological similarities cHL. broad spectrum produce pleiotropic effects include enhanced response. kindly Ricerche. M.Szy., P.J., A.S.-C. conceived wrote manuscript; M.Szo., M.P.S., obtained samples; A.M.T. revised M.Szy. S.D. experiments. S2PIM virus) 2 (PMLBCL) A: Effect PMLBCL NF-κB/STAT-dependent expression. (5 24 hours. Thereafter, real-time quantitative PCR. B: membrane 48 n = independent (A ∗P < 0.05, ∗∗P 0.01, ∗∗∗P 0.001.View Large Image ViewerDownload Hi-res Download (PPT) .docx (.02 MB) Help docx files S1