B-cell intrinsic regulation of antibody mediated immunity by histone H2A deubiquitinase BAP1.

Introduction: BAP1 is a deubiquitinase (DUB) of the Ubiquitin C-terminal Hydrolase (UCH) family that regulates gene expression and other cellular processes, through its direct catalytic activity on the repressive epigenetic mark histone H2AK119ub, as well as on several other substrates. BAP1 is also a highly important tumor suppressor, expressed and functional across many cell types and tissues. In recent work, we demonstrated a cell intrinsic role of BAP1 in the B cell lineage development in murine bone marrow, however the role of BAP1 in the regulation of B cell mediated humoral immune response has not been previously explored. Methods and results: In the current study, we demonstrate that a B-cell intrinsic loss of BAP1 in activated B cells in the Bap1 fl/fl Cγ1-cre murine model results in a severe defect in antibody production, with altered dynamics of germinal centre B cell, memory B cell, and plasma cell numbers. At the cellular and molecular level, BAP1 was dispensable for B cell immunoglobulin class switching but resulted in an impaired proliferation of activated B cells, with genome-wide dysregulation in histone H2AK119ub levels and gene expression. Conclusion and discussion: In summary, our study establishes the B-cell intrinsic role of BAP1 in antibody mediated immune response and indicates its central role in the regulation of the genome-wide landscapes of histone H2AK119ub and downstream transcriptional programs of B cell activation and humoral immunity.

AFF3, a susceptibility factor for autoimmune diseases, is a molecular facilitator of immunoglobulin class switch recombination.

Immunoglobulin class switch recombination (CSR) plays critical roles in controlling infections and inflammatory tissue injuries. Here, we show that AFF3, a candidate gene for both rheumatoid arthritis and type 1 diabetes, is a molecular facilitator of CSR with an isotype preference. Aff3-deficient mice exhibit low serum levels of immunoglobulins, predominantly immunoglobulin G2c (IgG2c) followed by IgG1 and IgG3 but not IgM. Furthermore, Aff3-deficient mice show weak resistance to Plasmodium yoelii infection, confirming that Aff3 modulates immunity to this pathogen. Mechanistically, the AFF3 protein binds to the IgM and IgG1 switch regions via a C-terminal domain, and Aff3 deficiency reduces the binding of AID to the switch regions less efficiently. One AFF3 risk allele for rheumatoid arthritis is associated with high mRNA expression of AFF3, IGHG2, and IGHA2 in human B cells. These findings demonstrate that AFF3 directly regulates CSR by facilitating the recruitment of AID to the switch regions.

AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

AID in Antibody Diversification: There and Back Again.

Activation-Induced cytidine Deaminase (AID) initiates affinity maturation and isotype switching by deaminating deoxycytidines within immunoglobulin genes, leading to somatic hypermutation (SHM) and class switch recombination (CSR). AID thus potentiates the humoral response to clear pathogens. Marking the 20th anniversary of the discovery of AID, we review the current understanding of AID function. We discuss AID biochemistry and how error-free forms of DNA repair are co-opted to prioritize mutagenesis over accuracy during antibody diversification. We discuss the regulation of DNA double-strand break (DSB) repair pathways during CSR. We describe genomic targeting of AID as a multilayered process involving chromatin architecture, cis- and trans-acting factors, and determining mutagenesis - distinct from AID occupancy at loci that are spared from mutation.

Immunoregulatory effects of Lurbinectedin in chronic lymphocytic leukemia.

Despite significant therapeutic improvements chronic lymphocytic leukemia (CLL) remains an incurable disease and there is a persistent pursuit of new treatment alternatives. Lurbinectedin, a selective inhibitor of active transcription of protein-coding genes, is currently in phase II/III clinical trials for solid tumors such as small-cell lung cancer (SCLC). In this study, we aimed to evaluate the activity of Lurbinectedin on circulating mononuclear cells from CLL patients and to determine whether Lurbinectedin could affect the cross-talk between B-CLL cells and the tumor microenvironment. We found that Lurbinectedin induced a dose- and time-dependent death in all cell types evaluated, with B cells, monocytes and monocytic myeloid derived suppressor cells (Mo-MDSC) being the most susceptible populations. At sub-apoptotic doses, Lurbinectedin decreased the expression of CCR7 in B-CLL cells and impaired their migration towards CCL19 and CCL21. Furthermore, low concentrations of Lurbinectedin stimulated the synthesis of pro-IL1ß in monocytes and nurse-like cells, without inducing the inflammasome activation. Altogether, these results indicate that Lurbinectedin might have antitumor activity in CLL due to its direct action on leukemic cells in combination with its effects on the tumor microenvironment. Our findings encourage further investigation of Lurbinectedin as a potential therapy for CLL.

Ibrutinib therapy downregulates AID enzyme and proliferative fractions in chronic lymphocytic leukemia.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination of the immunoglobulin genes. As a trade-off for its physiological function, AID also contributes to tumor development through its mutagenic activity. In chronic lymphocytic leukemia (CLL), AID is overexpressed in the proliferative fractions (PFs) of the malignant B lymphocytes, and its anomalous expression has been associated with a clinical poor outcome. Recent preclinical data suggested that ibrutinib and idelalisib, 2 clinically approved kinase inhibitors, increase AID expression and genomic instability in normal and neoplastic B cells. These results raise concerns about a potential mutagenic risk in patients receiving long-term therapy. To corroborate these findings in the clinical setting, we analyzed AID expression and PFs in a CLL cohort before and during ibrutinib treatment. We found that ibrutinib decreases the CLL PFs and, interestingly, also reduces AID expression, which correlates with dampened AKT and Janus Kinase 1 signaling. Moreover, although ibrutinib increases AID expression in a CLL cell line, it is unable to do so in primary CLL samples. Our results uncover a differential response to ibrutinib between cell lines and the CLL clone and imply that ibrutinib could differ from idelalisib in their potential to induce AID in treated patients. Possible reasons for the discrepancy between preclinical and clinical findings, and their effect on treatment safety, are discussed.

LPL protein in Chronic Lymphocytic Leukaemia have different origins in Mutated and Unmutated patients. Advances for a new prognostic marker in CLL.

Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.

S100-A9 protein in exosomes from chronic lymphocytic leukemia cells promotes NF-κB activity during disease progression.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by accumulation of clonal B lymphocytes, resulting from a complex balance between cell proliferation and apoptotic death. Continuous crosstalk between cancer cells and local/distant host environment is required for effective tumor growth. Among the main actors of this dynamic interplay between tumoral cells and their microenvironment are the nano-sized vesicles called exosomes. Emerging evidence indicates that secretion, composition, and functional capacity of exosomes are altered as tumors progress to an aggressive phenotype. In CLL, no data exist exploring the specific changes in the proteomic profile of plasma-derived exosomes from patients during disease evolution. We hereby report for the first time different proteomic profiles of plasma exosomes, both between indolent and progressive CLLs as well as within the individual patients at the onset of disease and during its progression. Next, we focus on the changes of the exosome protein cargoes, which are found exclusively in patients with progressive CLL after disease progression. The alterations in the proteomic cargoes underline different networks specific for leukemia progression related to inflammation, oxidative stress, and NF-κB and phosphatidylinositol 3-kinase/AKT pathway activation. Finally, our results suggest a preponderant role for the protein S100-A9 as an activator of the NFκB pathway during CLL progression and suggest that the leukemic clone can generate an autoactivation loop through S100-A9 expression, NF-κB activation, and exosome secretion. Collectively, our data propose a new pathway for NF-κB activation in CLL and highlight the importance of exosomes as extracellular mediators promoting tumor progression in CLL.