Glucocorticoids-Resistant Leukemic B-Cells Undergo a Phenotypic Change That Increases Sensitivity to SRC/ABL Inhibition

Abstract Glucocorticoids (GCs) have a crucial role in the treatment of B-cell acute lymphoblastic leukemia (B-ALL). GCs resistance is considered a strong prognostic marker of relapse, which occurs in about 20% of pediatric B-ALL patients. GCs exert therapeutic effects by inducing cell cycle arrest and eventually, cell death. However, their precise mechanisms of action and resistance are not fully understood. We have previously demonstrated the importance of developmental state in treatment failure in B-ALL (Good Z Nat Med 2018 and Bendall SC Cell 2014), specifically at the developmental transition between pro-B to pre-B cells. Using this developmental framework, we examined the effect of GCs on healthy and malignant early B cells progenitors to better understand mediators of GC resistance. To understand the transcriptional programs mediating early B-cell differentiation, we sorted three B-cell developmental populations (pre-pro-B, pro-B/pre-B I and pre-B II) and performed RNA-seq analysis. Ingenuity pathway analysis revealed a coordinated upregulation of B cell developmental genes and Glucocorticoid Receptor (GCR) pathway genes in healthy pre-B I cells, suggesting a role of GCs in healthy B-cell development. We confirmed expression of the GCR in healthy pre-B I cells by mass cytometry (CyTOF). Further, in vitro treatment of healthy B-cells with GCs (dexamethasone, dex 1 uM) demonstrated pre-B I cycling cells to be most sensitive to GC-induced cell death. Given the importance of GCs in B-ALL treatment, we investigated the GCR pathway and its relationship with B-cell development in 18 B-ALL primary samples (14 diagnosis, 4 relapse; including Ph+=1; MLL rearranged=2; TEL-AML translocated=3). Samples were profiled by CyTOF with a 40-antibody panel including surface markers, signaling molecules, transcription factors, apoptosis and cell cycle molecules. To better comprehend the relation of GCR pathway with BCR signaling, primary cells were analyzed after 48 hours of treatment with dex (1uM), SRC/ABL inhibitor dasatinib (das, 100 nM) or their combination. Similar to results obtained in healthy B-cells, we found the highest expression of GCR in the pre B-I cells in all patients (p<0.001). Interestingly, samples from relapse or diagnostic high-risk groups demonstrated the highest expression of GCR in this cell population. As expected, dex induced cell cycle arrest and cell death in almost all the samples (88%), while two samples were completely unresponsive. Moreover, combined treatment resulted in increased cell death in 29% of patients, compared to dex or das alone. Interestingly, we also observed consistent patterns of phenotypic modulation in dex-resistant cells and to understand how these were related to B-cell development we performed developmental classification (Good Z Nat Med 2018) of leukemic cells in dex-treated versus untreated conditions. This analysis revealed a large decrease of early B-cell progenitors and corresponding increase of more mature subsets of either B and non-B cells (false discovery rate, FDR<0.001), suggesting a differentiating effect of GCs. This phenomenon was even more prominent when we performed, for some primary samples (n=4), longer culture up to six days. Moreover, dex-resistant cells showed aberrant activation of prpS6 and pCREB that was blunted by the combination with das resulting in a reversion of the phenotype and killing of the majority of the cells. To determine if this effect was directly related to GCR expression, we compared results between a GCR+ cell line (NALM6) and GCR- cell line (REH). Consistent with the primary samples, the combination of dex with das more effectively killed NALM6 cells yet the REH were unresponsive to either treatment. However, overexpressing GCR in REH cells reversed dex-resistance, also becoming more sensitive to the combined treatment with das. Similar to primary cells, NALM6 and REH GCR+ cells showed a phenotypic change when treated with dex, that will be further investigated to clarify the relationship between the dex-induced phenotype and its apoptotic effect. In summary, these data suggest a dual role of glucocorticoids on early B-cells; mediating both apoptosis and differentiation in relationship to cell cycle status and GCR level. These findings may carry therapeutic implications and suggest cell types vulnerable to BCR signaling inhibition as a therapeutic intervention to overcome GCs resistance. Disclosures Nolan: Akoya Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Download Full-text

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3840 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3840-3847 ◽

Cited By ~ 44

Author(s):

H M Chen ◽

L M Boxer

Keyword(s):

Cell Death ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Regulatory Region ◽

Simian Virus 40 ◽

Simian Virus ◽

Cell Development ◽

B Cell Development ◽

Negative Regulators

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

Download Full-text

A Novel Molecular Complex Expressed on Immature B Cells: A Possible Role in T Cell-Independent B Cell Development

Developmental Immunology ◽

10.1155/1996/21973 ◽

1996 ◽

Vol 5 (1) ◽

pp. 67-78 ◽

Cited By ~ 5

Author(s):

Philip J. Griebel ◽

Paolo Ghia ◽

Ulf Grawunder ◽

Giorgio Ferrari

Keyword(s):

Bone Marrow ◽

Cell Death ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Molecular Complex ◽

Cell Development ◽

B Cell Development ◽

Lymphoid Tissues

To identify surface molecules that may play a role in regulating ileal Peyer's patch (PP) B cell growth, we generated monoclonal antibodies (mAbs) and then selected them for a unique reactivity with ileal PP B cells. Flow cytometric analysis identified a mAb (SIC4.8R) that labeled 97% of ileal and 50–60% of jejunal PP sIgM+B cells. SIC4.8R also labeled a subpopulation of cortical thymocytes but few B or T cells in other lymphoid tissues, including bone marrow. Immunohistochemistry revealed intense SIC4.8R staining of B cells in the cortex of ileal PP follicles. SIC4.8R also labeled bovine PP B cells, a murine pro-B cell line, and pre-B cells in human bone marrow. Protein chemistry revealed that a structurally similar molecular complex was expressed on sheep ileal PP B cells and thymocytes and murine pro-B cells. Addition of soluble SIC4.8R to cultured ileal PP B cells reduced apoptotic cell death, elevated proliferative responses, partially inhibited anti-Ig-induced cell death, and induced IL-4 responsiveness. In contrast, soluble SIC4.8R had an antiproliferative effect on a mouse pro-B cell line. Finally, SIC4.8R labeling declined following the stimulation of ileal PP B cells with CD40 ligand. In conclusion, the present investigation determined that SIC4.8R identified a novel molecular complex that is expressed at several stages of T cell-independent B cell development in a variety of mammalian species. This observation confirmed that PP B cells are developmentally distinct from other B cell populations in sheep and suggested that the bone marrow may not be a site of B lymphopoiesis in young lambs.

Download Full-text

E2F4 Plays a Critical Role in Early B-Cell Development.

Blood ◽

10.1182/blood.v104.11.318.318 ◽

2004 ◽

Vol 104 (11) ◽

pp. 318-318

Author(s):

Clayton Smith ◽

Michelle Glozak ◽

Maura Gasparetto ◽

Rachel Rempel ◽

Jos Domens ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Cell Cycle Progression ◽

Critical Role ◽

Cell Development ◽

B Cell Development ◽

Immunoglobulin Gene ◽

B Cell Differentiation ◽

Cycle Progression

Abstract The E2Fs are important mediators of cell cycle control, DNA synthesis and apoptosis in many cell types. Recently E2F4 has been shown to play a role in hematopoietic cell growth and development (Rempel et al. Mol Cell, 6 p293, 2000). Here we report the effects of loss of E2F4 specifically on B-cell development. E2F4−/− mice have a partial block in early B-cell development prior to immunoglobulin gene rearrangement. The block is intrinsic to B-cell progenitors rather than secondary to micro-environmental effects since it occurs following transplant of E2F4−/− marrow into wild type recipients. Increases in apoptosis and abnormalities in cell cycle progression were found in B220+CD43+ B-cells of E2F4−/− mice indicating that E2F4 plays an important role in these processes in early B-cells. Expression of a variety of genes important in B-cell development including E2A, RAG, IL-7, EBF and Pax-5 were decreased in early E2F4−/− B-cells. In contrast, Id1 and Id2, regulators of a variety of genes critical to B-cell development, were relatively over-expressed in early E2F4−/− B-cells while Id3 was relatively under-expressed in these cells. E2F binding sites were identified in the Id2 and Id3 promoters and E2F4 was found to directly bind to these promoters in splenic B-cells. These findings suggest that E2F4 may also regulate early B-cell development by directly and indirectly modulating expression of the genes critical to B-cell differentiation. Together, these observations indicate that E2F4 is a critical mediator of early B-cell development via its effects on multiple pathways including those involved with apoptosis, cell cycle progression and differentiation. These findings also suggest that the E2Fs may serve to link cell survival and proliferation pathways to differentiation pathways in early B-cells and perhaps other cells aswell.

Download Full-text

Polypyrimidine Tract Binding Proteins are essential for B cell development

10.1101/769141 ◽

2019 ◽

Author(s):

Elisa Monzón-Casanova ◽

Louise S. Matheson ◽

Kristina Tabbada ◽

Kathi Zarnack ◽

Christopher W. J. Smith ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Binding Protein ◽

Cell Development ◽

B Cell Development ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein ◽

Cell Ontogeny

AbstractPolypyrimidine Tract Binding Protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.

Download Full-text

Glucocorticoids Regulate the Splicing Factor MBNL1, a Potential Control Point for B-Cell Specification

Blood ◽

10.1182/blood-2019-131322 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2478-2478

Author(s):

Miles A. Pufall ◽

Mimi Fang ◽

Robert K. Bradley ◽

Chew Guo-Liang ◽

Ross Lanier ◽

...

Keyword(s):

Cell Death ◽

B Cells ◽

B Cell ◽

Research Funding ◽

Rna Binding ◽

Control Point ◽

Cell Development ◽

B Cell Development ◽

Splicing Factor ◽

Cell Specification

Glucocorticoids, such as dexamethasone and prednisone, are mainstays in the treatment of lymphoid malignancies and autoimmune diseases. To understand the mechanism of how glucocorticoids induce cell death, we integrated a comprehensive, genome-wide shRNA screen with differential gene expression analysis in B-cell acute lymphoblastic leukemia (B-ALL). In addition to activating pro-apoptosis genes and repressing anti-apoptosis genes, we found that repression of B-cell development genes contributes to dexamethasone-induced cell death in B-ALL. Similarly, elevated glucocorticoid levels brought on by stress in stroke victims causes lymphocytopenia by inducing of apoptosis in immature B-cells, but not hemopoietic stem cells or mature B-cells. Additionally, chronic stress has been associated with immune suppression and increased risk for autoimmune disease. Together these findings suggest that glucocorticoids play a normal role in B-cell development, and that elevated glucocorticoid levels in response to stress can alter that development. A control point in this mechanism may be MBNL1. MBNL1 is a sequence-specific RNA binding factor that alters mRNA splicing, stability, and localization. In this work, we show that glucocorticoid suppression of MBNL1 contributes to induction of cell death in B-ALL. Deletion of MBNL1 by CRISPR/Cas9 in the B-ALL cell line NALM-6 induces accumulation and depletion of mRNA levels for hundreds of genes. A large number of B-cell specification genes, including CD19, CD79A, SPI1, and LEF1, are repressed upon MBNL1 deletion, whereas the most highly activated gene is CD34, which is a marker for the less mature lymphoid progenitor cells. Depletion of MBNL1 also changed the splicing of B-cell development genes, including quantitative depletion of LEF1 exon 6. These data suggest that depletion of MBNL1 induces de-differentiation of B-cell precursors. Consistent with this model, an analysis of gene expression in differentiating B-cells revealed a rapid elevation of MBNL1 expression during B-cell specification (after the lymphoid precursor stage), and a concomitant repression of CELF2, an RNA-binding splicing factor that has been shown to oppose MBNL1. We therefore propose that glucocorticoids play a physiological role in B-cell development by restraining B-cell specification through repression of key genes, including MBNL1. Figure Disclosures Tasian: Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corportation: Research Funding.

Download Full-text

Polypyrimidine tract-binding proteins are essential for B cell development

eLife ◽

10.7554/elife.53557 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Elisa Monzón-Casanova ◽

Louise S Matheson ◽

Kristina Tabbada ◽

Kathi Zarnack ◽

Christopher WJ Smith ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Binding Protein ◽

Cell Development ◽

B Cell Development ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein ◽

Cell Ontogeny

Polypyrimidine tract-binding protein 1 (PTBP1) is a RNA-binding protein (RBP) expressed throughout B cell development. Deletion of Ptbp1 in mouse pro-B cells results in upregulation of PTBP2 and normal B cell development. We show that PTBP2 compensates for PTBP1 in B cell ontogeny as deletion of both Ptbp1 and Ptbp2 results in a complete block at the pro-B cell stage and a lack of mature B cells. In pro-B cells PTBP1 ensures precise synchronisation of the activity of cyclin dependent kinases at distinct stages of the cell cycle, suppresses S-phase entry and promotes progression into mitosis. PTBP1 controls mRNA abundance and alternative splicing of important cell cycle regulators including CYCLIN-D2, c-MYC, p107 and CDC25B. Our results reveal a previously unrecognised mechanism mediated by a RBP that is essential for B cell ontogeny and integrates transcriptional and post-translational determinants of progression through the cell cycle.

Download Full-text

The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs

Blood ◽

10.1182/blood.v112.11.2465.2465 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2465-2465

Author(s):

Wanda P. Blanton ◽

Fangnian Wang ◽

Hongsheng Liu ◽

Paul Romesser ◽

Douglas Faller ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Brdu Incorporation ◽

Cell Compartment ◽

Hematopoietic Stem

Abstract Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.

Download Full-text