P623: IBRUTINIB REGULATES MIR-181A/B VIA C-FOS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Abstract

Background: B-cell receptor (BCR) signalling controls B-cell survival and proliferation, and its deregulation has a pathogenic role in Chronic Lymphocytic Leukemia (CLL), a B-cell malignancy. Currently, several BCR signalling inhibitors are used in therapy, among them the Ibrutinib. We previously reported that the BCR-signalling inhibition mediated by Ibrutinib leads to miR-181a/b activation; those miRNAs are significant biomarkers of CLL progression, and their downregulation is associated to a poor prognosis; furthermore, low levels of miR-181a/b impair several anti-apoptotic proteins, contributing to cell survival. BCR signalling also controls c-Fos/AP-1 pathway in pre-B cells. Recently, we suggested the involvement of c-Fos in miR-181b expression, however the mechanism by which this regulation is exerted is still not explored. Moreover, the involvement of c-Fos in miR-181a/b transcription during Ibrutinib is still not clear. Aims: Assess the impact of Ibrutinib on c-Fos expression; assess the involvement of the transcription factor c-Fos in miR-181a/b transcription; assess the impact of miR-181a/b in CLL cells survival during Ibrutinib treatment. Methods: To assess the impact of Ibrutinb on c-Fos transcription, we evaluated its expression in CLL cells treated with Ibrutinib or vehicle. To assess the involvement of c-Fos in miR-181a/b transcription, we evaluated i) the expression of the miRs and of their primary transcripts in CLL cells overexpressing c-Fos, and ii) the ability of c-Fos to bind miR-181a/b host gene promoter by luciferase, ChIP and EMSA assays. To assess the impact of the miRs on CLL cells survival during Ibrutinib treatment, we performed MTT assay in CLL cells after miR-181a/b silencing and Ibrutinib treatment. Results: Considering that c-Fos was found to be controlled by BCR signalling in pre-B cells, we evaluated whether BCR signalling affect c-Fos also in CLL cells: we found that c-Fos expression increases in primary CLL cells after Ibrutinib treatment. Since miR-181a/b was up-regulated after Ibrutinib treatment in vitro and in vivo in CLL cells, and considering that a possible involvement of c-Fos in miR-181b expression was already suggested, we sought to identify if c-Fos was a direct regulator of miR-181a/b expression. We confirmed that c-Fos overexpression affects miR-181b levels in CLL patients, but not miR-181a; however, the dysregulation of the miR shows heterogeneity among patients. Indeed miR-181b expression increases in 3 and decreases in 2 of 7 patients. To better understand how c-Fos impact on miRs expression, we analysed both miR-181a and miR-181b at the transcriptional level in CLL and lymphoblastoid cell lines, finding that c-Fos negatively regulates the pri-miR-181a/b. To clarify the mechanism by which c-Fos regulates miR-181a/b transcription, we analysed the miRs host gene (MIR181A2HG) promoter. Since MIR181A2HG promoter shows two consensus sequences for the transcription factor c-Fos, we studied whether c-Fos binds these regions. We found that c-Fos binds at MIR181A2HG promoter by recognizing the two consensus sequences. Accordingly, after their deletion, the ability of c-Fos to recognize the promoter was overcome. Finally, since miR-181a and miR-181b are involved in cell death, we assessed whether those miRNAs participate in CLL cells death induced by Ibrutinib: we found that the silencing of both miRNAs improves CLL cell survival during Ibrutinib treatment. Summary/Conclusion: c-Fos is a downstream target of BCR signalling and a direct regulator of miR-181a/b transcription in CLL. MiR-181a/b are effectors of Ibrutinib-induced CLL cells death.