Consequences of oncogenic CARD11 signalling for B cells

Diffuse large B cell lymphoma is the most common form of Non Hodgkin’s lymphoma, accounting for forty percent of cases. Gain of function CARD11 mutations have been found in approximately thirteen percent of DLBCL patients. CARD11 functions as a scaffold protein and relays signals from the antigen receptor to the JNK and NFkB signaling pathways, which contribute to the survival, proliferation and differentiation of lymphocytes. The consequences of acquiring gain of function CARD11 mutations in normal B lymphocytes, in isolation from the other approximately 30 mutations that accumulate on average in lymphoma cells, had not been previously addressed. By introducing lymphoma CARD11 mutations into antigen activated B lymphocytes, we found that gain of function CARD11 mutations result in proliferation, growth and survival in vitro. The CARD11 mutants also increased signalling to NFkB and JNK pathways to varying degrees. Moreover, when the genetically modified B cells were transferred in vivo to investigate the consequences of the acquisition of the CARD11Mut10 version of CARD11, we observed that the B cells proliferate rapidly and differentiate into plasmablasts. CARD11Mut10 induced BLIMP1 mediated plasmablast differentiation and autocrine production of IL6, which contributed to differentiation into plasmablasts. However, this initial rapid population expansion was self-limiting: it peaked at day 10, and by 20 days the numbers of CARD11Mut10 expressing B cells decreased drastically. The induction of the NFkB negative regulator A20 contributed to limiting the expansion and accumulation of CARD11Mut10 expressing B cells. By introducing CARD11 mutations from human lymphomas into antigen activated mature B lymphocytes in mice, we tested the hypothesis that lymphoma somatic mutations corrupt the normal response to self-antigens. Here, we show that all lymphoma derived CARD11 mutations tested, but not NFkB activating mutations in Ikbkb, block self-antigen induced death and cooperate with chronic antigen receptor stimulation to induce extensive T cell independent proliferation, BLIMP1 mediated plasmablast differentiation and autoantibody secretion. Our findings demonstrate that regulation of CARD11 signalling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B cells, and that mutations in this switch represent a powerful initiator for aberrant B cell responses in vivo. Recently, patients with germline activating mutations in CARD11 were reported. These patients are characterised by a B cell lymphoproliferative disorder, with B cell expansion with NFkB and T cell anergy. We show that E127G CARD11, discovered in patients with germline CARD11 mutations, increased NFkB signalling and, akin to lymphoma derived CARD11 mutants, E127G CARD11 protected the B cells from death and induced accumulation of self reactive B cells. However, there was no apparent expansion of B cells in mice expressing an ENU induced germline CARD11M365K mutation. In vitro assays with B cells showed that CARD11M365K has stronger NFkB and growth inducing effects than CARD11E127G. Together, these results reveal an intriguing difference between the effects of overactive CARD11 signalling in humans and mice. The availability of an ENU induced CARD11M365K mutant mouse strain presents opportunities to further study the effects of overactive CARD11 in lymphocytes

Emerging targets in human lymphoma: targeting the MYD88 mutation

B cell neoplasms co-opt the molecular machinery of normal B cells for their survival. Technological advances in cancer genomics has significantly contributed to uncovering the root cause of aggressive lymphomas, revealing a previously unknown link between TLR signaling and B cell neoplasm. Recurrent oncogenic mutations in MYD88 have been found in 39% of the activated B cell-like subtype of diffuse large B cell lymphoma (ABC DLBCL). Interestingly, 29% of ABC DLBCL have a single amino acid substitution of proline for the leucine at position 265 (L265P), and the exact same variant has also been identified in a number of lymphoid malignancies. The MYD88 L265P variant was recently identified in 90% of Wadenstrom's macroglobulinemia patients. These recent developments warrant the need for novel diagnostic tools as well as targeted therapeutics. In this review, we discuss the physiological functions of MYD88 and focus on its role in B cell lymphomas, evaluating the potential for targeting oncogenic MYD88 in lymphoma

Toll-Like Receptors and Cancer: MYD88 Mutation and Inflammation

Pattern recognition receptors (PRRs) expressed on immune cells are crucial for the early detection of invading pathogens, in initiating early innate immune response and in orchestrating the adaptive immune response. PRRs are activated by specific pathogen-associated molecular patterns that are present in pathogenic microbes or nucleic acids of viruses or bacteria. However, inappropriate activation of these PRRs, such as the Toll-like receptors (TLRs), due to genetic lesions or chronic inflammation has been demonstrated to be a major cause of many hematological malignancies. Gain-of-function mutations in the TLR adaptor protein MYD88 found in 39% of the activated B cell type of diffuse large B cell lymphomas and almost 100% of Waldenström's macroglobulinemia further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signaling pathways, thus the constitutive activation of the transcription factor NFκB to promote cell survival and proliferation. These recent insights into TLR pathway driven malignancies warrant the need for a better understanding of TLRs in cancers and the development of novel anti-cancer therapies targeting TLRs. This review focuses on TLR function and signaling in normal or inflammatory conditions, and how mutations can hijack the TLR signaling pathways to give rise to cancer. Finally, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumor effects

Synergistic cooperation and crosstalk betweenMYD88L265Pand mutations that dysregulate CD79B and surface IgM

CD79B andMYD88mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences ofCD79BandMYD88L265Pmutations individually and combined in normal activated mouse B lymphocytes.CD79Bmutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressingMYD88L265Pdecreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect ofMYD88L265PIn B cells chronically stimulated by self-antigen,CD79BandMYD88L265Pmutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation byMYD88L265Pand provide an explanation for the co-occurrence ofMYD88andCD79Bmutations in lymphomas

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8-dendritic cells require the intramembrane endopeptidase SPPL2A

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.R01 AI052127/AI/NIAID NIH HHS/United States U19 AI100627/AI/NIAID NIH HHS/United States Medical Research Council/United Kingdom Wellcome Trust/United Kingdo

Toll-like receptors and cancer: MYD88 mutation and inflammation

Pattern recognition receptors (PRRs) expressed on immune cells are crucial for the early detection of invading pathogens, in initiating early innate immune response and in orchestrating the adaptive immune response. PRRs are activated by specific pathogen-associated molecular patterns (PAMPs) that are present in pathogenic microbes or nucleic acids of viruses or bacteria. However, inappropriate activation of these PRRs, such as the Toll-like receptors (TLRs), due to genetic lesions or chronic inflammation has been demonstrated to be a major cause of many haematological malignancies. Gain-of-function mutations in the TLR adaptor protein MYD88 found in 39% of the activated B cell type of diffuse large B cell lymphomas (ABC-DLBCL) and almost 100% of Waldenström’s macroglobulinemia (WM) further highlight the involvement of TLRs in these malignancies. MYD88 mutations result in the chronic activation of TLR signalling pathways, thus the constitutive activation of the transcription factor NFκB to promote cell survival and proliferation. These recent insights into TLR pathway driven malignancies warrant the need for a better understanding of TLRs in cancers and the development of novel anti-cancer therapies targeting TLRs. This review focuses on Toll-like receptors function and signalling in normal or inflammatory conditions, and how mutations can also hijack the TLR signalling pathways to give rise to cancer. Lastly, we discuss how potential therapeutic agents could be used to restore normal responses to TLRs and have long lasting anti-tumour effects

DeepSNVMiner: a sequence analysis tool to detect emergent, rare mutations in subsets of cell populations

Background. Massively parallel sequencing technology is being used to sequence highly diverse populations of DNA such as that derived from heterogeneous cell mixtures containing both wild-type and disease-related states. At the core of such molecule tagging techniques is the tagging and identification of sequence reads derived from individual input DNA molecules, which must be first computationally disambiguated to generate read groups sharing common sequence tags, with each read group representing a single input DNA molecule. This disambiguation typically generates huge numbers of reads groups, each of which requires additional variant detection analysis steps to be run specific to each read group, thus representing a significant computational challenge. While sequencing technologies for producing these data are approaching maturity, the lack of available computational tools for analysing such heterogeneous sequence data represents an obstacle to the widespread adoption of this technology.\ud \ud Results. Using synthetic data we successfully detect unique variants at dilution levels of 1 in a 1,000,000 molecules, and find DeeepSNVMiner obtains significantly lower false positive and false negative rates compared to popular variant callers GATK, SAMTools, FreeBayes and LoFreq, particularly as the variant concentration levels decrease. In a dilution series with genomic DNA from two cells lines, we find DeepSNVMiner identifies a known somatic variant when present at concentrations of only 1 in 1,000 molecules in the input material, the lowest concentration amongst all variant callers tested.\ud \ud Conclusions. Here we present DeepSNVMiner; a tool to disambiguate tagged sequence groups and robustly identify sequence variants specific to subsets of starting DNA molecules that may indicate the presence of a disease. DeepSNVMiner is an automated workflow of custom sequence analysis utilities and open source tools able to differentiate somatic DNA variants from artefactual sequence variants that likely arose during DNA amplification. The workflow remains flexible such that it may be customised to variants of the data production protocol used, and supports reproducible analysis through detailed logging and reporting of results. DeepSNVMiner is available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/DeepSNVMiner [see Related URLs].\u

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8⁻ dendritic cells require the intramembrane endopeptidase SPPL2A

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases