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Chronic lymphocytic leukemia

Overexpression of CD49d in trisomy 12 chronic lymphocytic leukemia patients is mediated by IRF4 through induction of IKAROS

Leukemia volume 33, pages 1278–1302 (2019)Cite this article

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In chronic lymphocytic leukemia (CLL), homing and retention of leukemic cells inside tissue niches represent crucial steps of disease progression. CLL trafficking from bloodstream into microenvironments is actively coordinated by transient interactions with endothelium through adhesion molecules and chemokines that trigger integrin activation, inducing firm adhesion and transendothelial migration into tissues. This process allows leukemic cells to invade the microenvironmental niches and generate a favorable soil for their survival, proliferation, and clonal evolution. CD49d, the α-chain of α4β1 (VLA-4) integrin heterodimer, has a pivotal role in CLL migration and retention into lymph nodes and bone marrow. VLA-4 mediates CLL interaction with accessory cells and tissue matrix by binding to its ligands vascular cell adhesion molecule-1 (VCAM-1) and fibronectin [1]. CD49d is a strong negative prognosticator in CLL, expressed at high levels in about 40% of CLL patients showing aggressive clinical course and shorter overall survival [2]. Increased levels of CD49d were reported to identify those patients under ibrutinib treatment with reduced lymphocytosis, inferior nodal response, and shorter progression-free survival [3]. Of interest, CD49d is almost universally expressed in CLL cases harboring trisomy 12 aberration (Tri12 CLL). Among the wide clinico-biological heterogeneity of CLL patients, Tri12 CLL represents a distinctive subset (about 15% of cases) characterized by the prevalence of nodal presentation, pronounced lymphadenopathy, and by higher rates of cell proliferation, disease progression, and Richter syndrome transformation [4, 5]. Tri12 CLL cells have an increased ability to migrate towards lymph nodes and reside inside tissue niches [6]. These patients show a significant abbreviated redistribution of B lymphocytes during ibrutinib treatment probably due to increased integrin-mediated signaling [7]. The reason why CLL patients harboring trisomy 12 abnormality are characterized by a more pronounced expression of CD49d and tropism toward lymph node is still under investigation. IRF4 is a member of the IRF family of transcription factors that exerts critical functions in different cell types of the immune system. In mature B cells, different IRF4 concentrations underlie the generation of alternative fate in cells localized in secondary lymphoid organs, promoting at lower levels the formation of germinal center reaction and class switch recombination or, conversely inducing, at higher concentrations, terminal plasma cell differentiation [8]. In CLL setting, a causal relationship between low levels of IRF4 and the development of CLL was demonstrated in two different mouse models, suggesting a possible pathogenic role of IRF4 in CLL [9, 10]. B cells deficient of IRF4 show an enrichment of genes involved in cell migration and homing, in particular of VLA-4 [11].

Given the high expression of CD49d, Tri12 CLL cells were able to adhere more firmly to plate-bound VCAM-1 compared to no Tri12 CLL cells (Fig. 1b). We wondered if low expression of IRF4 can regulate the adhesive properties of Tri12 leukemic cells to microenvironmental cells. Purified Tri12 CLL cells (n = 6) were transfected with an IRF4 plasmid vector or an empty vector pCMV6-entry and allowed to adhere to HS-5 stromal cell layer. As shown in Figure S1, transfection with IRF4 vector determined a significant decrease of Tri12 CLL adhesion to stromal cells (P < 0.01). We linked this reduction of adhesion to a modulation of CD49d expression in CLL cells. After transfection with IRF4 or empty vector, we analyzed the expression of CD49d in Tri12 CLL. As shown in Fig. 1c, the forced expression of IRF4  was able to revert the expression of CD49d inducing its downregulation in all the samples analyzed (P < 0.01). The reduction of CD49d expression induced by restoration of IRF4 levels in Tri12 CLL cells reflected a decreased ability of IRF4-transfected CLL cells to adhere to VCAM-1 (Fig. 1d).

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Acknowledgements

Funding

This work was supported by Associazione Italiana per la Ricerca sul Cancro and Fondazione Cariplo (TRIDEO 16923 to R M), AIRC IG14376 and PRIN 2015 ZMRFEA_002 (to RM). SF was supported by FIRC/AIRC Triennal Fellowship 16430, Milan, Italy and now by Fondazione Umberto Veronesi, Milan, Italy; Ricerca Finalizzata Giovani Ricercatori 2011–2012, Ministero della Salute (GR-2011-02349282 to RM) (GR-2011-02346826 to AZ), Rome, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

RM and SF conceived the research, coordinated the research, and interpreted the results; SF analyzed the results and wrote the manuscript; SF performed the in vitro experiments; SF acquired and analyzed flow cytometric data; SB and PZ performed some molecular analyses; SF performed the statistical analyses; AZ provided protein samples for some western blot evaluation; RM supervised the workflow, and revised the results and the manuscript critically. VG, ML, and RM revised and approved the final version of the paper.

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  1. These authors contributed equally: Roberto Marasca, Rossana Maffei

Authors and Affiliations

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

    Stefania Fiorcari, Stefania Benatti, Patrizia Zucchini, Mario Luppi, Roberto Marasca & Rossana Maffei

  2. Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy

    Antonella Zucchetto & Valter Gattei

  3. Hematology Unit, Azienda Ospedaliero-Universitaria of Modena-Policlinico, Modena, Italy

    Rossana Maffei

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  1. Stefania Fiorcari
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Fiorcari, S., Benatti, S., Zucchetto, A. et al. Overexpression of CD49d in trisomy 12 chronic lymphocytic leukemia patients is mediated by IRF4 through induction of IKAROS. Leukemia 33, 1278–1302 (2019). https://doi.org/10.1038/s41375-018-0296-5

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