KDM6A, a histone demethylase, regulates stress hematopoiesis and early B-cell differentiation.

Histone methylases and demethylases regulate gene expression programs in hematopoiesis. The molecular function of the demethylase KDM6A in normal hematopoiesis and, in particular, for the hematopoietic stem and progenitor cell (HSPC) compartment remains only partially understood. Female but not male Kdm6a-/- HSPCs were functionally impaired in adoptive transfer experiments as well as upon proliferative stress induced by 5-fluorouracil. Loss of Kdm6a affected primarily early B cells and erythroid and myeloid progenitor cells with respect to both number and function. Global gene expression analyses revealed a shared altered gene signature in Kdm6a-/- pro-B and pre-B cells that is also present in HSPCs, supporting that altered B-cell differentiation in Kdm6a-/- animals is already initiated in HSPCs. Interestingly, loss of KDM6A did not affect the global level of methylation of H3K27, its presumed target, in hematopoietic cells. Our data indicate a critical role for KDM6A in the regulation of hematopoietic differentiation and differentiation-specific gene expression programs, with a prominent role in early B-cell differentiation that is likely independent of H3K27 methylation status.

Regulation of B Lymphocyte Development by Histone H2A Deubiquitinase BAP1.

BAP1 is a deubiquitinase (DUB) of the Ubiquitin C-terminal Hydrolase (UCH) family that regulates gene expression and other cellular processes, via deubiquitination of histone H2AK119ub and other substrates. BAP1 is an important tumor suppressor in human, expressed and functional across many cell-types and tissues, including those of the immune system. B lymphocytes are the mediators of humoral immune response, however the role of BAP1 in B cell development and physiology remains poorly understood. Here we characterize a mouse line with a selective deletion of BAP1 within the B cell lineage (Bap1fl/fl mb1-Cre) and establish a cell intrinsic role of BAP1 in the regulation of B cell development. We demonstrate a depletion of large pre-B cells, transitional B cells, and mature B cells in Bap1fl/fl mb1-Cre mice. We characterize broad transcriptional changes in BAP1-deficient pre-B cells, map BAP1 binding across the genome, and analyze the effects of BAP1-loss on histone H2AK119ub levels and distribution. Overall, our work establishes a cell intrinsic role of BAP1 in B lymphocyte development, and suggests its contribution to the regulation of the transcriptional programs of cell cycle progression, via the deubiquitination of histone H2AK119ub.

Phospholipid flippases enable precursor B cells to flee engulfment by macrophages.

ATP11A and ATP11C, members of the P4-ATPases, are flippases that translocate phosphatidylserine (PtdSer) from the outer to inner leaflet of the plasma membrane. Using the W3 T lymphoma cell line, we found that Ca2+ ionophore-induced phospholipid scrambling caused prolonged PtdSer exposure in cells lacking both the ATP11A and ATP11C genes. ATP11C-null (ATP11C-/y ) mutant mice exhibit severe B-cell deficiency. In wild-type mice, ATP11C was expressed at all B-cell developmental stages, while ATP11A was not expressed after pro-B-cell stages, indicating that ATP11C-/y early B-cell progenitors lacked plasma membrane flippases. The receptor kinases MerTK and Axl are known to be essential for the PtdSer-mediated engulfment of apoptotic cells by macrophages. MerTK-/- and Axl-/- double deficiency fully rescued the lymphopenia in the ATP11C-/y bone marrow. Many of the rescued ATP11C-/y pre-B and immature B cells exposed PtdSer, and these cells were engulfed alive by wild-type peritoneal macrophages, in a PtdSer-dependent manner. These results indicate that ATP11A and ATP11C in precursor B cells are essential for rapidly internalizing PtdSer from the cell surface to prevent the cells' engulfment by macrophages.

The endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 controls a critical checkpoint in B cell development in mice.

Humoral immunity involves multiple checkpoints that occur in B cell development, maturation, and activation. The pre-B-cell receptor (pre-BCR) is expressed following the productive recombination of the immunoglobulin heavy-chain gene, and sSignalsing through the pre-BCR are required for the differentiation of pre-B cells into immature B cells. However, the molecular mechanisms controlling the pre-BCR expression and signaling strength remain undefined. Herein, we probed the role of the endoplasmic reticulum-associated, stress-activated E3 ubiquitin ligase HMG-CoA reductase degradation 1 (Hrd1) in B cell differentiation. Using mice with a specific Hrd1 deletion in pro-B cells and subsequent B cell developmental stages, we showed that the E3 ubiquitin ligase Hrd1 governs a critical checkpoint during B cell development. We observed that Hrd1 is required for degradation of the pre-BCR complex during the early stage of B cell development. As a consequence, loss of Hrd1 in the B cell lineage resulted in increased pre-BCR expression levels and a developmental defect in the transition from large to small pre-B cells. This defect, in turn, resulted in reduced fewer mature B cells in bone marrow and peripheral lymphoid organs. Our results revealed a novel critical role of Hrd1 in controlling a critical checkpoint in B cell-mediated immunity and suggest that Hrd1 may functioning as an E3 ubiquitin ligase of the pre-BCR complex.

Histone acetyltransferase p300/CBP-associated factor is an effective suppressor of secretory immunoglobulin synthesis in immature B cells.

The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.

[Expression of FLT3-ITD oncogene confers mice progenitor B-cells BAF3 sensitivity to the ribonuclease binase cytotoxic action].

Acute myeloid leukemia is the most common acute leukemia affecting adults, and its incidence increases with age. Along with chromosomal translocations in leukemic cells mutations in the genes of receptor tyrosine kinases KIT and FLT3 were found with a high frequency. Here we show that transgenic progenitor of B-cells BAF3/FLT3-ITD are much more sensitive to the ribonuclease binase cytotoxic effects than the original BAF3 cells. The principal difference between BAF3/FLT3-ITD and the original BAF3 cells is the expression of FLT3-ITD oncogene, which leads to a change in the normal cell signaling pathways. Earlier, we described a similar effect for the cytotoxic action of binase on Kasumi-1 and FDC-P1-N822K cells, which express the activated KIT-N822K oncogene. Increased binase cytotoxicity toward the cells, expressing FLT3-ITD oncogene, suggests that, as in the case of FDC-P1 cells, transduced by KIT oncogene, the expression of an activated oncogene determines the sensitivity of cells to binase.

Acat1 gene ablation in mice increases hematopoietic progenitor cell proliferation in bone marrow and causes leukocytosis.

OBJECTIVE: To investigate the role of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in hematopoiesis. APPROACH AND RESULTS: ACAT1 converts cellular cholesterol to cholesteryl esters for storage in multiple cell types and is a potential drug target for human diseases. In mouse models for atherosclerosis, global Acat1 knockout causes increased lesion size; bone marrow transplantation experiments suggest that the increased lesion size might be caused by ACAT1 deficiency in macrophages. However, bone marrow contains hematopoietic stem cells that give rise to cells in myeloid and lymphoid lineages; these cell types affect atherosclerosis at various stages. Here, we test the hypothesis that global Acat1(-/-) may affect hematopoiesis, rather than affecting macrophage function only, and show that Acat1(-/-) mice contain significantly higher numbers of myeloid cells and other cells than wild-type mice. Detailed analysis of bone marrow cells demonstrated that Acat1(-/-) causes a higher proportion of the stem cell-enriched Lin(-)Sca-1(+)c-Kit(+) population to proliferate, resulting in higher numbers of myeloid progenitor cells. In addition, we show that Acat1(-/-) causes higher monocytosis in Apoe(-/-) mouse during atherosclerosis development. CONCLUSIONS: ACAT1 plays important roles in hematopoiesis in normal mouse and in Apoe(-/-) mouse during atherosclerosis development.

B-cell lymphopoiesis is regulated by cathepsin L.

Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL(nkt/nkt) mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSL (nkt/nkt) mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSL (nkt/nkt) mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSL (nkt/nkt) mice. Besides, BM B-cell emigration to the spleen was increased in CTSL (nkt/nkt) mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSL (nkt/nkt) mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.

BCL6 breaks occur at different AID sequence motifs in Ig-BCL6 and non-Ig-BCL6 rearrangements.

BCL6 translocations are common in B-cell lymphomas and frequently have chromosomal breaks in immunoglobulin heavy chain (IgH) switch regions, suggesting that they occur during class-switch recombination. We analyze 120 BCL6 translocation breakpoints clustered in a 2156-bp segment of BCL6 intron 1, including 62 breakpoints (52%) joined to IgH, 12 (10%) joined to Ig light chains, and 46 (38%) joined to non-Ig partners. The BCL6 breaks in Ig-BCL6 translocations prefer known activation-induced cytosine deaminase (AID) hotspots such as WGCW and WRC (W = A/T, R = A/G), whereas BCL6 breaks in non-Ig rearrangements occur at CpG/CGC sites in addition to WGCW. Unlike previously identified CpG breaks in pro-B/pre-B-cell translocations, the BCL6 breaks do not show evidence of recombination activating gene or terminal deoxynucleotidyl transferase activity. Both WGCW/WRC and CpG/CGC breaks at BCL6 are most likely initiated by AID in germinal center B-cells, and their differential use suggests subtle mechanistic differences between Ig-BCL6 and non-Ig-BCL6 rearrangements.

Constitutive Akt1 signals attenuate B-cell receptor signaling and proliferation, but enhance B-cell migration and effector function.

Ligation of the BCR induces a complex signaling network that involves activation of Akt, a family of serine/threonine protein kinases associated with B-cell development, proliferation, and tumor formation. Here, we analyzed the effect of enhanced Akt1 signals on B-cell maturation and function. Unexpectedly, we found that peripheral B cells overexpressing Akt1 were less responsive to BCR stimuli. This correlated with a decrease in Ca(2+) -mobilization and proliferation, in an impaired activation of Erk1/2 and mammalian target of rapamycin (mTOR) kinases and poor mobilization of NFATc1 and NF-κB/p65 factors. In contrast, activation of STAT5 and migration of B cells toward the chemokine SDF1α was found to be enhanced. Akt1 Tg mice showed an altered maturation of peritoneal and splenic B1 B cells and an enhanced production of IgG1 and IgG3 upon immunization with the T-cell independent Ag TNP-Ficoll. Furthermore, mice homo-zygous for Tg Akt1 showed a severe block in the maturation of B-cell precursors in BM and a strong enrichment of plasma cells in spleen. Altogether, our data reveal that enhanced Akt1 signals modify BCR signaling strength and, thereby, B-cell development and effector function.

Suppressive effect of maslinic acid on PMA-induced protein kinase C in human B-lymphoblastoid cells.

Protein kinase C (PKC) has been implicated in carcinogenesis and displays variable expression profiles during cancer progression. Studies of dietary phytochemicals on cancer signalling pathway regulation have been conducted to search for potent signalling regulatory agents. The present study was designed to evaluate any suppressive effect of maslinic acid on PKC expression in human B-lymphoblastoid cells (Raji cells), and to identify the PKC isoforms expressed. Effects of maslinic acid on PKC activity were determined using a PepTag assay for non-radioactive detection of PKC. The highest expression in Raji cells was obtained at 20 nM PMA induced for 6 hours. Suppressive effects of maslinic acid were compared with those of four PKC inhibitors (H- 7, rottlerin, sphingosine, staurosporine) and two triterpenes (oleanolic acid and ursolic acid). The IC50 values achieved for maslinic acid, staurosporine, H-7, sphingosine, rottlerin, ursolic acid and oleanolic acid were 11.52, 0.011, 0.767, 2.45, 5.46, 27.93 and 39.29 µM, respectively. Four PKC isoforms, PKC ßI, ßII, δ, and ζ, were identified in Raji cells via western blotting. Maslinic acid suppressed the expression of PKC ßI, δ, and ζ in a concentration-dependent manner. These preliminary results suggest promising suppressive effects of maslinic acid on PKC activity in Raji cells. Maslinic acid could be a potent cancer chemopreventive agent that may be involved in regulating many downstream signalling pathways that are activated through PKC receptors.

Genetic and functional abnormalities of the melatonin biosynthesis pathway in patients with bipolar disorder.

Patients affected by bipolar disorder (BD) frequently report abnormalities in sleep/wake cycles. In addition, they showed abnormal oscillating melatonin secretion, a key regulator of circadian rhythms and sleep patterns. The acetylserotonin O-methyltransferase (ASMT) is a key enzyme of the melatonin biosynthesis and has recently been associated with psychiatric disorders such as autism spectrum disorders and depression. In this paper, we analysed rare and common variants of ASMT in patients with BD and unaffected control subjects and performed functional analysis of these variants by assaying the ASMT activity in their B-lymphoblastoid cell lines. We sequenced the coding and the regulatory regions of the gene in a discovery sample of 345 patients with BD and 220 controls. We performed an association study on this discovery sample using common variants located in the promoter region and showed that rs4446909 was significantly associated with BD (P= 0.01) and associated with a lower mRNA level (P< 10(-4)) and a lower enzymatic activity (P< 0.05) of ASMT. A replication study and a meta-analysis using 480 independent patients with BD and 672 controls confirmed the significant association between rs4446909 and BD (P= 0.002). These results correlate with the general lower ASMT enzymatic activity observed in patients with BD (P= 0.001) compared with controls. Finally, several deleterious ASMT mutations identified in patients were associated with low ASMT activity (P= 0.01). In this study, we determined how rare and common variations in ASMT might play a role in BD vulnerability and suggest a general role of melatonin as susceptibility factor for BD.

RhoA of the Rho family small GTPases is essential for B lymphocyte development.

RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA(flox/flox) mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19(Cre/+) significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA(-/-) B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development.

RAG-induced DNA double-strand breaks signal through Pim2 to promote pre-B cell survival and limit proliferation.

Interleukin 7 (IL-7) promotes pre-B cell survival and proliferation by activating the Pim1 and Akt kinases. These signals must be attenuated to induce G1 cell cycle arrest and expression of the RAG endonuclease, which are both required for IgL chain gene rearrangement. As lost IL-7 signals would limit pre-B cell survival, how cells survive during IgL chain gene rearrangement remains unclear. We show that RAG-induced DNA double-strand breaks (DSBs) generated during IgL chain gene assembly paradoxically promote pre-B cell survival. This occurs through the ATM-dependent induction of Pim2 kinase expression. Similar to Pim1, Pim2 phosphorylates BAD, which antagonizes the pro-apoptotic function of BAX. However, unlike IL-7 induction of Pim1, RAG DSB-mediated induction of Pim2 does not drive proliferation. Rather, Pim2 has antiproliferative functions that prevent the transit of pre-B cells harboring RAG DSBs from G1 into S phase, where these DNA breaks could be aberrantly repaired. Thus, signals from IL-7 and RAG DSBs activate distinct Pim kinase family members that have context-dependent activities in regulating pre-B cell proliferation and survival.

Activation-induced cytidine deaminase mediates central tolerance in B cells.

The Aicda gene product, activation-induced cytidine deaminase (AID), initiates somatic hypermutation, class-switch recombination, and gene conversion of Ig genes by the deamination of deoxycytidine, followed by error-prone mismatch- or base-excision DNA repair. These processes are crucial for the generation of genetically diverse, high affinity antibody and robust humoral immunity, but exact significant genetic damage and promote cell death. In mice, physiologically significant AID expression was thought to be restricted to antigen-activated, mature B cells in germinal centers. We now demonstrate that low levels of AID in bone marrow immature and transitional B cells suppress the development of autoreactivity. Aicda(-/-) mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature and transitional B cells. In vitro, AID deficient immature/transitional B cells are significantly more resistant to anti-IgM-induced apoptosis than their normal counterparts. Thus, early AID expression plays a fundamental and unanticipated role in purging self-reactive immature and transitional B cells during their maturation in the bone marrow.

Activation-induced cytidine deaminase (AID) is required for B-cell tolerance in humans.

Impaired immune functions leading to primary immunodeficiencies often correlate with paradoxical autoimmune complications; patients with hyper-IgM syndromes who are deficient in activation-induced cytidine deaminase (AID), which is required for class-switch recombination and somatic hypermutation, are prone to develop autoimmune diseases. To investigate the impact of AID-deficiency on early B-cell tolerance checkpoints in humans, we tested by ELISA the reactivity of recombinant antibodies isolated from single B cells from AID-deficient patients. New emigrant/transitional and mature naive B cells from AID-deficient patients express an abnormal Ig repertoire and high frequencies of autoreactive antibodies, demonstrating that AID is required for the establishment of both central and peripheral B-cell tolerance. In addition, B-cell tolerance was further breached in AID-deficient patients as illustrated by the detection of anti-nuclear IgM antibodies in the serum of all patients. Thus, we identified a major and previously unsuspected role for AID in the removal of developing autoreactive B cells in humans.

Defective nonhomologous end joining blocks B-cell development in FLT3/ITD mice.

We have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.

Integrated microfluidic and imaging platform for a kinase activity radioassay to analyze minute patient cancer samples.

Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric (32)P-ATP-labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL-positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples.

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells.

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.

Expression of IL-5Ralpha on B-1 cell progenitors in mouse fetal liver and involvement of Bruton's tyrosine kinase in their development.

B-1 cells are a subset of B cells responsible for the production of natural antibodies. Although the amount of natural antibody is tightly regulated, how this regulation occurs remains unknown. We examined the expression of IL-5 receptor, a cytokine receptor critical for homeostatic proliferation of B-1 cells, on B-1 cell progenitors in the fetal liver. We identified B-1 progenitors expressing low levels of IL-5 receptor alpha chain (IL-5Ralpha) and eosinophil progenitors expressing higher levels of IL-5Ralpha in the fetal liver. Moreover, the number of these B-1 progenitors were significantly reduced in the fetuses of mice deficient in Bruton's tyrosine kinase (Btk), even though IL-5 and thymic stroma lymphopoietin signaling are intact in early B lineage cells in Btk-deficient mice. These data suggest that IL-5 is possibly involved in B-1 cell development and an uncharacterized, Btk-dependent regulatory signaling pathway is involved in unexpectedly early stages of B-1 cell differentiation.