Human pre-B Cell Research Articles

A novel mitochondrial pyruvate carrier inhibitor drives stem cell-like memory CAR T cell generation and enhances antitumor efficacy

Adoptive cell transfer with chimeric antigen receptor (CAR) expressing T cells can induce remarkable complete responses in cancer patients. Therapeutic success has been correlated with central and stem cell-like memory T cell subsets in the infusion product which are better able to drive efficient CAR T cell in vivo expansion and long-term persistence. We previously reported that inhibition of the mitochondrial pyruvate carrier (MPC) during mouse CAR T cell culture, induces a memory phenotype and enhances antitumor efficacy against melanoma. Here, we use a novel MPC inhibitor, MITO-66, which robustly induces a stem cell-like memory phenotype in CD19-CAR T cells generated from healthy donors and patients with relapsed/refractory B cell malignancies. MITO-66-conditioned CAR T cells were superior in controlling human pre-B cell acute lymphoblastic leukemia in mice. Following adoptive cell transfer, MITO-66-conditioned CAR T cells maintained a memory phenotype and protected cured mice against tumor rechallenge. Furthermore, in an in vivo B cell leukemia stress model, CD19-CAR T cells generated in the presence of MITO-66 largely outperformed clinical-stage AKT and PI-3Kδ inhibitors. Thus, we provide compelling preclinical evidence that MPC inhibition with MITO-66 during CAR T cell manufacturing dramatically enhances their antitumor efficacy, thereby paving the way to clinical translation.

Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842.

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2A-PBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2A-PBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPC) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1-induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2APBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish.

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression.

Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines.

Remodelling of Ca2+ homeostasis is linked to enlarged endoplasmic reticulum in secretory cells

The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca2+ store in the cell, we characterised the Ca2+ homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca2+ leak from ER, Ca2+ storage capacity and store-operated Ca2+ entry were significantly enlarged in the secretory RPMI 8226 cell line. Using an immunoglobulin M heavy chain producing HeLa cell model, we found that the enlarged Ca2+ storage capacity and Ca2+ leak from ER are linked to ER expansion. Our data delineates a developmental remodelling of Ca2+ homeostasis in professional secretory cells in which a high Sec61-mediated Ca2+ leak and, thus, a high Ca2+ turnover in the ER is backed up by enhanced store-operated Ca2+ entry.

Mechanistic basis for chromosomal translocations at the E2A gene and its broader relevance to human B cell malignancies.

SUMMARYAnalysis of translocation breakpoints in human B cell malignancies reveals that DNA double-strand breaks at oncogenes most frequently occur at CpG sites located within 20–600 bp fragile zones and depend on activation-induced deaminase (AID). AID requires single-stranded DNA (ssDNA) to act, but it has been unclear why or how this region transiently acquires a ssDNA state. Here, we demonstrate the ssDNA state in the 23 bp E2A fragile zone using several methods, including native bisulfite DNA structural analysis in live human pre-B cells. AID deamination within the E2A fragile zone does not require but is increased upon transcription. High C-string density, nascent RNA tails, and direct DNA sequence repeats prolong the ssDNA state of the E2A fragile zone and increase AID deamination at overlapping AID hotspots that contain the CpG sites at which breaks occur in patients. These features provide key insights into lymphoid fragile zones generally.

Reciprocal Regulation of BCL6 and MLL Expression As a Vulnerability in MLL -Rearranged Acute Lymphoblastic Leukemia

Background: Chromosomal translocations involving the Mixed Lineage Leukemia gene MLL account for ~70% of infant acute lymphoblastic leukemias (ALLs; Ayton and Cleary, 2001) and are associated with dismal prognosis. These findings underscore the importance of understanding the molecular mechanisms underlying disease progression of MLL-rearranged (MLLr) leukemia for the development of novel therapeutics. Recently, an integrative epigenomic analysis has identified the transcriptional repressor and proto-oncogene BCL6 as a transcriptional target in MLL-AF4 -driven pre-B ALL (Geng et al., 2012). BCL6 functions as a mediator of drug resistance to tyrosine kinase inhibitors in Ph+ ALL (Duy et al., 2011). Here, we tested the hypothesis that BCL6 is required for MLLr leukemia and represents a therapeutic target in MLLr pre-B ALL.Results: Western blot analysis of a panel of patient-derived pre-B ALL cells (MLLr : n= 7; ETV6-RUNX1 : n= 2; BCR-ABL1 : n= 1; not otherwise specified: n= 4) and normal CD19+ B cells (n= 3) revealed that BCL6 levels were elevated in MLL r ALL samples. Furthermore, immunohistochemistry staining in bone marrow biopsies from ALL patients (n= 72) showed that BCL6 levels were upregulated in MLLr cases. Expression of MLL-ENL or MLL-AF4 fusion protein increased Bcl6 protein levels (~10-fold) in murine pre-B cells. Moreover, ChIP-seq analysis of human MLLr pre-B ALL cell lines using antibodies against MLL, AF4 and ENL demonstrated direct binding to the BCL6 promoter. Studying methylation status revealed that the BCL6 promoter was hypomethylated in patients with MLLr pre-B ALL compared to patients with other subtypes of pre-B ALL (including BCR-ABL1, ETV6-RUNX 1 and hyperdiploid) as well as normal pre-B cells from healthy donors (St. Jude, n= 132, Figueroa, 2014). Consistent with these observations, multivariate analysis of high risk pre-B ALL patients showed that high BCL6 levels in combination with MLL rearrangements were associated with the worst clinical outcome (COG P9906). Collectively, our findings demonstrated that BCL6 levels are elevated in MLLr ALL, and that expression levels of MLL and BCL6 can serve as predictors for risk stratification in ALL.Interestingly, expression of wild-type MLL was positively correlated with that of BCL6 in both pediatric (COG P9906) and adult (ECOG E2993) pre-B ALL. Furthermore, multivariate analysis comparing MLLHighBCL6High patients with MLLLowBCL6Low patients showed that high expression levels of both genes correlated with poor clinical outcome (COG P9906). These findings prompted us to study whether MLL regulates BCL6 expression, and vice versa . Cre-mediated deletion of Mll abrogated imatinib-mediated induction of Bcl6 in BCR-ABL1 -driven pre-B ALL, indicating that Mll is required for upregulation of Bcl6 expression. While genetic inactivation of Bcl6 as well as treatment with the BCL6-specific retro-inverso peptide-inhibitor (RI-BPI) decreased Mll expression, doxycycline-induced expression of Bcl6 upregulated Mll expression. Combining chromatin immunoprecipitation and DNA microarray (ChIP-on-chip), we found that BCL6 was recruited to the MLL promoter in pre-B ALL. Taken together, these findings support our proposed scenario of reciprocal regulation of MLL and BCL6 expression.Notably, ChIP-seq analyses of MLLr pre-B ALL cells showed that there are 268 common gene targets of both MLL and BCL6. Among the common targets include cell cycle checkpoint regulators (CDKN1B, ETS2, BMI1, RB1), apoptosis facilitator BIM, and tumor suppressor TET2, indicating that BCL6 is a key regulator of the transcriptional program in MLLr pre-B ALL. In support of an essential role of BCL6 in MLLr ALL, expression of a dominant negative BCL6 mutant in patient-derived MLLr pre-B ALL cells induced rapid depletion from cell culture in competitive-growth assays. Furthermore, RI-BPI treatment resulted in cell cycle arrest, impaired colony forming ability and inhibited leukemia initiation in transplant recipient mice. Importantly, RI-BPI in combination with standard chemotherapy Vincristine exerted more potent effects on killing MLLr pre-B ALL cells than either agent alone.Conclusions: Our findings suggest that aberrant BCL6 expression levels are important for disease progression, and that pharmacological inhibition of BCL6 augments responsiveness to standard chemotherapy in the treatment of MLLr pre-B ALL. DisclosuresHurtz:Incyte: Research Funding. Armstrong:Cyteir: Consultancy; Imago: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

HOX GENE EXPRESSION IN HUMAN B-CELL PROGENITOR LEUKEMIA CELL LINES EXPRESSING E2A-PBX1 ONCOGENE

Introduction. Acute lymphoblastic leukemia (ALL) is diagnosed mainly in children (2/3 of diseases) making this type of leukemia one of the most common oncological diseases among children. Oncogenes are involved in the development of ALL, in particular the product of chromosomes 1 and 19 translocation, the oncogene E2A-PBX1 that codes for E2A-PBX1 chimeric oncoprotein with strong transcription activation properties as well as oncogenes of HOX family, mainly HOXA and HOXB cluster genes. E2A-PBX1 chimeric oncoprotein and НОХА proteins are associated in vivo with factors participating in epigenetic regulation of gene expression such as chromatin modifying and remodeling enzymes that partially determines their oncogenic properties. In previous studies we obtained data indicating genetic interactions of E2A-PBX1 and НОХ genes participating in leukemia development.The aim of this research was to confirm the role of Е2А – РВХ1 oncogene in the activation of the expression of НОХА cluster genes coding for the proteins with high oncogenic potential.Materials and methods. The objects of the study were four B cell progenitor (pre-B) leukemia cell lines: RCH-ACV, KASUMI-2, 697 and NALM-6. Standard polymerase chain reaction (PCR) was used for the identification of chromosome 1 and 19 translocation product, E2A-PBX1 oncogene and its expression. Method of reverse transcription coupled with quantitative polymerase chain reaction (Q-RT-PCR) was used for the analysis of 11 HOXA cluster genes expression.Results. It is demonstrated that E2A-PBX1 oncogene is present and expressed in three studied human pre-B leukemia cell lines, RCH-ACV, KASUMI-2 and 697, while its expression is absent in NALM-6 cell line. High expression of 7 from 11 HOXА cluster genes is revealed in RCH-ACV, KASUMI-2 and 697 cell lines expressing E2A-PBX1 oncogene, whereas NALM-6 cell line, that does not express E2A-PBX1 oncogene, also does not express HOXA genes except low expression of two genes from this cluster.Conclusions. The data obtained in this study demonstrate that RCH-ACV, KASUMI-2 and 697 human leukemia pre-B cell lines, containing and expressing Е2А-РВХ1 oncogene, also express most of HOXA genes (7 genes of 11 genes) at high level in contrast to control NALM-6 cell line that does not comprise Е2А-РВХ1 oncogene and almost does not express НОХА genes. Therefore, the results of this study suggest the participation of strong transcriptional activator, chimeric oncoprotein Е2А-РВХ1, associated with chromatin modifying and remodeling enzymes, in the expression activation of HOXA cluster genes that also possess high oncogenic potential.

MTA2/NuRD Regulates B Cell Development and Cooperates with OCA-B in Controlling the Pre-B to Immature B Cell Transition

SUMMARYThe NuRD complex contains both chromatin remodeling and histone deacetylase activities. Mice lacking the MTA2 subunit of NuRD show developmental defects in pro-B, pre-B, immature B, and marginal zone B cells, and abnormal germinal center B cell differentiation during immune responses. Mta2 inactivation also causes a derepression of Igll1 and VpreB1 genes in pre-B cells. Furthermore, MTA2/NuRD interacts directly with AIOLOS/IKAROS and shows a striking overlap with AIOLOS/IKAROS target genes in human pre-B cells, suggesting a functional interdependence between MTA2/NuRD and AIOLOS. Mechanistically, MTA2 deficiency in mice leads to increased H3K27 acetylation at both Igll1 and VpreB1 promoters. Gene profiling analyses also identify distinct MTA2-dependent transcription programs in pro-B and pre-B cells. In addition, we find a strong synergy between MTA2 and OCA-B in repressing Igll1 and VpreB1 at the pre-B cell stage, and in regulating both the pre-B to immature B transition and splenic B cell development.

CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

Acrofolione A and B, acetophenone dimers from Acronychia pendunculata, induce an apoptotic effect on human NALM-6 pre-B cell leukaemia cells.

We investigated the apoptotic activities of acrofolione A (1) and B (2) isolated from Acronychia pedunculata against a human pre-B cell leukaemia cell line (NALM-6) to explore the apoptosis-related signalling molecules targeted by 1 and 2. The apoptosis effects of 1 and 2 in NALM-6 cells were investigated by TUNEL staining, annexin V, mitochondria membrane potential and caspase 3/7 activity. We carried out a protein array to explore the signalling molecules involved in apoptosis comprehensively. Acrofolione A (1) suppressed the growth of NALM-6, K562 and HPB-ALL cells (IC50 16.7 ± 1.9, 17.9 ± 0.3 and 10.1 ± 0.2 μm, respectively) more effectively than acrofolione B (2). Both compounds time-dependently increased the number of NALM-6 cells with abnormal nuclei, and increased the number of annexin V-positive cells and decreased the mitochondrial membrane potential of NALM-6 cells. Acrofolione A (1) markedly elevated caspase 3/7 activity and increased the number of TUNEL-positive cells. Cells treated with either compound showed enhanced expression of cleaved PARP and cleaved caspase 3 and 7, and reduced survivin protein levels. Acrofolione A (1) and B (2) may be useful in the treatment of various types of leukaemia.

TACI Isoforms Regulate Ligand Binding and Receptor Function.

TACI signals activate B cell proliferation, isotype switch and antibody production in both normal immunity and autoimmune states. In contrast to murine TACI, the human TACI gene undergoes alternative splicing to produce short and long isoforms (TACI-S and TACI-L). In previous studies, we showed that transduction of the short, but not long isoform, into murine B cells or human pre-B cells lacking TACI, caused them to become transcriptional and morphologically identical to plasma cells. These data suggest that the expression of different isoforms in humans provides unique controls on B cell maturation. In these studies we show that TACI-S and TACI-L form complexes in a ligand-independent manner, not dependent on a single extracellular domain. Both TACI isoforms are detectable in the endosomal cellular compartment where they co-localize with MyD88, TRAF6, and the activated 65 kDa form of TLR9, depending on a conserved intracellular TACI sequence. In contrast to TACI-L expressing cells, or cells bearing both isoforms, TACI-S binds ligands BAFF and APRIL with substantially greater affinity and promotes enhanced NF-kB activation. Using isoform-specific monoclonal antibodies, we show that while TACI-L is predominant as a surface receptor surface on human B cells, significantly more TACI-S is noted in the intracellular compartment and also in marginal zone, isotype switched and plasmablast in resting B cells. TACI-S is increased in tonsillar B cells and also in the intracellular compartment of activated peripheral B cells. These data shows that alternative splicing of the human TACI gene leads to two isoforms both of which intersect with MyD88 and TRAF6 and form complexes with TLR9, but the two isoforms have different ligand binding capacities, subcellular locations and activation capabilities.

Hesperidin inhibits insulin-induced phosphoinositide 3-kinase/Akt activation in human pre-B cell line NALM-6.

It has been shown that hesperidin induces apoptosis in NALM-6 cells through inhibition of nuclear factor-kappa B (NF-κB) activation. To investigate the effect of hesperidin on inhibition of NF-κB activation through blocking phosphoinositide 3-kinase (PI3K)/Akt pathway as a main target in cancer treatment, in NALM-6 cells. NALM-6 cells were incubated with two concentrations of hesperidin (25, 50 μM) in the presence or absence of insulin (100 nM), as a potent activator of Akt. The cytotoxic activity of hesperidin was determined by 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptotic death was measured by ELISA test using cell death detection ELISA Plus kit. To assay the effect of hesperidin on Akt pathway, the phosphorylation levels of Akt, inhibitor of kappa B alpha (IκBα), and glycogen synthase kinase-3 beta (GSK-3β) and expression level of IκB kinase alpha (IKKα) were determined by Western blot analysis. Hesperidin (both concentrations) significantly reduced cells survival in the presence and absence of insulin compared to untreated cells in a time-dependent manner (P < 0.05). Hesperidin also significantly increased apoptosis in NALM-6 cells even in hyperinsulinemia condition (P < 0.0001). Hesperidin inhibited insulin-induced phosphorylation and activation of Akt, IκBα, and GSK-3β and decreased expression of IKKα. The results of this study demonstrated that cytotoxic and proapoptotic actions of hesperidin are partly mediated through the suppression of PI3K3/Akt/IKK signaling pathway. So, hesperidin might act as a chemotherapeutic agent by targeting cell survival pathways.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

CREB knockdown inhibits growth and induces apoptosis in human pre-B acute lymphoblastic leukemia cells through inhibition of prosurvival signals.

A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation. The gene expression levels of prosurvival signals Bcl-2, Mcl-1, Bcl-xL, survivin and XIAP were down-regulated upon CREB suppression. These findings indicate a critical role for CREB in proliferation, survival, and apoptosis of BCP-ALL cells. The data also suggest that CREB could possibly serve as potential therapeutic target in BCP-ALL.