Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chronic lymphocytic leukemia

IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK

Leukemia volume 35, pages 1330–1343 (2021)Cite this article

Subjects

Abstract

Interferon regulatory factor 4 (IRF4) is a transcriptional regulator of immune system development and function. Here, we investigated the role of IRF4 in controlling responsiveness to B-cell receptor (BCR) stimulation in chronic lymphocytic leukemia (CLL). We modulated IRF4 levels by transfecting CLL cells with an IRF4 vector or by silencing using small-interfering RNAs. Higher IRF4 levels attenuated BCR signaling by reducing AKT and ERK phosphorylation and calcium release. Conversely, IRF4 reduction improved the strength of the intracellular cascade activated by BCR engagement. Our results also indicated that IRF4 negatively regulates the expression of the spleen tyrosine kinase SYK, a crucial protein for propagation of BCR signaling, and the zinc finger DNA-binding protein IKAROS. We modulated IKAROS protein levels both by genetic manipulation and pharmacologically by treating CLL cells with lenalidomide and avadomide (IMIDs). IKAROS promoted BCR signaling by reducing the expression of inositol 5-phosphatase SHIP1. Lastly, IMIDs induced IRF4 expression, while down-regulating IKAROS and interfered with survival advantage mediated by BCR triggering, also in combination with ibrutinib. Overall, our findings elucidate the mechanism by which IRF4 tunes BCR signaling in CLL cells. Low IRF4 levels allow an efficient transmission of BCR signal throughout the accumulation of SYK and IKAROS.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Low IRF4 levels allow an efficient intracellular transmission of BCR signaling.
Fig. 2: IRF4 modulates the expression of SYK and IKAROS protein.
Fig. 3: Accumulation of IKAROS in CLL cells is regulated by IRF4 and promotes BCR signaling.
Fig. 4: IKAROS tunes BCR activation by regulating SHIP1.
Fig. 5: IMIDs up-regulate IRF4 expression in CLL cells.
Fig. 6: Lenalidomide induces IRF4 expression by promoting IFN-γ secretion.
Fig. 7: IMIDs interfere with BCR-mediated CLL survival, acting also in combination with ibrutinib.
Fig. 8: Graphical representation of IRF4 control of BCR signaling.

Similar content being viewed by others

References

  1. Lu R. Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol. 2008;29:487–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ma S, Pathak S, Trinh L, Lu R. Interferon regulatory factors 4 and 8 induce the expression of Ikaros and Aiolos to down-regulate pre-B-cell receptor and promote cell-cycle withdrawal in pre-B-cell development. Blood. 2008;111:1396–403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ochiai K, Maienschein-Cline M, Simonetti G, Chen J, Rosenthal R, Brink R, et al. Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4. Immunity. 2013;38:918–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sciammas R, Shaffer AL, Schatz JH, Zhao H, Staudt LM, Singh H. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity. 2006;25:225–36.

    Article  CAS  PubMed  Google Scholar 

  5. Di Bernardo MC, Crowther-Swanepoel D, Broderick P, Webb E, Sellick G, Wild R, et al. A genome-wide association study identifies six susceptibility loci for chronic lymphocytic leukemia. Nat Genet. 2008;40:1204–10.

    Article  PubMed  Google Scholar 

  6. Crowther-Swanepoel D, Broderick P, Ma Y, Robertson L, Pittman AM, Price A, et al. Fine-scale mapping of the 6p25.3 chronic lymphocytic leukaemia susceptibility locus. Hum Mol Genet. 2010;19:1840–5.

    Article  CAS  PubMed  Google Scholar 

  7. Havelange V, Pekarsky Y, Nakamura T, Palamarchuk A, Alder H, Rassenti L, et al. IRF4 mutations in chronic lymphocytic leukemia. Blood. 2011;118:2827–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, Bahlo J, et al. Mutations driving CLL and their evolution in progression and relapse. Nature. 2015;526:525–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Puente XS, Beà S, Valdés-Mas R, Villamor N, Gutiérrez-Abril J, Martín-Subero JI, et al. Non-coding recurrent mutations in chronic lymphocytic leukaemia. Nature. 2015;526:519–24.

    Article  CAS  PubMed  Google Scholar 

  10. Nadeu F, Clot G, Delgado J, Martín-García D, Baumann T, Salaverria I, et al. Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia. Leukemia. 2018;32:645–53.

    Article  CAS  PubMed  Google Scholar 

  11. Chang C-C, Lorek J, Sabath DE, Li Y, Chitambar CR, Logan B, et al. Expression of MUM1/IRF4 correlates with clinical outcome in patients with B-cell chronic lymphocytic leukemia. Blood. 2002;100:4671–5.

    Article  CAS  PubMed  Google Scholar 

  12. Asslaber D, Qi Y, Maeding N, Steiner M, Denk U, Höpner JP, et al. B-cell specific IRF4 deletion accelerates Chronic Lymphocytic Leukemia development by enhanced tumor immune evasion. Blood. 2019;134:1717–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Shukla V, Ma S, Hardy RR, Joshi SS, Lu R. A role for IRF4 in the development of CLL. Blood. 2013;122:2848–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ma S, Shukla V, Fang L, Gould KA, Joshi SS, Lu R. Accelerated development of chronic lymphocytic leukemia in New Zealand Black mice expressing a low level of interferon regulatory factor 4. J Biol Chem. 2013;288:26430–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhong Y, Byrd JC. IRF4(−/−)Vh11 mice: a novel mouse model of CLL. Blood. 2013;122:2769–70.

    Article  CAS  PubMed  Google Scholar 

  16. Ten Hacken E, Burger JA. Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: Implications for disease pathogenesis and treatment. Biochim Biophys Acta. 2016;1863:401–13.

    Article  PubMed  Google Scholar 

  17. Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2011;118:4313–20.

    Article  CAS  PubMed  Google Scholar 

  18. Wiestner A. BCR pathway inhibition as therapy for chronic lymphocytic leukemia and lymphoplasmacytic lymphoma. Hematol Educ Program Am Soc Hematol Am Soc Hematol Educ Program. 2014;2014:125–34.

    Article  Google Scholar 

  19. Burger JA, Tedeschi A, Barr PM, Robak T, Owen C, Ghia P, et al. Ibrutinib as initial therapy for patients with chronic lymphocytic leukemia. N. Engl J Med. 2015;373:2425–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. O’Brien S, Jones JA, Coutre SE, Mato AR, Hillmen P, Tam C, et al. Ibrutinib for patients with relapsed or refractory chronic lymphocytic leukaemia with 17p deletion (RESONATE-17): a phase 2, open-label, multicentre study. Lancet Oncol. 2016;17:1409–18.

    Article  PubMed  Google Scholar 

  21. Byrd JC, Brown JR, O’Brien S, Barrientos JC, Kay NE, Reddy NM, et al. Ibrutinib versus ofatumumab in previously treated chronic lymphoid leukemia. N. Engl J Med. 2014;371:213–23.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Seiffert M, Stilgenbauer S, Döhner H, Lichter P. Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids. Leukemia. 2007;21:1977–83.

    Article  CAS  PubMed  Google Scholar 

  23. Maffei R, Fiorcari S, Vaisitti T, Martinelli S, Benatti S, Debbia G, et al. Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia. Oncotarget. 2017;8:90013–27.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Schwickert TA, Tagoh H, Gültekin S, Dakic A, Axelsson E, Minnich M, et al. Stage-specific control of early B cell development by the transcription factor Ikaros. Nat Immunol. 2014;15:283–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nera K-P, Alinikula J, Terho P, Narvi E, Törnquist K, Kurosaki T, et al. Ikaros has a crucial role in regulation of B cell receptor signaling. Eur J Immunol. 2006;36:516–25.

    Article  CAS  PubMed  Google Scholar 

  26. Uckun FM, Ma H, Zhang J, Ozer Z, Dovat S, Mao C, et al. Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros. Proc Natl Acad Sci USA. 2012;109:18072–7.

    Article  CAS  PubMed  Google Scholar 

  27. Alinikula J, Kohonen P, Nera K-P, Lassila O. Concerted action of Helios and Ikaros controls the expression of the inositol 5-phosphatase SHIP. Eur J Immunol. 2010;40:2599–607.

    Article  CAS  PubMed  Google Scholar 

  28. Rao N, Ghosh AK, Ota S, Zhou P, Reddi AL, Hakezi K, et al. The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation. EMBO J. 2001;20:7085–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Crowther-Swanepoel D, Corre T, Lloyd A, Gaidano G, Olver B, Bennett FL, et al. Inherited genetic susceptibility to monoclonal B-cell lymphocytosis. Blood. 2010;116:5957–60.

    Article  CAS  PubMed  Google Scholar 

  30. Budzyńska PM, Niemelä M, Sarapulov AV, Kyläniemi MK, Nera K-P, Junttila S, et al. IRF4 deficiency leads to altered BCR signalling revealed by enhanced PI3K pathway, decreased ship expression and defected cytoskeletal responses. Scand J Immunol. 2015;82:418–28.

    Article  PubMed  Google Scholar 

  31. Hoellenriegel J, Coffey GP, Sinha U, Pandey A, Sivina M, Ferrajoli A, et al. Selective, novel spleen tyrosine kinase (Syk) inhibitors suppress chronic lymphocytic leukemia B-cell activation and migration. Leukemia. 2012;26:1576–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yoshida T, Georgopoulos K. Ikaros fingers on lymphocyte differentiation. Int J Hematol. 2014;100:220–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Martinelli G, Iacobucci I, Storlazzi CT, Vignetti M, Paoloni F, Cilloni D, et al. IKZF1 (Ikaros) deletions in BCR-ABL1-positive acute lymphoblastic leukemia are associated with short disease-free survival and high rate of cumulative incidence of relapse: a GIMEMA AL WP report. J Clin Oncol J Am Soc Clin Oncol. 2009;27:5202–7.

    Article  CAS  Google Scholar 

  34. Iacobucci I, Iraci N, Messina M, Lonetti A, Chiaretti S, Valli E, et al. IKAROS deletions dictate a unique gene expression signature in patients with adult B-cell acute lymphoblastic leukemia. PLoS ONE 2012;7:e40934.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, et al. BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature. 2008;453:110–4.

    Article  CAS  PubMed  Google Scholar 

  36. Theocharides APA, Dobson SM, Laurenti E, Notta F, Voisin V, Cheng P-Y, et al. Dominant-negative Ikaros cooperates with BCR-ABL1 to induce human acute myeloid leukemia in xenografts. Leukemia. 2015;29:177–87.

    Article  CAS  PubMed  Google Scholar 

  37. Joshi I, Yoshida T, Jena N, Qi X, Zhang J, Van Etten RA, et al. Loss of Ikaros DNA-binding function confers integrin-dependent survival on pre-B cells and progression to acute lymphoblastic leukemia. Nat Immunol. 2014;15:294–304.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Fecteau J-F, Corral LG, Ghia EM, Gaidarova S, Futalan D, Bharati IS, et al. Lenalidomide inhibits the proliferation of CLL cells via a cereblon/p21(WAF1/Cip1)-dependent mechanism independent of functional p53. Blood. 2014;124:1637–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Krönke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343:301–5.

    Article  PubMed  Google Scholar 

  40. Fiorcari S, Benatti S, Zucchetto A, Zucchini P, Gattei V, Luppi M, et al. Overexpression of CD49d in trisomy 12 chronic lymphocytic leukemia patients is mediated by IRF4 through induction of IKAROS. Leukemia. 2019;33:1278–302.

    Article  PubMed  Google Scholar 

  41. Capece D, Zazzeroni F, Mancarelli MM, Verzella D, Fischietti M, Di Tommaso A, et al. A novel, non-canonical splice variant of the Ikaros gene is aberrantly expressed in B-cell lymphoproliferative disorders. PLoS ONE. 2013;8:e68080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Oliveira VC, de, Lacerda MP, de, Moraes BBM, Gomes CP, Maricato JT, Souza OF, et al. Deregulation of Ikaros expression in B-1 cells: New insights in the malignant transformation to chronic lymphocytic leukemia. J Leukoc Biol. 2019;106:581–94.

    Article  CAS  PubMed  Google Scholar 

  43. Thien CB, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol. 2001;2:294–307.

    Article  CAS  PubMed  Google Scholar 

  44. Hagner PR, Man H-W, Fontanillo C, Wang M, Couto S, Breider M, et al. CC-122, a pleiotropic pathway modifier, mimics an interferon response and has antitumor activity in DLBCL. Blood. 2015;126:779–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhang L-H, Kosek J, Wang M, Heise C, Schafer PH, Chopra R. Lenalidomide efficacy in activated B-cell-like subtype diffuse large B-cell lymphoma is dependent upon IRF4 and cereblon expression. Br J Haematol. 2013;160:487–502.

    Article  CAS  PubMed  Google Scholar 

  46. Maffei R, Colaci E, Fiorcari S, Martinelli S, Potenza L, Luppi M, et al. Lenalidomide in chronic lymphocytic leukemia: the present and future in the era of tyrosine kinase inhibitors. Crit Rev Oncol Hematol. 2016;97:291–302.

    Article  PubMed  Google Scholar 

  47. Duckworth A, Glenn M, Slupsky JR, Packham G, Kalakonda N. Variable induction of PRDM1 and differentiation in chronic lymphocytic leukemia is associated with anergy. Blood. 2014;123:3277–85.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) and Fondazione Cariplo (FC) (TRIDEO 16923 R.Maf., AIRC IG14376 R.Mar. FIRC/AIRC Triennal Fellowship 16430 S.F.), and Fondazione Umberto Veronesi (S.F.); Ricerca Finalizzata Giovani Ricercatori 2011–2012, Ministero della Salute (GR-2011-02349282- R.Maf.), Italy; PRIN 2015 ZMRFEA_002 (R.Mar), MIUR, Italy. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Hematology Unit, Department of Medical and Surgical Sciences, University of Modena and Reggio Emilia, Modena, Italy

    Rossana Maffei, Stefania Fiorcari, Stefania Benatti, Claudio Giacinto Atene, Silvia Martinelli, Patrizia Zucchini, Leonardo Potenza, Mario Luppi & Roberto Marasca

  2. Hematology Unit, Department of Oncology and Hematology, A.O.U of Modena, Policlinico, Modena, Italy

    Rossana Maffei

Authors
  1. Rossana Maffei
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefania Fiorcari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefania Benatti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudio Giacinto Atene
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Silvia Martinelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrizia Zucchini
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Leonardo Potenza
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Mario Luppi
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Roberto Marasca
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.Maf. conceived the research, coordinated the research, performed the statistical analyses and interpreted the results; R.Maf. performed the in vitro experiments; S.F. acquired and analyzed flow cytometric data; S.B performed molecular analyses; C.G.A. contributed to ChIP experiments and western blot analyses; S.M. and P.Z. managed the biological samples; R.Maf supervised the work-flow and wrote the manuscript; L.P., M.L. and R.Mar. revised and approved the final version of the manuscript.

Corresponding author

Correspondence to Rossana Maffei.

Ethics declarations

Conflict of interest

R.Mar. received research funding from Janssen and Gilead Sci and honoraria from Gilead Sci., Janssen, Abbvie, Roche and Shire. M.L received honoraria from Gilead Sci., MSD, Pfizer. R.Maf. has received a speaker fee from Abbvie. Other Authors declare that they have no competing interests. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maffei, R., Fiorcari, S., Benatti, S. et al. IRF4 modulates the response to BCR activation in chronic lymphocytic leukemia regulating IKAROS and SYK. Leukemia 35, 1330–1343 (2021). https://doi.org/10.1038/s41375-021-01178-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-021-01178-5

This article is cited by

Search

Advanced search

Quick links