Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

IRF4 expression is low in Philadelphia negative myeloproliferative neoplasms and is associated with a worse prognosis

Interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various hematologic malignancies. Its expression has been related to the negative regulation of myeloid-derived suppressor cells (MDSCs) and the polarization of anti-inflammatory M2 macrophages, thereby altering immunosurveillance and inflammatory mechanisms. An abnormal inflammatory status in the bone marrow microenvironment of myeloproliferative neoplasms (MPNs) has recently been demonstrated; moreover, in chronic myeloid leukemia a downregulated expression of IRF4 has been found. In this context, we evaluated the IRF4 expression in 119 newly diagnosed consecutive Philadelphia negative MPNs (Ph- MPNs), showing a low expression among the MPNs phenotypes with a more significant decrease in primary myelofibrosis patients. Lower IRF4 levels were associated with JAK2 + and triple negatives cases carrying the worst prognosis. Furthermore, the IRF4 levels were related to leukemic transformation and a shorter leukemia-free survival; moreover, the risk of myelofibrosis transformation in polycythemia vera and essential thrombocythemia patients was more frequent in cases with lower IRF4 levels. Overall, our study demonstrates an IRF4 dysregulated expression in MPNs patients and its association with a worse prognosis. Further studies could validate these data, to improve our knowledge of the MPNs pathogenesis and confirm the IRF4 role as a new prognostic factor.

Phosphorus and sulfur SAD phasing of the nucleic acid-bound DNA-binding domain of interferon regulatory factor 4

Pivotal to the regulation of key cellular processes such as the transcription, replication and repair of DNA, DNA-binding proteins play vital roles in all aspects of genetic activity. The determination of high-quality structures of DNA-binding proteins, particularly those in complexes with DNA, provides crucial insights into the understanding of these processes. The presence in such complexes of phosphate-rich oligonucleotides offers the choice of a rapid method for the routine solution of DNA-binding proteins through the use of long-wavelength beamlines such as I23 at Diamond Light Source. This article reports the use of native intrinsic phosphorus and sulfur single-wavelength anomalous dispersion methods to solve the complex of the DNA-binding domain (DBD) of interferon regulatory factor 4 (IRF4) bound to its interferon-stimulated response element (ISRE). The structure unexpectedly shows three molecules of the IRF4 DBD bound to one ISRE. The sole reliance on native intrinsic anomalous scattering elements that belong to DNA–protein complexes renders the method of general applicability to a large number of such protein complexes that cannot be solved by molecular replacement or by other phasing methods.

Interferon Regulatory Factor 4 Correlated With Immune Cells Infiltration Could Predict Prognosis for Patients With Lung Adenocarcinoma

BackgroundImmune related interferon regulatory factor 4 (IRF4) is a member of the IRF family, whereas the clinical significance and possible role of IRF4 in lung adenocarcinoma (LUAD) remains unclear. We aimed to investigate the role of IRF4 in predicting the prognosis of LUAD patients.MethodsUsing The Cancer Genome Atlas (TCGA) database and our immunohistochemical (IHC) cohort, we analyzed the correlation between IRF4 expression and clinical characteristics, and the prognostic value of IRF4 was also evaluated in LUAD. The potential biological functions of IRF4 in LUAD were analyzed by Gene Set Enrichment Analysis (GSEA). The relationship between IRF4 and immune cell infiltration were evaluated by TISIDB database and our own IHC cohort. In addition, an immune checkpoint inhibitor (ICI) treated cohort from Gene Expression Omnibus database was used to determine the role of IRF4 in LUAD patients with immunotherapy.ResultsWe found that either mRNA or protein expression level of IRF4 was significantly higher in LUAD than in normal tissues (P < 0.001). The elevate in IRF4 expression in LUAD was significantly associated with the earlier clinical stage (P = 0.002). Patients with LUAD and IRF4 high expression correlated with significant longer overall survival in both TCGA database (P < 0.05) and our IHC-cohort (P = 0.001). Our results also demonstrated that IRF4 could serve as an independent favorable prognostic factor in patients with LUAD. GSEA analysis indicated that high IRF4 expression group enriched with several immune-related pathways, such as B cell receptor signaling pathway, T cell receptor signaling pathway and cytokine-cytokine receptor interaction signaling pathway. In LUAD, IRF4 positively correlated with several different immune infiltrations including various B cells, CD8+ T cells and CD4+ T cells both in mRNA and protein levels. Additionally, we found that the expression of IRF4 was positively associated with PD-1 and PD-L1 mRNA expression levels, and IRF4 high expression predicted moderate better survival in LUAD with immunotherapy (P = 0.071).ConclusionsOur results suggested that IRF4 was associated with higher B cells and T cells infiltration levels and might be a favorable prognostic biomarker in LUAD patients, whereas the potential prognostic role of IRF4 in ICI-treated patients needed further exploration.

The epigenetic pioneer EGR2 initiates DNA demethylation in differentiating monocytes at both stable and transient binding sites

The differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.

Interferon Regulatory Factor 4 Regulates the Development of Polymorphonuclear Myeloid-Derived Suppressor Cells Through the Transcription of c-Myc in Cancer

The accumulation of myeloid-derived suppressor cells (MDSCs) is one of the major obstacles to achieve an appropriate anti-tumor immune response and successful tumor immunotherapy. MDSCs in tumor-bearing hosts are primarily polymorphonuclear (PMN-MDSCs). However, the mechanisms regulating the development of MDSCs remain poorly understood. In this report, we showed that interferon regulatory factor 4 (IRF4) plays a key role in the development of PMN-MDSCs, but not monocytic MDSCs. IRF4 deficiency caused a significant elevation of PMN-MDSCs and enhanced the suppressive activity of PMN-MDSCs, increasing tumor growth and metastasis in mice. Mechanistic studies showed that c-Myc was up-regulated by the IRF4 protein. Over-expression of c-Myc almost abrogated the effects of IRF4 deletion on PMN-MDSCs development. Importantly, the IRF4 expression level was negatively correlated with the PMN-MDSCs frequency and tumor development but positively correlated with c-Myc expression in clinical cancer patients. In summary, this study demonstrated that IRF4 represents a novel regulator of PMN-MDSCs development in cancer, which may have predictive value for tumor progression.

Structural determinants of the IRF4/DNA homodimeric complex

Interferon regulatory factor 4 (IRF4) is a key transcription factor (TF) in the regulation of immune cells, including B and T cells. It acts by binding DNA as both a homodimer and, in conjunction with other TFs, as a heterodimer. The choice of homo and heterodimeric/ DNA interactions is a critical aspect in the control of the transcriptional program and cell fate outcome. To characterize the nature of this interaction in the homodimeric complex, we have determined the crystal structure of the IRF4/ISRE homodimeric complex. We show that the complex formation is aided by a substantial DNA deformation with co-operative binding achieved exclusively through protein–DNA contact. This markedly contrasts with the heterodimeric form where DNA bound IRF4 is shown to physically interact with PU.1 TF to engage EICE1. We also show that the hotspot residues (Arg98, Cys99 and Asn102) contact both consensus and non-consensus sequences with the L1 loop exhibiting marked flexibility. Additionally, we identified that IRF4L116R, a mutant associated with chronic lymphocytic leukemia, binds more robustly to DNA thereby providing a rationale for the observed gain of function. Together, we demonstrate key structural differences between IRF4 homo and heterodimeric complexes, thereby providing molecular insights into IRF4-mediated transcriptional regulation.

Th17 Cell-Mediated Colitis Is Positively Regulated by Interferon Regulatory Factor 4 in a T Cell-Extrinsic Manner

Inflammatory bowel diseases (IBDs) are characterized by chronic, inflammatory gastrointestinal lesions and often require life-long treatment with immunosuppressants and repetitive surgical interventions. Despite progress in respect to the characterization of molecular mechanisms e.g. exerted by TNF-alpha, currently clinically approved therapeutics fail to provide long-term disease control for most patients. The transcription factor interferon regulatory factor 4 (IRF4) has been shown to play important developmental as well as functional roles within multiple immune cells. In the context of colitis, a T cell-intrinsic role of IRF4 in driving immune-mediated gut pathology is established. Here, we conversely addressed the impact of IRF4 inactivation in non-T cells on T cell driven colitis in vivo. Employing the CD4+CD25− naïve T cell transfer model, we found that T cells fail to elicit colitis in IRF4-deficient compared to IRF4-proficient Rag1−/− mice. Reduced colitis activity in the absence of IRF4 was accompanied by hampered T cell expansion both within the mesenteric lymph node (MLN) and colonic lamina propria (cLP). Furthermore, the influx of various myeloids, presumably inflammation-promoting cells was abrogated overall leading to a less disrupted intestinal barrier. Mechanistically, gene profiling experiments revealed a Th17 response dominated molecular expression signature in colon tissues of IRF4-proficient, colitic Rag1−/− but not in colitis-protected Rag1−/−Irf4−/− mice. Colitis mitigation in Rag1−/−Irf4−/− T cell recipients resulted in reduced frequencies and absolute numbers of IL-17a-producing T cell subsets in MLN and cLP possibly due to a regulation of conventional dendritic cell subset 2 (cDC2) known to impact Th17 differentiation. Together, extending the T cell-intrinsic role for IRF4 in the context of Th17 cell driven colitis, the provided data demonstrate a Th17-inducing and thereby colitis-promoting role of IRF4 through a T cell-extrinsic mechanism highlighting IRF4 as a putative molecular master switch among transcriptional regulators driving immune-mediated intestinal inflammation through both T cell-intrinsic and T cell-extrinsic mechanisms. Future studies need to further dissect IRF4 controlled pathways within distinct IRF4-expressing myeloid cell types, especially cDC2s, to elucidate the precise mechanisms accounting for hampered Th17 formation and, according to our data, the predominant mechanism of colitis protection in Rag1−/−Irf4−/− T cell receiving mice.