Diffuse large B-cell lymphoma (DLBCL), the most common form of lymphoma in adulthood, comprises multiple biologically and clinically distinct subtypes including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL1. Gene expression profile studies have shown that its most aggressive subtype, ABC-DLBCL, is associated with constitutive activation of the NF-kB transcription complex2. However, except for a small fraction of cases3, it remains unclear whether NF-kB activation in these tumors represents an intrinsic program of the tumor cell of origin or a pathogenetic event. Here we show that >50% of ABC-DLBCL and a smaller fraction of GCB-DLBCL carry somatic mutations in multiple genes, including negative (TNFAIP3/A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7/TAK1 and TNFRSF11A/RANK) regulators of NF-kB. Of these, the A20 gene, which encodes for a ubiquitin-modifying enzyme involved in termination of NF-kB responses, is most commonly affected, with ~30% of patients displaying biallelic inactivation by mutations and/or deletions. When reintroduced in cell lines carrying biallelic inactivation of the gene, A20 induced apoptosis and cell growth arrest, indicating a tumor suppressor role. Less frequently, missense mutations of TRAF2 and CARD11 produce molecules with significantly enhanced ability to activate NF-kB. Thus, our results demonstrate that NF-kB activation in DLBCL is caused by genetic lesions affecting multiple genes, whose loss or activation may promote lymphomagenesis by leading to abnormally prolonged NF-kB responses.

SUMMARY Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal centre B cell like (GCB) and activated B cell like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report, we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ~53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X a ), and in normal females, who carry one X a and one inactive X (X i ). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X i , XCI actually results in significant reductions in methylation at 7% of CGIs. Both intraand inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.[Supplemental material is available for this article.] X-chromosome inactivation (XCI) is a mechanism of dosage compensation that equalizes the expression of sex-linked genes between 46,XY males and 46,XX females (Lyon 1961). This results in the silencing of the majority of genes on one of the two X chromosomes in each somatic cell of females (Carrel and Willard 2005) and a transition to a heterochromatic state. XCI is accompanied by several epigenetic modifications to the inactive X (X i ), such as an accumulation of variant histones and transcripts from the XIST gene (Clemson et al. 1996;Costanzi and Pehrson 1998), a delay in replication timing (Willard and Latt 1976), and relocalization to the nuclear periphery (Barr and Bertram 1949).Several studies have shown that DNA methylation of the X i plays an important role in the maintenance of its inactive state. While the active X (X a ) and X i have very similar global levels of methylation (Bernadino et al. 1996), studies have shown that CpG islands (CGIs) have a tendency to be methylated on the X i and unmethylated on the X a (Tribioli et al. 1992;Hellman and Chess 2007). In contrast, the CGIs of genes escaping XCI often remain unmethylated on both the X i and X a (Weber et al. 2007). Mouse knockouts for the DNA methyltransferase enzyme Dnmt1 show defects in X inactivat...

SUMMARY Inducing graft acceptance without chronic immunosuppression remains an elusive goal in organ transplantation. Using an experimental transplantation mouse model, we demonstrate that local macrophage activation through dectin-1 and toll-like receptor 4 (TLR4) drives trained immunity-associated cytokine production during allograft rejection. We conducted nanoimmunotherapeutic studies and found that a short- term mTOR-specific high-density lipoprotein (HDL) nanobiologic treatment (mTORi-HDL) averted macrophage aerobic glycolysis and the epigenetic modifications underlying inflammatory cytokine production. The resulting regulatory macrophages prevented alloreactive CD8+ T cell-mediated immunity and promoted tolerogenic CD4+ regulatory T cell (Treg) expansion. To enhance therapeutic efficacy, we complemented the mTORi-HDL treatment with a CD40-TRAF6 specific nanobiologic (TRAF6i-HDL) that inhibits co-stimulation. This synergistic nanoimunnotherapy resulted in indefinite allograft survival. Together, we show that HDL- based nanoimmunotherapy can be employed to control macrophage function in vivo. Our strategy, focused on preventing inflammatory innate immune responses, provides a framework for developing targeted therapies that promote immunological tolerance.

SUMMARY Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8+ T cell immunity and promoted CD4+Foxp3+ Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN+ suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are the crucial events directing brain development. Here we show that the ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss and gain of function experiments for Huwe1 in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene in association with amplification of the N-myc locus. Our results indicate that Huwe1 is a master regulator of the balance between proliferation and neurogenesis in the developing brain and this pathway is subverted in malignant brain tumors.

Purpose: neoMONARCH assessed the biological effects of abemaciclib in combination with anastrozole in the neoadjuvant setting. Patients and Methods: Postmenopausal women with stage I-IIIB HR þ /HER2 À breast cancer were randomized to a 2-week lead-in of abemaciclib, anastrozole, or abemaciclib plus anastrozole followed by 14 weeks of the combination. The primary objective evaluated change in Ki67 from baseline to 2 weeks of treatment. Additional objectives included clinical, radiologic, and pathologic responses, safety, as well as gene expression changes related to cell proliferation and immune response. Results: Abemaciclib, alone or in combination with anastrozole, achieved a significant decrease in Ki67 expression and led to potent cell-cycle arrest after 2 weeks of treatment compared with anastrozole alone. More patients in the abemaciclib-containing arms versus anastrozole alone achieved complete cell-cycle arrest (58%/68% vs. 14%, P < 0.001). At the end of treatment, following 2 weeks lead-in and 14 weeks of combination therapy, 46% of intent-to-treat patients achieved a radiologic response, with pathologic complete response observed in 4%. The most common all-grade adverse events were diarrhea (62%), constipation (44%), and nausea (42%). Abemaciclib, anastrozole, and the combination inhibited cell-cycle processes and estrogen signaling; however, combination therapy resulted in increased cytokine signaling and adaptive immune response indicative of enhanced antigen presentation and activated T-cell phenotypes. Conclusions: Abemaciclib plus anastrozole demonstrated biological and clinical activity with generally manageable toxicities in patients with HR þ /HER2 À early breast cancer. Abemaciclib led to potent cell-cycle arrest, and in combination with anastrozole, enhanced immune activation.

Antimicrobial peptides (AMPs) are important components of the innate immune system of many species. These peptides are found in eukaryotes, including mammals, amphibians, insects and plants, as well as in prokaryotes. Other than having pathogen-lytic properties, these peptides have other activities like antitumor activity, mitogen activity, or they may act as signaling molecules. Their short length, fast and efficient action against microbes and low toxicity to mammals have made them potential candidates as peptide drugs. In many cases they are effective against pathogens that are resistant to conventional antibiotics. They can serve as natural templates for the design of novel antimicrobial drugs. Although there are vast amounts of data on natural AMPs, they are not available through one central resource. We have developed a comprehensive database (ANTIMIC, http://research.i2r. a-star.edu.sg/Templar/DB/ANTIMIC/) of known and putative AMPs, which contains approximately 1700 of these peptides. The database is integrated with tools to facilitate efficient extraction of data and their analysis at molecular level, as well as search for new AMPs. These tools include BLAST, PDB structure viewer and the Antimic profile module.