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.
Subscribe to Journal
Get full journal access for 1 year
only $41.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Lu R. Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol. 2008;29:487–92.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Shukla V, Ma S, Hardy RR, Joshi SS, Lu R. A role for IRF4 in the development of CLL. Blood. 2013;122:2848–55.
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.
Zhong Y, Byrd JC. IRF4(−/−)Vh11 mice: a novel mouse model of CLL. Blood. 2013;122:2769–70.
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.
Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia. Blood. 2011;118:4313–20.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Yoshida T, Georgopoulos K. Ikaros fingers on lymphocyte differentiation. Int J Hematol. 2014;100:220–9.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Thien CB, Langdon WY. Cbl: many adaptations to regulate protein tyrosine kinases. Nat Rev Mol Cell Biol. 2001;2:294–307.
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.
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.
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.
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.
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.
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.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
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 (2021). https://doi.org/10.1038/s41375-021-01178-5