MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

REGULAR ARTICLE MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells Serena Matis,1, * Anna Grazia Recchia,2,3, * Monica Colombo,1 Martina Cardillo,1,4 Marina Fabbi,5 Katia Todoerti,6 Sabrina Bossio,2,3 Sonia Fabris,6 Valeria Cancila,7 Rosanna Massara,1 Daniele Reverberi,1 Laura Emionite,8 Michele Cilli,8 Giannamaria Cerruti,9 Sandra Salvi,10 Paola Bet,10 Simona Pigozzi,10,11 Roberto Fiocca,10,11 Adalberto Ibatici,12 Emanuele Angelucci,12 Massimo Gentile,2,3 Paola Monti,13 Paola Menichini,13 Gilberto Fronza,13 Federica Torricelli,14 Alessia Ciarrocchi,14 Antonino Neri,15 Franco Fais,1,4 Claudio Tripodo,7 Fortunato Morabito,3,16,† Manlio Ferrarini,4,† and Giovanna Cutrona1,† 1 Molecular Pathology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 2 Hematology Unit AO of Cosenza, Cosenza, Italy; 3 Biothecnology Research Unit, AO, Cosenza, Italy; 4 Department of Experimental Medicine, University of Genoa, Genoa, Italy; 5 Biotherapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 6 Hematology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 7 Tumor Immunology Unit, Department of Health Sciences, University of Palermo School of Medicine, Palermo, Italy; 8 Animal Facility, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 9 Molecular Diagnostic Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 10 Pathology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 11 Department of Surgical and Diagnostic Sciences (DISC), University of Genoa, Genoa, Italy; 12 Hematology Unit and Transplant Center, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 13 Mutagenesis and Cancer Prevention Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy; 14 Laboratory of Translational Research, Azienda USL IRCCS di Reggio Emilia, Reggio Emilia, Italy; 15 Scientific Directorate, Azienda USL IRCCS di Reggio Emilia, Reggio Emilia, Italy; and 16 Hematology and Bone Marrow Transplant Unit, Hemato-Oncology Department, Augusta Victoria Hospital, East Jerusalem, Israel Key Points • • Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rβ1 chain requires cell stimulation via Low concentrations of miR-146b-5p have an adverse prognostic impact in CLL patients. miR-146b-5p controls IL-23 stimulation of CLL cells by negatively regulating the expression of the IL-12Rβ1 chain of the IL-23R complex. surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rβ1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3′ -UTR region of the IL-12Rβ1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rβ1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rβ1–positive CLL cells in the spleen. Our findings indicate that IL-12Rβ1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540. Submitted 9 July 2021; accepted 30 June 2022; prepublished online on Blood Advances First Edition 12 July 2022; final version published online xxx 2022. https:// doi.org/10.1182/bloodadvances.2021005726. *S.M. and A.G.R. are joint first authors. †F.M., M. Ferrarini, and G.C. are joint last authors. The data are deposited at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/) and 25 OCTOBER 2022 • VOLUME 6, NUMBER 20 are accessible through GEO Series accession number GSE40533. Contact the corresponding author for other forms of data sharing: . The full-text version of this article contains a data supplement. © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. 5593 Introduction different from TRAF6 (tumor necrosis factor receptor-associated factor 6) and IRAK1 control. MicroRNAs (miRNAs) represent a family of noncoding RNAs that prevent the translation and promote the degradation of specific mRNAs by binding to their 3′ -UTR.1,2 Several miRNAs have been implicated in the pathogenesis of chronic lymphocytic leukemia (CLL),3-5 a disease characterized by the accumulation of monoclonal CD5+CD19+ B cells in lymphoid organs and blood.6-9 In patients with 13q deletions (del[13q]), the most common cytogenetic lesion of CLL,10,11 the genes encoding the miR-15a/miR-16-1 cluster are targeted by the deletion.3,12-15 The downregulation of these regulatory miRNAs can lead to an increased expression of antiapoptotic molecules, which facilitate clonal expansion, inducing further transforming events.12-16 MiRNA expression profile studies have disclosed correlations between certain miRNA signatures and cytogenetic features and/or IGHV gene mutational status,17-19 which represent recognized prognostic markers of CLL. Finally, certain miRNA signatures are associated with disease progression and outcome4,17,20-22 or with the onset of a Richter transformation,23-25 a deadly condition characterized by the development of an aggressive lymphoma in CLL patients.7,26 Considerable evidence indicates that CLL clonal expansion is promoted by interactions with cells and cytokines from the microenvironment.46,47 Moreover, both miR-146a-5p and miR-146b-5p can regulate the release of and the response to cytokines.41,48,49 Based on these considerations, we hypothesized that miR-146b-5p was involved in the regulation of the interactions between CLL cells and the microenvironment. We focused on IL-23,50 a cytokine of the IL12 cytokine family, released primarily by dendritic cells, which is capable of driving T helper (Th) cell differentiation toward the Th17 cell subset.51 In a previous study, we found that IL-23 is instrumental in promotin (...truncated)