Piddock et al. Journal of Hematology & Oncology (2018) 11:66 https://doi.org/10.1186/s13045-018-0614-4 LETTER TO THE EDITOR Open Access Myeloma-derived macrophage inhibitory factor regulates bone marrow stromal cell-derived IL-6 via c-MYC 1 1 1 1 2 Rachel E. Piddock , Christopher R. Marlein , Amina Abdul-Aziz , Manar S. Shafat , Martin J. Auger , 1,2*† 1*† Kristian M. Bowles and Stuart A. Rushworth Abstract: Multiple myeloma (MM) remains an incurable malignancy despite the recent advancements in its treatment. The protective effects of the niche in which it develops has been well documented; however, little has been done to investigate the MM cell’s ability to ‘re-program’ cells within its environment to benefit disease progression. Here, we show that MM-derived macrophage migratory inhibitory factor (MIF) stimulates bone marrow stromal cells to produce the disease critical cytokines IL-6 and IL-8, prior to any cell-cell contact. Furthermore, we provide evidence that this IL-6/8 production is mediated by the transcription factor cMYC. Pharmacological inhibition of cMYC in vivo using JQ1 led to significantly decreased levels of serum IL-6—a highly positive prognostic marker in MM patients. Conclusions: Our presented findings show that MM-derived MIF causes BMSC secretion of IL-6 and IL-8 via BMSC cMYC. Furthermore, we show that the cMYC inhibitor JQ1 can reduce BMSC secreted IL-6 in vivo, irrespective of tumor burden. These data provide evidence for the clinical evaluation of both MIF and cMYC inhibitors in the treatment of MM. Keywords: Myeloma, MIF, cMYC, BMSC, Stromal, IL-6, IL-8, Bone marrow Despite significant recent advancements made in the We and others have found that MM cells have signifi- treatment of multiple myeloma (MM), relapse remains cantly elevated MIF gene expression and secreted pro- inevitable and the disease presently remains incurable. tein levels  (Additional file 1: Figure S1). MIF KD This is attributable, in part, to the highly protective na- resulted in lower MM proliferation in BMSC co-culture ture of the BM micro-environment niche in which the (Additional file 1: Figure S2C) alongside reduced tumor malignant plasma cells proliferate. Macrophage migra- burden and improved overall survival in vivo (Fig. 1a, b). tory inhibitory factor (MIF) is a cytokine associated with To investigate the effects of MIF secretion by MM on its various roles  and is rapidly developing a pro-tumoral microenvironment, we used cytokine arrays to establish identity . Elevated MIF levels are described in MM if cytokine changes occur when MM cells are cultured and have been implicated in MM bone marrow homing with primary BMSC. Elevated levels of IL-6/8 were de- and chemotherapy resistance ; however, the adaptive tected in co-culture experiments when compared with ei- effect that MM-derived MIF has on the tumor micro- ther BMSC or MM monoculture arrays (Fig. 1c, d); no MIF environment is not yet defined. Here, we investigate the was detectedinBMSCculturedalone. Cytokinearray function of MM-derived MIF in the MM microenviron- analysis of the supernatant from MIF-stimulated BMSCs ment by examining its effects on bone marrow stromal confirmed this IL-6/IL-8 secretion (Fig. 1e, f)and was cells (BMSC). quantified via ELISA (Additional file 2)inFig. 1g.Pre- treatment of BMSC with the MIF inhibitor ISO-1 signifi- cantly reduced the MIF induction of IL-6/IL-8 by BMSC * Correspondence: email@example.com; firstname.lastname@example.org (Fig. 1h). BMSC expressed all three known receptors for Kristian M. Bowles and Stuart A. Rushworth contributed equally to this work. MIF (CXCR4, CXCR2 and CD74); however, only blocking Department of Molecular Haematology, Norwich Medical School, The University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Piddock et al. Journal of Hematology & Oncology (2018) 11:66 Page 2 of 4 a b *** P=0.0005 Con MIF KD 0 204060 Day c d Monoculture Co-culture ** 2 * BM-MSC MM Co-Culture EGF IL-6 IL-8 MPO VEGF e f Control 4 * +MIF IL-6 IL-8 800 25 g h Control Control +MIF +MIF 600 +ISO MIF + ISO 10 * IL-6 IL-8 IL-6 IL-8 Fig. 1 MM derived MIF is pro-tumoral and drives BMSC IL-6 and IL-8. 1 × 10 MM.1S-luc cells (ShE control n = 10, and ShMIF n = 7) were injected via the tail vein of 6–8-week-old NSG mice. a Mice were monitored weekly by bioluminescent imaging. b Kaplan-Meier curve showing survival, analyzed using Mantel Cox regression. c Representative (n = 3) Human XL cytokine array output after a 24-h incubation in either mono or co-culture, cell supernatant was used for analysis. d Graphical representation of c—values for BMSC and MM monoculture intensities were added together and were analyzed against co-culture experiment signal intensity using HL++ image software which show differences in several key cytokines. e, f BMSC were stimulated with 100 ng/mL of human recombinant MIF and incubated for 24 h; supernatant was used for assay. Representative (n =3) image of cytokine array (e) and subsequent graphical representation (f)ofanalysisusing HL++ software. g Primary BMSC (n = 4) were stimulated with 100 ng/mL recombinant human MIF for 6 h after which IL-6 and IL-8 protein excretion was analyzed by ELISA. h Primary BMSC (n = 4) were incubated with/ without 10μg ISO-1 and then stimulated with 100 ng/mL recombinant human MIF for 6 h. IL-6 and IL-8 transcriptional levels were then analyzed by RT-PCR Cytokine conc. (pg/mL) +100ng MIF Control Relative fold change in density Percentage Survival Relative fold change in density Relative fold chance in mRNA Piddock et al. Journal of Hematology & Oncology (2018) 11:66 Page 3 of 4 CD74 inhibited MIF-Induced IL6/8 (Additional file 1: 7 days of treatment with JQ1 was predicted to have no Figure S3A&B). measurable effect on tumor burden  and was selected An inhibitor panel was used to screen for potential to control for the effects of tumor burden on MM-MIF pathways responsible for MIF-induced BMSC-derived secretion and subsequent BMSC IL-6 expression. We IL-6/IL-8 expression, and we found that JQ1 inhibited found no difference in MM burden between groups MIF induced IL-6 and IL-8 (Fig. 2a, b) mRNA in BMSC. (Fig. 2d, f). Nevertheless, murine IL-6 was signifi- We then determined if JQ1 could regulate BMSC pro- cantly reduced in the JQ1-treated animals (carrying tumoral interleukin production in-vivo. Although BMSC human MM) compared to control animals (Fig. 2e). are often cyto-protective in the context of anti-MM Despite MIF’s association with hematological malig- therapy, others have observed that the sensitivity of MM nancies, previous work has focused on the effects of cell lines to JQ1 was unchanged by the presence of HS-5 MIF on the malignant cells rather than the supportive . Following MM engraftment (Fig. 2d), mice were ran- cells of the microenvironment . Here, we show that domized and treated for 5 days with I.P injections of MM-derived MIF is pro-tumoral through induction of 50 mg/kg JQ1 or alternatively vehicle control. Under BMSC-derived IL-6/8. IL-6 is central to MM a b 30 30 IL-6 Control IL-8 Control 25 25 MIF treated MIF treated 20 20 15 15 10 10 5 5 0 0 Con BZ PS Len JNK JQ1 Con BZ PS Len JNK JQ1 e f 8x10 6x10 4x10 2x10 Fig. 2 MM-derived MIF regulates bone marrow stromal cell-derived IL-6 and IL-8 via cMYC. a, b BMSC cells were pretreated with various drugs (bortezomib 10 nM, PS341 100 nM, Lenolidiomide 500 nM, JNKV 10uM, and JQ1 500 nM) for 30 mins and then activated with MIF for 2 h. RNA was extracted, and transcriptional levels of IL-6 (a) or IL-8 (b) was analyzed. c Schematic showing JQ1 in vivo experiment. 1 × 10 U266 cells were injected via the tail vein of NGS mice (n = 8). Following a 2-week engraftment period, mice were treated with 50 mg/kg JQ1 or vehicle control daily for 5 days, after which all mice were sacrificed. d Mice were monitored pre and post treatment with JQ1 by bioluminescent imaging which was quantified (f) by ImageJ densitometry. e ELISA data showing murine IL-6 serum concentration following JQ1 treatment. Baseline levels of murine IL-6 were non-detectable in mice without MM Relative fold change in mRNA Relative Tumor Burden (AU) Relative fold change in mRNA Piddock et al. Journal of Hematology & Oncology (2018) 11:66 Page 4 of 4 pathogenesis and primarily comes from the BMSC in essential knowledge and reagents; REP, KMB and SAR wrote the paper. All authors read and approved the final manuscript. the tumor micro-environment . Here, we place IL-6 downstream of MIF-induced BMSC activation [6, 7]. IL- Ethics approval and consent to participate 8 expression in BMSC, which has been shown to parallel Informed consent was given in accordance with the Declaration of Helsinki and under approval from the Health Research Authority of the National MM disease progression  and positively influence os- Health Service, United Kingdom (07/H0310/146). teoclastogenesis in MM , was increased in BMSC in All animal experiments were performed in accordance with UK Home Office response to MIF. Furthermore, the BET-bromodomain and University of East Anglia Animal Welfare Ethics Review Board regulations. inhibitor JQ1 significantly decreased IL-6/8 secretion in Consent for publication MIF-stimulated BMSC. In vivo use of JQ1 significantly Informed consent has been obtained from all patients (see above). reduced levels of murine IL-6 in the serum [5, 10, 11]. Competing interests Taken together, this suggests that JQ1 is exerting anti- The authors declare that they have no competing interests. MM activity, in part, through a direct effect on BMSC via the inhibition of BMSC IL-6 (and IL-8) synthesis. Publisher’sNote This in turn could explain why BMSC do not appear to Springer Nature remains neutral with regard to jurisdictional claims in offer MM protection from JQ1 therapy. published maps and institutional affiliations. Author details Additional files Department of Molecular Haematology, Norwich Medical School, The University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK. Department of Haematology, Norfolk and Norwich University Hospitals NHS Additional file 1: Figure S1. (A) Relative transcriptional levels of MIF Trust, Colney Lane, Norwich NR4 7UY, UK. expression in B cells, T cells, non-malignant plasma cells and CD138+ purified primary MM cells were normalized to Beta-Actin (n = 5). (B) MIF Received: 21 February 2018 Accepted: 6 May 2018 ELISA data showing the increase in MIF extracellular protein levels in CD138+ purified primary MM cells and MM cell lines in comparison to other cell types (n = 5). Figure S2. (A) MM.1 s cells were transduced with References lentivirus targeted to MIF or control shRNA for 96 h. RNA was extracted 1. Lue H, Kleemann R, Calandra T, Roger T, Bernhagen J. Macrophage and MIF mRNA expression was analyzed via RT-PCR to confirm KD. (B) migration inhibitory factor (MIF): mechanisms of action and role in disease. Cells described in (A) were cultured for 24 h in fresh media after which Microbes Infect. 2002;4(4):449–60. the supernatant was analyzed for MIF via ELISA. (C) MM.1S MIF KD cells 2. O'Reilly C, Doroudian M, Mawhinney L, Donnelly SC. Targeting MIF in were co-cultured with primary BMSC for 96 h, after which MM cells were cancer: therapeutic strategies, current developments, and future analyzed using Cell Titre Glo (CTG), normalized to a ShE control. Figure S3. opportunities. Med Res Rev. 2016;36(3):440–60. PubMed PMID: 26777977. (A) BMSC from MM patients was analyzed for CXCR4, CXCR2 and CD74 Epub 2016/01/19. eng using flow cytometry and compared to isotype control. (B) BMSC were 3. Zheng Y, Wang Q, Li T, Qian J, Lu Y, Li Y, et al. Role of myeloma-derived MIF preincubated with either AMD3100 (10 μM), SB225002 (100 nM) and in myeloma cell adhesion to bone marrow and chemotherapy response. anti-CD74 (10 μg/ml). BMSC were then stimulated with recombinant J Natl Cancer Inst. 2016;108(11):djw131. MIF (100 ng/ml) for 2 h. RNA was extracted and analyzed for IL-6 4. Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, et al. BET and IL-8 expression using Real-time PCR. (PPTX 178 kb) bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell. Additional file 2: Supplementary methods. Methods section. (DOCX 19 kb) 2011;146(6):904–17. PubMed PMID: PMC3187920 5. Mahtouk K, Moreaux J, Hose D, Reme T, Meissner T, Jourdan M, et al. Growth factors in multiple myeloma: a comprehensive analysis of their Abbreviations expression in tumor cells and bone marrow environment using Affymetrix BMSC: Bone marrow stromal cell; IL: Interleukin; MIF: Macrophage migratory microarrays. BMC Cancer. 2010;10:198. PubMed PMID: 20465808. Pubmed inhibitory factor; MM: Multiple myeloma; NSG: NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ Central PMCID: PMC2882921. Epub 2010/05/15. eng 6. Klein B, Zhang X, Lu Z, Bataille R. Interleukin-6 in human multiple myeloma. Acknowledgements Blood. 1995;85(4):863–72. The authors wish to thank The Norwich Research Park (NRP), BBSRC, The 7. Hilbert DM, Kopf M, Mock BA, Kohler G, Rudikoff S. Interleukin 6 is essential National Institutes for Health Research (UK), The Big C, and The Rosetrees for in vivo development of B lineage neoplasms. J Exp Med. 1995;182(1): Trust for funding. Additionally, we are grateful to Professor Richard Ball and 243–8. PubMed PMID: 7790819. Pubmed Central PMCID: PMC2192088. Epub Iain Sheriffs, Norwich tissue bank (UK) for help with sample collection. pCDH- 1995/07/01. eng luciferase-T2A-mCherry was kindly gifted from Prof. Dr. med. Irmela Jeremias, 8. Kline M, Donovan K, Wellik L, Lust C, Jin W, Moon-Tasson L, et al. Cytokine Helmholtz Zentrum München, Munchen, Germany. and chemokine profiles in multiple myeloma; significance of stromal interaction and correlation of IL-8 production with disease progression. Funding Leuk Res. 2007;31(5):591–8. REP receives funding from the Norwich Research Park doctoral training 9. Bendre MS, Montague DC, Peery T, Akel NS, Gaddy D, Suva LJ. Interleukin-8 program partnership, which is supported by the BBSRC. CRM is funded by stimulation of osteoclastogenesis and bone resorption is a mechanism for the Rosetrees Trust, MSS by The Big C, and MJA and KMB are supported by the increased osteolysis of metastatic bone disease. Bone. 2003;33(1):28–37. the Norwich and Norfolk University Hospital. SAR receives funding from the 10. Jourdan M, Mahtouk K, Veyrune J-l, Couderc G, Fiol G, Redal N, et al. University of East Anglia. Delineation of the roles of paracrine and autocrine interleukin-6 (IL-6) in myeloma cell lines in survival versus cell cycle. A possible model for the Availability of data and materials cooperation of myeloma cell growth factors. Eur Cytokine Netw. 2005;16(1): The datasets supporting the conclusions of this article are included within 57–64. this article and additional files. 11. Dankbar B, Padró T, Leo R, Feldmann B, Kropff M, Mesters RM, et al. Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell Authors’ contributions interactions in multiple myeloma. Blood. 2000;95(8):2630–6. REP, AA, KMB and SAR designed the research; REP, MSS and SAR performed the research; REP and CRM carried out in vivo work; MJA and KMB provided
Journal of Hematology & Oncology – Springer Journals
Published: May 16, 2018
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