CARM1 inhibition reduces histone acetyltransferase activity causing synthetic lethality in CREBBP/EP300-mutated lymphomas.

Somatic mutations affecting CREBBP and EP300 are a hallmark of diffuse large B-cell lymphoma (DLBCL). These mutations are frequently monoallelic, within the histone acetyltransferase (HAT) domain and usually mutually exclusive, suggesting that they might affect a common pathway, and their residual WT expression is required for cell survival. Using in vitro and in vivo models, we found that inhibition of CARM1 activity (CARM1i) slows DLBCL growth, and that the levels of sensitivity are positively correlated with the CREBBP/EP300 mutation load. Conversely, treatment of DLBCLs that do not have CREBBP/EP300 mutations with CARM1i and a CBP/p300 inhibitor revealed a strong synergistic effect. Our mechanistic data show that CARM1i further reduces the HAT activity of CBP genome wide and downregulates CBP-target genes in DLBCL cells, resulting in a synthetic lethality that leverages the mutational status of CREBBP/EP300 as a biomarker for the use of small-molecule inhibitors of CARM1 in DLBCL and other cancers.

PTIP promotes chromatin changes critical for immunoglobulin class switch recombination.

Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here, we showed that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3)-MLL4 complex display impaired trimethylation of histone 3 at lysine 4 (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus, leading to defective immunoglobulin class switching. We also showed that PTIP accumulation at DSBs contributes to class switch recombination (CSR) and genome stability independently of Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR and suggest a nonredundant role for the MLL3-MLL4 complex in altering antibody effector function.

Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8.

53BP1 is a well-known mediator of the cellular response to DNA damage. Two alternative mechanisms have been proposed to explain 53BP1's interaction with DNA double-strand breaks (DSBs), one by binding to methylated histones and the other via an RNF8 E3 ligase-dependent ubiquitylation pathway. The formation of RNF8 and 53BP1 irradiation-induced foci are both dependent on histone H2AX. To evaluate the contribution of the RNF8-dependent pathway to 53BP1 function, we generated RNF8 knockout mice. We report that RNF8 deficiency results in defective class switch recombination (CSR) and accumulation of unresolved immunoglobulin heavy chain-associated DSBs. The CSR DSB repair defect is milder than that observed in the absence of 53BP1 but similar to that found in H2AX(-/-) mice. Moreover, similar to H2AX but different from 53BP1 deficiency, RNF8(-/-) males are sterile, and this is associated with defective ubiquitylation of the XY chromatin. Combined loss of H2AX and RNF8 does not cause further impairment in CSR, demonstrating that the two genes function epistatically. Importantly, although 53BP1 foci formation is RNF8 dependent, its binding to chromatin is preserved in the absence of RNF8. This suggests a two-step mechanism for 53BP1 association with chromatin in which constitutive loading is dependent on interactions with methylated histones, whereas DNA damage-inducible RNF8-dependent ubiquitylation allows its accumulation at damaged chromatin.

Notch1 engagement by Delta-like-1 promotes differentiation of B lymphocytes to antibody-secreting cells.

Notch signaling regulates B and T lymphocyte development and T cell effector class decision. In this work, we tested whether Notch activity affects mature B cell activation and differentiation to antibody-secreting cells (ASC). We show increased frequency of ASC in cultures of splenic B cells activated with LPS or anti-CD40 when provided exogenous Notch ligand Delta-like-1 (Dll1). Our results indicate that Notch-Dll1 interaction releases a default pathway that otherwise inhibits Ig secretion upon B cell activation. Thus, Dll1 enhanced spontaneous Ig secretion by naturally activated marginal zone B and B1 cells and reversed the inhibition of ASC differentiation mediated by B cell receptor crosslinking during LPS. Moreover, suppression of Notch signaling in B cell expression of either a dominant-negative mutant form of Mastermind-like 1 or a null mutation of Notch1 not only prevented Dll1-mediated enhancement of ASC differentiation but also reduced dramatically LPS-induced Ig secretion. Finally, we show that Dll1 and Jagged-1 are differentially expressed in discrete areas of the spleen, and that the effect of Notch engagement on Ig secretion is ligand-specific. These results indicate that Notch ligands participate in the definition of the mature B cell microenvironment that influences their terminal differentiation.

Virulence factors in food, clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.