miR-28-based combination therapy impairs aggressive B cell lymphoma growth by rewiring DNA replication.

Diffuse large B cell lymphoma (DLBCL) is the most common aggressive B cell lymphoma and accounts for nearly 40% of cases of B cell non-Hodgkin lymphoma. DLBCL is generally treated with R-CHOP chemotherapy, but many patients do not respond or relapse after treatment. Here, we analyzed the therapeutic potential of the tumor suppressor microRNA-28 (miR-28) for DLBCL, alone and in combination with the Bruton's tyrosine kinase inhibitor ibrutinib. Combination therapy with miR-28 plus ibrutinib potentiated the anti-tumor effects of monotherapy with either agent by inducing a specific transcriptional cell-cycle arrest program that impairs DNA replication. The molecular actions of miR-28 and ibrutinib synergistically impair DNA replication by simultaneous inhibition of origin activation and fork progression. Moreover, we found that downregulation of the miR-28-plus-ibrutinib gene signature correlates with better survival of ABC-DLBCL patients. These results provide evidence for the effectiveness of a new miRNA-based ibrutinib combination therapy for DLBCL and unveil the miR-28-plus-ibrutinib gene signature as a new predictor of outcome in ABC-DLBCL patients.

MYC directly transactivates CR2/CD21, the receptor of the Epstein-Barr virus, enhancing the viral infection of Burkitt lymphoma cells.

MYC is an oncogenic transcription factor dysregulated in about half of total human tumors. While transcriptomic studies reveal more than 1000 genes regulated by MYC, a much smaller fraction of genes is directly transactivated by MYC. Virtually all Burkitt lymphoma (BL) carry chromosomal translocations involving MYC oncogene. Most endemic BL and a fraction of sporadic BL are associated with Epstein-Barr virus (EBV) infection. The currently accepted mechanism is that EBV is the BL-causing agent inducing MYC translocation. Herein we show that the EBV receptor, CR2 (also called CD21), is a direct MYC target gene. This is based on several pieces of evidence: MYC induces CR2 expression in both proliferating and arrested cells and in the absence of protein synthesis, binds the CR2 promoter and transactivates CR2 in an E-box-dependent manner. Moreover, using mice with conditional MYC ablation we show that MYC induces CR2 in primary B cells. Importantly, modulation of MYC levels directly correlates with EBV's ability of infection in BL cells. Altogether, in contrast to the widely accepted hypothesis for the correlation between EBV and BL, we propose an alternative hypothesis in which MYC dysregulation could be the first event leading to the subsequent EBV infection.

microRNA Fine-Tuning of the Germinal Center Response.

Germinal centers (GCs) are complex multicellular structures in which antigen-specific B cells undergo the molecular remodeling that enables the generation of high-affinity antibodies and the differentiation programs that lead to the generation of plasma-antibody-secreting cells and memory B cells. These reactions are tightly controlled by a variety of mechanisms, including the post-transcriptional control of gene expression by microRNAs (miRNAs). Through the development of animal models with B cell-specific modified miRNA expression, we have contributed to the understanding of the role of miRNAs in the regulation of GC responses and in B cell neoplasia. Here, we review recent advances in the understanding of the role of miRNAs in the regulation of B cell and T follicular helper physiology during the GC response and in the diseases associated to GC response dysregulation.

Interplay between UNG and AID governs intratumoral heterogeneity in mature B cell lymphoma.

Most B cell lymphomas originate from B cells that have germinal center (GC) experience and bear chromosome translocations and numerous point mutations. GC B cells remodel their immunoglobulin (Ig) genes by somatic hypermutation (SHM) and class switch recombination (CSR) in their Ig genes. Activation Induced Deaminase (AID) initiates CSR and SHM by generating U:G mismatches on Ig DNA that can then be processed by Uracyl-N-glycosylase (UNG). AID promotes collateral damage in the form of chromosome translocations and off-target SHM, however, the exact contribution of AID activity to lymphoma generation and progression is not completely understood. Here we show using a conditional knock-in strategy that AID supra-activity alone is not sufficient to generate B cell transformation. In contrast, in the absence of UNG, AID supra-expression increases SHM and promotes lymphoma. Whole exome sequencing revealed that AID heavily contributes to lymphoma SHM, promoting subclonal variability and a wider range of oncogenic variants. Thus, our data provide direct evidence that UNG is a brake to AID-induced intratumoral heterogeneity and evolution of B cell lymphoma.

miRNA-Based Therapies in B Cell Non-Hodgkin Lymphoma.

Non-Hodgkin lymphoma (NHL) is a diverse class of hematological cancers, many of which arise from germinal center (GC)-experienced B cells. Thus GCs, the sites of antibody affinity maturation triggered during immune responses, also provide an environment that facilitates B cell oncogenic transformation. miRNAs provide attractive and mechanistically different strategies to treat these malignancies based on their potential for simultaneous modulation of multiple targets. Here, we discuss the scientific rationale for miRNA-based therapeutics in B cell neoplasias and review recent advances that may help establish a basis for novel candidate miRNA-based therapies for B cell-NHL (B-NHL).

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

OBJECTIVE: microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE-/- mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE-/- mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk. CONCLUSIONS: Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.

Frequent mutations in the amino-terminal domain of BCL7A impair its tumor suppressor role in DLBCL.

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.

Transfer of extracellular vesicle-microRNA controls germinal center reaction and antibody production.

Intercellular communication orchestrates effective immune responses against disease-causing agents. Extracellular vesicles (EVs) are potent mediators of cell-cell communication. EVs carry bioactive molecules, including microRNAs, which modulate gene expression and function in the recipient cell. Here, we show that formation of cognate primary T-B lymphocyte immune contacts promotes transfer of a very restricted set of T-cell EV-microRNAs (mmu-miR20-a-5p, mmu-miR-25-3p, and mmu-miR-155-3p) to the B cell. Transferred EV-microRNAs target key genes that control B-cell function, including pro-apoptotic BIM and the cell cycle regulator PTEN. EV-microRNAs transferred during T-B cognate interactions also promote survival, proliferation, and antibody class switching. Using mouse chimeras with Rab27KO EV-deficient T cells, we demonstrate that the transfer of small EVs is required for germinal center reaction and antibody production in vivo, revealing a mechanism that controls B-cell responses via the transfer of EV-microRNAs of T-cell origin. These findings also provide mechanistic insight into the Griscelli syndrome, associated with a mutation in the Rab27a gene, and might explain antibody defects observed in this pathogenesis and other immune-related and inflammatory disorders.

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

miR-28 regulates the germinal center reaction and blocks tumor growth in preclinical models of non-Hodgkin lymphoma.

Non-Hodgkin lymphoma comprises a variety of neoplasms, many of which arise from germinal center (GC)-experienced B cells. microRNA-28 (miR-28) is a GC-specific miRNA whose expression is lost in numerous mature B-cell neoplasms. Here we show that miR-28 regulates the GC reaction in primary B cells by impairing class switch recombination and memory B and plasma cell differentiation. Deep quantitative proteomics combined with transcriptome analysis identified miR-28 targets involved in cell-cycle and B-cell receptor signaling. Accordingly, we found that miR-28 expression diminished proliferation in primary and lymphoma cells in vitro. Importantly, miR-28 reexpression in human Burkitt (BL) and diffuse large B-cell lymphoma (DLBCL) xenografts blocked tumor growth, both when delivered in viral vectors or as synthetic, clinically amenable, molecules. Further, the antitumoral effect of miR-28 is conserved in a primary murine in vivo model of BL. Thus, miR-28 replacement is uncovered as a novel therapeutic strategy for DLBCL and BL treatment.

In vivo conditional deletion of HDAC7 reveals its requirement to establish proper B lymphocyte identity and development.

Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro-B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development.

miR-217 is an oncogene that enhances the germinal center reaction.

microRNAs are a class of regulators of gene expression that have been shown critical for a great number of biological processes; however, little is known of their role in germinal center (GC) B cells. Although the GC reaction is crucial to ensure a competent immune response, GC B cells are also the origin of most human lymphomas, presumably due to bystander effects of the immunoglobulin gene remodeling that takes place at these sites. Here we report that miR-217 is specifically upregulated in GC B cells. Gain- and loss-of-function mouse models reveal that miR-217 is a positive modulator of the GC response that increases the generation of class-switched antibodies and the frequency of somatic hypermutation. We find that miR-217 down-regulates the expression of a DNA damage response and repair gene network and in turn stabilizes Bcl-6 expression in GC B cells. Importantly, miR-217 overexpression also promotes mature B-cell lymphomagenesis; this is physiologically relevant as we find that miR-217 is overexpressed in aggressive human B-cell lymphomas. Therefore, miR-217 provides a novel molecular link between the normal GC response and B-cell transformation.

Regulation of B-cell development and function by microRNAs.

MicroRNAs (miRNAs) have emerged as a new class of gene expression regulators whose functions influence a myriad of biological processes, from developmental decisions through immune responses and numerous pathologies, including cancer and autoimmunity. miRNAs are small RNA molecules that drive post-transcriptional negative regulation of gene expression by promoting the degradation or translational block of their target mRNAs. Here, we review some of the data relating to the role of miRNAs in the regulation of the B-cell lineage, with a special focus on results obtained in vivo. We start by giving a general overview of miRNA activity, including the issue of target specificity and the experimental approaches more widely used to analyze the function of these molecules. We then go on to discuss the function of miRNAs during B-cell differentiation in the bone marrow and in the periphery as well as during the humoral immune response. Finally, we describe a few examples of the contribution of miRNAs, both as oncogenes and tumor suppressors, to the development of B-cell neoplasias.

UNG shapes the specificity of AID-induced somatic hypermutation.

Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.

MicroRNAs prevent the generation of autoreactive antibodies.

MicroRNAs have been shown to be critical for a number of aspects of immune system regulation and function. Here, we have examined the role of microRNAs in terminal B cell differentiation by analyzing Cd19-Cre(ki/+) Dicer1(fl/fl) mice. We found that in the absence of Dicer, the transitional and marginal zone (MZ) B cell compartments were overrepresented and follicular (FO) B cell generation was impaired. microRNA analysis revealed that miR185, a microRNA overexpressed in FO cells, dampened B cell receptor (BCR) signaling through Bruton tyrosine kinase downregulation. Dicer-deficient B cells had a skewed BCR repertoire with hallmarks of autoreactivity, which correlated with high titers of autoreactive antibodies in serum and autoimmune features in females. Together, our results reveal a crucial role for microRNAs in late B cell differentiation and in the establishment of B cell tolerance.

MicroRNA activity in B lymphocytes.

Gene expression regulation by miRNAs has been reported to control key aspects of B cell differentiation and function (Chen et al., Science 303:83-86, 2004; Xiao et al., Cell 131:146-159, 2007; O'Carroll et al., Genes Dev. 21:1999-2004, 2007; Koralov et al. Cell 132:860-874, 2008; Rodriguez et al., Science 316:608-611, 2007; Costinean et al., Proc Natl Acad Sci USA 103:7024-7029, 2006; Thai et al., Science 316:604-608, 2007; Vigorito et al., Immunity 27:847-859, 2007; Dorsett et al., Immunity 28:630-638, 2008; Teng et al., Immunity 28:621-629, 2008; de Yebenes et al., J Exp Med 205:2199-2206, 2008; He et al., Nature 435:828-833, 2005; Ventura et al. Cell 132:875-886, 2008; Xiao et al., Nat Immunol 9:405-414, 2008). In this chapter, we describe the methodology used to perform a functional screening of a miRNA library to identify miRNAs relevant for mature B cell function in primary mouse B cells. These procedures include the construction of a miRNA library and the expression of individual miRNA clones in spleen B cells, as well as the description of functional assays used to determine the impact of miRNA expression on several aspects of B cell function, such as proliferation, apoptosis, and class switch recombination.

Haploinsufficiency of activation-induced deaminase for antibody diversification and chromosome translocations both in vitro and in vivo.

The humoral immune response critically relies on the secondary diversification of antibodies. This diversification takes places through somatic remodelling of the antibody genes by two molecular mechanisms, Class Switch Recombination (CSR) and Somatic Hypermutation (SHM). The enzyme Activation Induced Cytidine Deaminase (AID) initiates both SHM and CSR by deaminating cytosine residues on the DNA of immunoglobulin genes. While crucial for immunity, AID-catalysed deamination is also the triggering event for the generation of lymphomagenic chromosome translocations. To address whether restricting the levels of AID expression in vivo contributes to the regulation of its function, we analysed mice harbouring a single copy of the AID gene (AID(+/-)). AID(+/-) mice express roughly 50% of normal AID levels, and display a mild hyperplasia, reminiscent of AID deficient mice and humans. Moreover, we found that AID(+/-) cells have an impaired competence for CSR and SHM, which indicates that AID gene dose is limiting for its physiologic function. We next evaluated the impact of AID reduction in AID(+/-) mice on the generation of chromosome translocations. Our results show that the frequency of AID-promoted c-myc/IgH translocations is reduced in AID(+/-) mice, both in vivo and in vitro. Therefore, AID is haploinsufficient for antibody diversification and chromosome translocations. These findings suggest that limiting the physiologic levels of AID expression can be a regulatory mechanism that ensures an optimal balance between immune proficiency and genome integrity.

miR-181b negatively regulates activation-induced cytidine deaminase in B cells.

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

Oncogenic events triggered by AID, the adverse effect of antibody diversification.

The generation of an efficient immune response depends on highly refined mechanisms of antibody diversification. Two of these mechanisms, somatic hypermutation (SHM) and class switch recombination (CSR), are initiated by activation-induced cytidine deaminase (AID) upon antigen stimulation of mature B cells. AID deaminates cytosines on the DNA of Ig genes thereby generating a lesion that can be processed into a mutation (SHM) or a DNA double-strand break followed by a recombination reaction (CSR). A number of mechanisms are probably responsible for regulating AID function, such as transcriptional regulation, subcellular localization, post-transcriptional modifications and target specificity, but the issue remains of how unwanted DNA damage is fully prevented. Most lymphocyte neoplasias are originated from mature B cells and harbour hallmark chromosome translocations of lymphomagenic potential, such as the c-myc/IgH translocations found in Burkitt lymphomas. It has been recently shown that such translocations are initiated by AID and that ataxia-telangiectasia mutated, p53 and ARF provide surveillance mechanisms to prevent these aberrations. In addition, evidence is accumulating that AID expression can be induced in B cells independently of the germinal centre environment, such as in response to some viral infections, and occasionally in non-B cells, at least in certain inflammation-associated neoplasic situations. The most recent findings on AID expression and function and their relevance to the generation of oncogenic lesions will be discussed.

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates.

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.