Interferon Regulatory Factor 4 Orchestrates the Response to B-Cell Receptor in Chronic Lymphocytic Leukemia Cells By Regulating Ikaros

Abstract

Chronic lymphocytic leukemia (CLL) accounts for about 30% of all adult leukemias and is the most common hematologic malignancy in the Western countries. Interferon regulatory factor 4 (IRF4, also known as MUM1) is a transcriptional regulator of immune system development and function. Different IRF4 concentrations underlie the generation of alternative cell fate in mature B cells localized in secondary lymphoid organs, i.e. IRF4 “kinetic control” model. IRF4 at lower levels promotes germinal center formation and class switch recombination, whereas at higher levels IRF4 inhibits Bcl-6 and induces Blimp-1 to facilitate plasma cell development. Recent studies suggest a possible role of IRF4 in CLL pathogenesis. In New Zealand Black (NZB) IRF4+/- mouse model, CLL development is dramatically accelerated and IRF4+/- CLL cells showed hyper-responsiveness to B-cell receptor (BCR) activation.In the present study, we investigated the role of IRF4 in controlling CLL responsiveness to BCR stimulation.We first inspected IRF4 expression in a cohort of 225 CLL, finding reduced levels of expression in almost all cases. Then, we modulated IRF4 levels in vitro in leukemic cells collected from CLL patients by transfecting cells using Human IRF4 pCMV6-Entry vector or by silencing with siRNA strategy. CLL cells were then stimulated with F(AB')2 fragment to human IgM (5FCµ). Higher levels of IRF4 attenuated BCR signaling as assessed by a reduction in AKT and ERK phosphorylation and calcium release. Conversely, IRF4 silencing improved CLL responsiveness to BCR stimulation. The expression levels of IgM, CD79A and CD79B were not affected by IRF4 modulation. Ikaros (IKZF1), a zinc finger, DNA-binding protein that regulates gene transcription and chromatin remodeling, is a master regulator of lymphocyte development. Our results indicate that IRF4 induction in CLL cells down-regulates IKZF1 levels throughout post-transcriptional mechanisms. We wondered if IKZF1 may mediate BCR activation in CLL cells. To demonstrate this hypothesis, we interfered with IKZF1 by silencing strategies and pharmacologically by treating CLL cells with IMIDs, lenalidomide and avadomide (CC-122). We found that IKZF1 down-regulation attenuates BCR signaling by inducing the expression of the inositol 5-phosphatase SHIP, which dephosphorylates PIP3 blocking AKT activation. Concomitantly, IRF4 up-regulation promotes ZAP70 degradation. These effects were also obtained by treating CLL cells with both lenalidomide and avadomide, which up-regulated IRF4 expression while reducing IKZF1 levels. Both IMIDs interfered with BCR response and mediated apoptosis in CLL cells in combination with ibrutinib.Overall, the results elucidate the mechanism by which IRF4 regulates BCR responsiveness in CLL cells. Low IRF4 levels maintains a permissive state in CLL cells, allowing an effective transmission of BCR signals throughout the accumulation of IKZF1. DisclosuresNo relevant conflicts of interest to declare.