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Philadelphia-chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is characterized by reciprocal chromosomal translocation between chromosome 9 and 22, leading to the expression of constitutively active oncogenic BCR–ABL1 fusion protein. CXC chemokine receptor 4 (CXCR4) is essential for the survival of BCR–ABL1-transformed mouse pre-B cells, as the deletion of CXCR4 induces death in these cells. To investigate whether CXCR4 inhibition also effectively blocks BCR–ABL1-transformed cell growth in vitro, in this study, we explored an array of peptide-based inhibitors of CXCR4. The inhibitors were optimized derivatives of EPI-X4, an endogenous peptide antagonist of CXCR4. We observed that among all the candidates, EPI-X4 JM#170 (referred to as JM#170) effectively induced cell death in BCR–ABL1-transformed mouse B cells but had little effect on untransformed wild-type B cells. Importantly, AMD3100, a small molecule inhibitor of CXCR4, did not show this effect. Treatment with JM#170 induced transient JNK phosphorylation in BCR–ABL1-transformed cells, which in turn activated the intrinsic apoptotic pathway by inducing cJun, Bim, and Bax gene expressions. Combinatorial treatment of JM#170 with ABL1 kinase inhibitor Imatinib exerted a stronger killing effect on BCR–ABL1-transformed cells even at a lower dose of Imatinib. Surprisingly, JM#170 actively killed Sup-B15 cells, a BCR–ABL1+ human ALL cell line, but had no effect on the BCR–ABL1− 697 cell line. This suggests that the inhibitory effect of JM#170 is specific for BCR–ABL1+ ALL. Taken together, JM#170 emerges as a potent novel drug against Ph+ ALL. [ABSTRACT FROM AUTHOR]

Simple Summary: Chronic lymphocytic leukemia (CLL) is the most prevalent among adult leukemias. Over the years, several research efforts discovered a lot of intricate details about the cause of the disease, its mechanism, and the prognostic factors that help to understand the progression and outcome of the disease. Mutations in the immunoglobulin gene sequences in B cells are the most important prognostic factor for CLL. The cells having no to very less mutations show aggressive disease, while those having more mutations are either fairly indolent or non-aggressive. In this review, we discussed the current gain of knowledge about these mutations and their effects in the overall disease pathology. Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation of CD5+ CD19+ malignant B cells. Autonomous ligand-independent B-cell signaling is a key process involved in the development of CLL pathogenesis. Together with other cytogenetic alterations, mutations in the immunoglobulin heavy chain variable (IGHV) gene act as a prognostic marker for CLL, with mutated CLL (M-CLL) being far more indolent than unmutated CLL (U-CLL). Recent studies highlight the role of a specific light chain mutation, namely, IGLV3-21R110G, in the development and prognosis of CLL. Such a mutation increases the propensity of homotypic BCR–BCR interaction, leading to cell autonomous signaling. In this article, we review the current findings on immunoglobulin gene sequence mutations as a potential risk factor for developing CLL. [ABSTRACT FROM AUTHOR]

The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA -deficient mice. The RhoA -deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA -deficient cells with RhoA or Foxo1 , a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function. [ABSTRACT FROM AUTHOR]

Abstract Background HIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator. Results We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD. Conclusions Taken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a role in transcription deregulation observed in HD.

In contrast to other B‐cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co‐express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3‐kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten‐deficient B cells expressing knock‐ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten‐deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten‐deficient B cells downregulate BCR expression and become unresponsive to further BCR‐mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD‐deficient B cells after immunization with trinitrophenyl‐ovalbumin (TNP‐Ova), a commonly used antigen for T‐cell‐dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T‐cell‐dependent antibody responses. Synopsis: By activation of Ig gene rearrangement and receptor editing, Pten controls antibody diversity and the development of self‐tolerant B cells. Pten also controls the development of follicular B cellsand their responsiveness towards complex antigen. Pten is required for receptor editing but not for anergy in B cells.Increased IgD BCR expression results in mature B cells that are controlled by soluble antigen whileresponsive to antigencomplexes.Pten activates IgD BCR expression and selective B cell responsiveness through FoxO1.IgD regulates the quality of immune responses by efficiently directing B cells into GC reactions. [ABSTRACT FROM AUTHOR]

The random gene segment rearrangement during B cell development ensures Ab repertoire diversity. Because this process might generate autoreactive specificities, it has been proposed that stringent selection mechanisms prevent the development of autoreactive B cells. However, conventional assays to identify autoreactive B cells usually employ in vitro-generated Abs, which differ from membrane-bound BCRs. In this study, we used a cell-based assay to investigate the autoreactivity of membrane-bound BCRs derived from different B cell developmental stages of human peripheral blood. Contrasted to soluble Ab counterparts, only a few of the tested BCRs were autoreactive, although the cell-based assay sensitively detects feeble Ag recognition of a germline-reverted murine BCR that was selected after OVA immunization of mice, whereas conventional assays failed to do so. Together, these data suggest that proper identification of autoreactive B cells requires the membrane-bound BCR, as the soluble Ab may largely differ from its BCR counterpart in Ag binding. [ABSTRACT FROM AUTHOR]

Increased poly glutamine (polyQ) stretch at N-terminal of Huntingtin (HTT) causes Huntington’s disease. HTT interacts with large number of proteins, although the preference for such interactions with wild type or mutated HTT protein remains largely unknown. HYPK, an intrinsically unstructured protein chaperone and interactor of mutant HTT was found to interact with myeloid leukemia factor 1 (MLF1) and 2 (MLF2). To identify the role of these two proteins in mutant HTT mediated aggregate formation and toxicity in a cell model, both the proteins were found to preferentially interact with the mutated N-terminal HTT. They significantly reduced the number of cells containing mutant HTT aggregates and subsequent apoptosis in Neuro2A cells. Additionally, in FRAP assay, mobile fraction of mutant HTT aggregates was increased in the presence of MLF1 or MLF2. Further, MLF1 could release transcription factors like p53, CBP and CREB from mutant HTT aggregates. Moreover, in HeLa cell co-expressing mutant HTT exon1 and full length MLF1, p53 was released from the aggregates, leading to the recovery of the expression of the GADD45A transcript, a p53 regulated gene. Taking together, these results showed that MLF1 and MLF2 modulated the formation of aggregates and induction of apoptosis as well as the expressions of genes indirectly. [ABSTRACT FROM AUTHOR]

Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/ NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain- derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP 1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. [ABSTRACT FROM AUTHOR]

Summary Microglia as tissue macrophages contribute to the defense and maintenance of central nervous system (CNS) homeostasis. Little is known about the epigenetic signals controlling microglia function in vivo . We employed constitutive and inducible mutagenesis in microglia to delete two class I histone deacetylases, Hdac1 and Hdac2. Prenatal ablation of Hdac1 and Hdac2 impaired microglial development. Mechanistically, the promoters of pro-apoptotic and cell cycle genes were hyperacetylated in absence of Hdac1 and Hdac2, leading to increased apoptosis and reduced survival. In contrast, Hdac1 and Hdac2 were not required for adult microglia survival during homeostasis. In a mouse model of Alzheimer’s disease, deletion of Hdac1 and Hdac2 in microglia, but not in neuroectodermal cells, resulted in a decrease in amyloid load and improved cognitive impairment by enhancing microglial amyloid phagocytosis. Collectively, we report a role for epigenetic factors that differentially affect microglia development, homeostasis, and disease that could potentially be utilized therapeutically. [ABSTRACT FROM AUTHOR]

A considerable proportion of peripheral B cells is autoreactive, and it is unclear how the activation of such potentially harmful cells is regulated. In this study, we show that the different activation thresholds or IgM and IgD BCRs adjust B cell activation to the diverse requirements during development. We rely on the autoreactive 3-83 model BCR to generate and analyze mice expressing exclusively autoreactive IgD BCRs on two different backgrounds that determine two stages of autoreactivity, depending on the presence or absence of the cognate Ag. By comparing these models with IgM-expressing control mice, we found that, compared with IgM, IgD has a higher activation threshold in vivo, as it requires autoantigen to enable normal B cell development, including allelic exclusion. Our data indicate that IgM provides the high sensitivity required during early developmental stages to trigger editing of any autoreactive specificities, including those enabling weak interaction with autoantigen. In contrast, IgD has the unique ability to neglect weakly interacting autoantigens while retaining reactivity to higher-affinity Ag. This IgD function enables mature B cells to ignore autoantigens while remaining able to efficiently respond to foreign threats., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21 R110 ), we show that IGLV3-21 R110 -expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21 * 01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21 * 01 -expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21 R110 -expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21 R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors., Competing Interests: Competing interest statement: H.J. is a cofounder of AVA LifeScience GmbH that has filed patents on antibodies recognizing structures involved in autonomous BCR signaling., (Copyright © 2020 the Author(s). Published by PNAS.)

Expression of the membrane-bound form of the immunoglobulin (Ig) as part of the antigen receptor is indispensable for both the development and the effector function of B cells. Among five known isotypes, IgM and IgD are the common B cell antigen receptors (BCRs) that are co-expressed in naïve B cells. Despite having identical antigen specificity and being associated with the same signaling heterodimer Igα/Igβ (CD79a/CD79b), IgM and IgD-BCR isotypes functionally differ from each other in the manner of antigen binding, the formation of isolated nanoclusters and in their interaction with co-receptors such as CD19 and CXCR4 on the plasma membrane. With recent developments in experimental techniques, it is now possible to investigate the nanoscale organization of the BCR and better understand early events of BCR engagement. Interestingly, the cytoskeleton network beneath the membrane controls the BCR isotype-specific organization and its interaction with co-receptors. BCR triggering results in reorganization of the cytoskeleton network, which is further modulated by isotype-specific signals from co-receptors. For instance, IgD-BCR is closely associated with CXCR4 on mature B cells and this close proximity allows CXCR4 to employ the BCR machinery as signaling hub. In this review, we discuss the functional specificity and nanocluster assembly of BCR isotypes and the consequences of cross-talk between CXCR4 and IgD-BCR. Furthermore, given the role of BCR and CXCR4 signaling in the development and survival of leukemic B cells, we discuss the consequences of the cross-talk between CXCR4 and the BCR for controlling the growth of transformed B cells.

Microglia constitute a highly specialized network of tissue-resident immune cells that is important for the control of tissue homeostasis and the resolution of diseases of the CNS. Little is known about how their spatial distribution is established and maintained in vivo. Here we establish a new multicolor fluorescence fate mapping system to monitor microglial dynamics during steady state and disease. Our findings suggest that microglia establish a dense network with regional differences, and the high regional turnover rates found challenge the universal concept of microglial longevity. Microglial self-renewal under steady state conditions constitutes a stochastic process. During pathology this randomness shifts to selected clonal microglial expansion. In the resolution phase, excess disease-associated microglia are removed by a dual mechanism of cell egress and apoptosis to re-establish the stable microglial network. This study unravels the dynamic yet discrete self-organization of mature microglia in the healthy and diseased CNS.

Background: HIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator., Results: We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD., Conclusions: Taken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a role in transcription deregulation observed in HD.

Intraspecific strains of Pythium aphanidermatum induced resistance in ginger against rhizome rot and activated biosynthesis of selected host proteins. Pre-inoculation of plants with IR strain (avirulent) or co-inoculation with SR2 (virulent) caused significant reduction in disease severity. Analysis of protein profiles of ginger leaves of inoculated and non-inoculated plants by SDS-PAGE and Image Master VDS-ID Gel Analysis version : 3.0 revealed that some specific defence proteins/stress proteins increased in inoculated plants. Five such proteins having molecular weight 56, 32, 27, 18 and 14 kDa were detected in leaves of plant treated with IR + SR2 strains. On the contrary, mycelial protein profiles and submerged growth of strains were studied separately and together. Mycelia of IR, SR2 and IR + SR2 exhibited 26, 23 and 25 protein bands, respectively although, 21 bands were common between IR and SR2. Growth of SR2 in synthetic medium was much higher than that of IR, but the growth of two strains together was lower than SR2 alone. To characterise strains, their differential growth response to DL-beta-aminobutyric acid (BABA), a known defence activator of ginger was also tested. Results suggested that at least 5 specific defence proteins/stress proteins were involved in microbially induced resistance in ginger and inducer strains were distinct in their specific protein profiles and sensitivity to BABA.