FOXM1 Mediates Drug-Resistance and Represents a Therapeutic Target in Pre-B Acute Lymphoblastic Leukemia

Abstract Background & Significance: Pre-B acute lymphoblastic leukemia (ALL) emerges in virtually all cases from B cell precursors that are arrested at the pre-B cell receptor checkpoint. In a gene expression survey of early B cell development, we found specific upregulation of FOXM1 at this developmental stage. FOXM1 belongs to the forkhead box transcription factor family and is a key regulator of cell growth by promoting cell cycle progression and has been implicated in progression of solid tumors. Therefore, we characterized the function and regulation of FOXM1 in normal B cell development as well as in pre-B ALL. Results: First, we verified the upregulation of FOXM1 during B cell development by qRT-PCR on sorted human and murine B cell progenitor populations. Then, we crossed Mb1-Cre tg mice to a Foxm1 conditional knockout mouse model (Foxm1fl/fl) and analyzed the early B cell populations according to the Hardy fractions. Despite the observed high expression of Foxm1 mRNA in fraction C’ and D, Foxm1 deletion did not alter B cell development. In order to investigate a potential role of FOXM1 in transformed B cells, we compared FOXM1 protein levels in patient-derived pre-B ALL samples with healthy B cells and B cell precursors and found 10-60-fold higher expression in the transformed B cell progenitors. To evaluate a potential predictive value of FOXM1 levels in patient-derived ALL samples, we measured FOXM1 mRNA levels at the time of diagnosis which strikingly correlate with risk stratification of ALL (intermediate-risk ALL n=31 vs. high risk ALL n=21; P=7.3e-5; BFM-REZ 2002). To further study the function and regulation of FOXM1, we cultured murine B cell precursors in the presence of IL7 and induced transformation with a retroviral BCR-ABL1 expression vector. BCR-ABL1 expression increased levels of FOXM1 compared to the normal IL7-dependent pre-B cells. Short-term inhibition of BCR-ABL1 did not affected protein levels of FOXM1. However, after 4 days of tyrosine kinase inhibition (TKI) treatment, FOXM1 protein levels were significantly downregulated in a dose-dependent manner. BCL2 overexpression prevented apoptosis induction by TKI but FOXM1 downregulation was retained. In addition, we found evidence that inactivation of FOXO factors by the PI3K/AKT pathway contributes to high FOXM1 expression in Ph+ALL. Overexpression of a constitutively active form of Akt to prevent activation of FOXO factors in the presence of TKI abrogated FOXM1 downregulation. Similarly, BCR-ABL1+ ALL derived from FOXO3a knockout mice prevented TKI-mediated FOXM1 reduction. Overexpression of a constitutively active form of FOXO3a but not FOXO1 significantly reduces levels of FOXM1 expression. In line with this, we found a significant inverse correlation of FOXM1 with FOXO3A mRNA levels in Ph+ ALL patients from the ECOG E2993 trial. However, the requirement of long-term treatment indicates, that, in addition to FOXO3a activation, epigenetic regulation of the FOXM1 promoter downstream of BCR-ABL1 is required. Consistent with this finding, the FOXM1 promoter region was found to be de-methylated in a large fraction of ALL. In order to further study FOXM1 function, we transduced pre-B cells derived from Foxm1fl/fl mice with BCR-ABL1 and with an inducible ERT2-Cre vector. Deletion of Foxm1 in BCR-ABL1-driven leukemia decreases cell viability, colony formation, and proliferative capacity in vitro as well as leukemia formation in vivo. FOXM1-deleted ALL cells revealed a strikingly higher sensitivity towards TKI-treatment compared to the control cells in Imatinib dose-response curves (IC50 EV:420 nM vs IC50 Cre-ERT2: 160 nM) as well as annexin V staining. We identified the ROS scavenger Catalase as a critical target of FOXM1 in mediating this drug resistance. As potential therapeutic agents to target FOXM1, we evaluated the effects of a previously described ARF peptide and the natural occurring antibiotic Thiostrepton. Both bind FOXM1 and inhibit its function as shown by reduced mRNA expression of FOXM1 target genes (Cyclin B1, PLK1, AURKB) and induced apoptosis in ALL and prolonged survival of patient-derived ALL transplant recipient mice. Conclusion: We have identified a critical function of the transcription factor FOXM1 in mediating proliferation and drug-resistance in B cell lineage ALL, but not in normal B cell progenitors and validated FOXM1 as a therapeutic target in a large fraction of drug-resistant B cell lineage ALL. Disclosures No relevant conflicts of interest to declare.

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Precursor B Cell Acute Lymphoblastic Leukemia Induces Pro-Leukemic Changes in the Bone Marrow Microenvironment That Contribute to Therapeutic Resistance

Blood ◽

10.1182/blood.v120.21.1466.1466 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1466-1466

Author(s):

Christopher D Chien ◽

Elizabeth D Hicks ◽

Paul P Su ◽

Haiying Qin ◽

Terry J Fry

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Bone Marrow Microenvironment ◽

Therapeutic Resistance

Abstract Abstract 1466 Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although cure rates for this disease are approximately 90%, ALL remains one of the leading causes cancer-related deaths in children. Thus, new treatments are needed for those patients that do not respond to or recur following standard chemotherapy. Understanding the mechanisms underlying resistance of pediatric ALL to therapy offers one approach to improving outcomes. Recent studies have demonstrated the importance of communication between cancer cells and their microenvironment and how this contributes to the progression and therapeutic resistance but this has not been well studied in the context of ALL. Since the bone marrow is presumed to be the site of initiation of B precursor ALL we set out in our study to determine how ALL cells utilize the bone marrow milieu in a syngeneic transplantable model of preB cell ALL in immunocompetent mice. In this model, intravenously injected preB ALL develops first in the bone marrow, followed by infiltration into the spleen, lymph node, and liver. Using flow cytometry to detect the CD45.2 isoform following injection into B6CD45.1+ congenic recipients, leukemic cells can be identified in the bone marrow as early as 5 days after IV injection with a sensitivity of 0.01%-0.1%. The pre-B ALL line is B220+/CD19+/CD43+/BP1+/IL-7Ralpha (CD127)+/CD25-/Surface IgM-/cytoplasmic IgM+ consistent with a pre-pro B cell phenotype. We find that increasing amounts of leukemic infiltration in the bone marrow leads to an accumulation of non-malignant developing B cells at stages immediately prior to the pre-pro B cell (CD43+BP1-CD25-) and a reduction in non-malignant developing pre B cells at the developmental stage just after to the pre-pro B cell stage (CD43+BP1+CD25+). These data potentially suggest occupancy of normal B cell developmental niches by leukemia resulting in block in normal B cell development. Further supporting this hypothesis, we find significant reduction in early progression of ALL in aged (10–12 month old) mice known to have a deficiency in B cell developmental niches. We next explored whether specific factors that support normal B cell development can contribute to progression of precursor B cell leukemia. The normal B cell niche has only recently been characterized and the specific contribution of this niche to early ALL progression has not been extensively studied. Using a candidate approach, we examined the role of specific cytokines such as Interleukin-7 (IL-7) and thymic stromal lymphopoietin (TSLP) in early ALL progression. Our preB ALL line expresses high levels of IL-7Ralpha and low but detectable levels of TLSPR. In the presence of IL-7 (0.1 ng/ml) and TSLP (50 ng/ml) phosphSTAT5 is detectable indicating that these receptors are functional but that supraphysiologic levels of TSLP are required. Consistent with the importance of IL-7 in leukemia progression, preliminary data demonstrates reduced lethality of pr-B cell ALL in IL-7 deficient mice. Overexpression of TSLP receptor (TSLPR) has been associated with high rates of relapse and poor overall survival in precursor B cell ALL. We are currently generating a TSLPR overepressing preBALL line to determine the effect on early ALL progression and are using GFP-expressing preB ALL cells to identify the initial location of preB ALL occupancy in the bone marrow. In conclusion, or model of early ALL progression provides insight into the role of the bone marrow microenvironment in early ALL progression and provides an opportunity to examine how these microenvironmental factors contribute to therapeutic resistance. Given recent advances in immunotherapy for hematologic malignancies, the ability to study this in an immunocompetent host will be critical. Disclosures: No relevant conflicts of interest to declare.

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Long noncoding RNAs in B-cell development and activation

Blood ◽

10.1182/blood-2015-11-680843 ◽

2016 ◽

Vol 128 (7) ◽

pp. e10-e19 ◽

Cited By ~ 48

Author(s):

Tiago F. Brazão ◽

Jethro S. Johnson ◽

Jennifer Müller ◽

Andreas Heger ◽

Chris P. Ponting ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Cell Lineage ◽

Noncoding Rnas ◽

Cell Development ◽

B Cell Development ◽

Long Noncoding Rnas ◽

Human B Cells

AbstractLong noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia.

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Dysregulation of PAX5 causes uncommitted B cell development and tumorigenesis in mice

10.1101/2021.01.29.428877 ◽

2021 ◽

Author(s):

Brigette Boast ◽

Kaiyue Helian ◽

T. Daniel Andrews ◽

Xi Li ◽

Vicky Cho ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Protein Function ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Causal Effects ◽

Stop Mutation ◽

Cell Acute Lymphoblastic Leukemia ◽

Key Pathways

AbstractPAX5 is the master transcription factor controlling B cell identity. In humans, mutations in PAX5 account for 30% of B cell acute lymphoblastic leukemia (B-ALL) cases. Investigating the causal effects of PAX5 mutations has however been difficult due to the premature lethality of Pax5−/− mice. Here we describe a novel mouse strain with a premature STOP mutation in Pax5 (Y351*) that produces a truncated protein and reduction in protein function, yet still allows for some B cell development to occur. A population of uncommitted and multipotent CD19+B220− B cells develops in the bone marrow of homozygous mice leading to the development of B-ALL. We show that the tumors frequently acquire secondary mutations in Jak3, and Ptpn11 highlighting key pathways interacting with PAX5 during malignant transformation. Analysis of the PAX5Y351* mice provide insight not only into the functional consequence of reduced PAX5 activity on B cell development and identity, but also provides an avenue in which to study PAX5-driven B-ALL in mice.One Sentence SummaryReduction in PAX5 function in mice induces the development of uncommitted B cells that have multipotent and malignant potential.

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Immunoglobulin Heavy Chain (IgH) Knockout Inhibits Proliferation of Pre-BCR+ B-Cell Acute Lymphoblastic Leukemia (B-ALL) Via a FOXO1 and MYC Dependent Mechanism

Blood ◽

10.1182/blood.v124.21.287.287 ◽

2014 ◽

Vol 124 (21) ◽

pp. 287-287

Author(s):

Stefan Koehrer ◽

Ondrej Havranek ◽

R Eric Davis ◽

Felix Seyfried ◽

Greg Coffey ◽

...

Keyword(s):

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Total Protein ◽

Lymphoblastic Leukemia ◽

Cell Development ◽

B Cell Development ◽

Protein Levels ◽

Bcr Signaling ◽

Cell Acute Lymphoblastic Leukemia

Abstract A pivotal step during B-cell development is the expression of the precursor B-cell receptor (pre-BCR) by pre-B lymphocytes (cyto-Igµ+, surface-IgM-). The pre-BCR represents an immature form of the BCR and consists of two immunoglobulin heavy chains (IgH), two surrogate light chains (SLC) and the signal transducing adapter proteins Igα and Igβ. A functional pre-BCR drives proliferation of pre-B-cells, ensuring their further differentiation into mature B-cells. By immunophenotype, ~20% of B-cell acute lymphoblastic leukemia (B-ALL) cases originate from the pre-B-cell stage (pre-B-ALL) of lymphocyte development and might therefore also express the pre-BCR. In view of the importance of pre-BCR signaling for normal pre-B-cell development, we hypothesize that it is exploited by pre-B-ALL for malignant growth and proliferation. A hallmark of active pre-BCR signaling is the continuous internalization of pre-BCRs, resulting in low pre-BCR surface expression. Using this phenotype of active pre-BCR signaling (low pre-BCR expression and high phosphorylation of the pre-BCR associated kinases LYN and SYK), we identified pre-BCR+ ALL cell lines (RCH-ACV, SMS-SB and Nalm6) and xenograft expanded patient samples. To study the role of the pre-BCR in these cells, we rendered RCH-ACV and SMS-SB pre-BCR null by using CRISPR/CAS9 gene editing with guide RNAs specific for the hypervariable region (recombined V, D, and J segments) of their expressed IgH allele. As identified by flow cytometry for the pre-BCR, deficient RCH-ACV and SMS-SB cells exhibited reduced viability and impaired proliferation when compared to their pre-BCR+ controls (Figure 1). Pre-BCR- cells showed reduced baseline phosphorylation of CD19, VAV1 and AKT. Interestingly, BTK and ERK phosphorylation were not affected. These results provide evidence for the dependency of pre-BCR+ ALL on pre-BCR signaling and suggest selective involvement of the PI3K-AKT pathway. We also investigated the effects of pharmacological pre-BCR inhibition by treating pre-BCR+ and pre-BCR- ALL cell lines and xenograft expanded primary patient samples with PRT318, a small-molecule inhibitor of spleen tyrosine kinase (SYK). In pre-BCR+ ALL PRT318 blocked cell proliferation and selectively inhibited AKT phosphorylation, thus mimicking the effects of IgH knockout. Pre-BCR- ALL cells were resistant to PRT318. Key effectors of the pre-BCR during normal B-cell development are FOXO transcription factors. In line with this, we found reduced FOXO1 phosphorylation and increased FOXO1 total protein levels after IgH knockout as well as after treatment with PRT318. This was accompanied by an increase in the FOXO1 transcriptional targets p27 and BLNK, suggesting increased FOXO1 transcriptional activity in response to the inhibition of pre-BCR signaling. To study the contribution of FOXO1 to the effects of IgH knockout and SYK inhibition more thoroughly, we expressed constitutively active FOXO1 (FOXO1-3A) in the pre-BCR+ ALL cell line RCH-ACV and consequently assessed its effects on cell proliferation and protein expression. Similar to IgH knockout and PRT318, FOXO1-3A reduced cell proliferation and increased p27 and BLNK protein levels, confirming FOXO1 as an important downstream target of pre-BCR signaling in B-ALL. To identify additional effectors of the pre-BCR in B-ALL we performed gene expression profiling (GEP) to compare pre-BCR+ and pre-BCR- cells of RCH-ACV and SMS-SB. Gene set enrichment analysis (GSEA) showed that IgH knockout resulted in significant enrichment for gene sets associated with down-modulation of MYC activity. This was confirmed by Western blot analysis of MYC total protein levels, and consistent with the finding of reduced MYC protein in PRT318-treated and FOXO1-3A-expressing pre-BCR+ cells, all indicating that pre-BCR signaling modulates MYC activity through a mechanism involving SYK and FOXO1. In conclusion, we provide evidence for the dependence of certain B-ALL subgroups on pre-BCR signaling. According to our data this is mainly due to pre-BCR-induced inactivation of FOXO1 and the subsequent deregulation of MYC. Importantly, pharmacological inhibition of pre-BCR signaling with the SYK inhibitor PRT318 completely reversed these effects, therefore providing a rationale for the use of SYK inhibitors in pre-BCR+ subgroups of B-ALL. Figure 1: Figure 1 Figure 1. Disclosures Coffey: Portola Pharmaceuticals: Employment, Equity Ownership.

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A Fail-safe Mechanism for Negative Selection of Isotype-switched B Cell Precursors Is Regulated by the Fas/FasL Pathway

Journal of Experimental Medicine ◽

10.1084/jem.20030357 ◽

2003 ◽

Vol 198 (10) ◽

pp. 1609-1619 ◽

Cited By ~ 25

Author(s):

Jane Seagal ◽

Efrat Edry ◽

Zohar Keren ◽

Nira Leider ◽

Ofra Benny ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Peripheral Tolerance ◽

Developmental Pathway ◽

Isotype Switching ◽

T Cell Help ◽

B Cell Precursors

In B lymphocytes, immunoglobulin (Ig)M receptors drive development and construction of naive repertoire, whereas IgG receptors promote formation of the memory B cell compartment. This isotype switching process requires appropriate B cell activation and T cell help. In the absence of T cell help, activated B cells undergo Fas-mediated apoptosis, a peripheral mechanism contributing to the establishment of self-tolerance. Using Igμ-deficient μMT mouse model, where B cell development is blocked at pro-B stage, here we show an alternative developmental pathway used by isotype-switched B cell precursors. We find that isotype switching occurs normally in B cell precursors and is T independent. Ongoing isotype switching was found in both normal and μMT B cell development as reflected by detection of IgG1 germline and postswitch transcripts as well as activation-induced cytidine deaminase expression, resulting in the generation of IgG-expressing cells. These isotype-switched B cells are negatively selected by Fas pathway, as blocking the Fas/FasL interaction rescues the development of isotype-switched B cells in vivo and in vitro. Similar to memory B cells, isotype-switched B cells have a marginal zone phenotype. We suggest a novel developmental pathway used by isotype-switched B cell precursors that effectively circumvents peripheral tolerance requirements. This developmental pathway, however, is strictly controlled by Fas/FasL interaction to prevent B cell autoimmunity.

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Regulation of B-cell development by BCAP and CD19 through their binding to phosphoinositide 3-kinase

Blood ◽

10.1182/blood-2007-08-109769 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1497-1503 ◽

Cited By ~ 96

Author(s):

Yuichi Aiba ◽

Megumi Kameyama ◽

Tetsuo Yamazaki ◽

Thomas F. Tedder ◽

Tomohiro Kurosaki

Keyword(s):

B Cells ◽

B Cell ◽

Active Form ◽

Cell Development ◽

B Cell Development ◽

Activation Mechanism ◽

Developmental Defect ◽

Akt Activation ◽

Pi3k Activation ◽

Phosphoinositide 3 Kinase

Abstract Despite the importance of phosphoinositide 3-kinase (PI3K) in B-cell development, its activation mechanism still remains elusive. In this study, we show that deletion of both BCAP and CD19 leads to an almost complete block of BCR-mediated Akt activation and to severe defects in generation of immature and mature B cells. The YXXM motifs in BCAP and CD19 are crucial for regulating B-cell development in that mutation of these motifs abrogated their ability to induce BCR-mediated Akt activation as well as to promote B-cell development. Furthermore, the developmental defect in CD19−/−BCAP−/− B cells was partly relieved by introducing a constitutively active form of PI3K or PDK1. Together, our data suggest that BCAP and CD19 have complementary roles in BCR-mediated PI3K activation, thereby, at least in part, contributing to B-cell development.

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Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20101947 ◽

2011 ◽

Vol 208 (6) ◽

pp. 1135-1149 ◽

Cited By ~ 92

Author(s):

Lynn M. Heltemes-Harris ◽

Mark J.L. Willette ◽

Laura B. Ramsey ◽

Yi Hua Qiu ◽

E. Shannon Neeley ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Active Form ◽

Cell Development ◽

B Cell Development ◽

Leukemic Cells ◽

Small Perturbations ◽

Potential Tumor Suppressor

As STAT5 is critical for the differentiation, proliferation, and survival of progenitor B cells, this transcription factor may play a role in acute lymphoblastic leukemia (ALL). Here, we show increased expression of activated signal transducer and activator of transcription 5 (STAT5), which is correlated with poor prognosis, in ALL patient cells. Mutations in EBF1 and PAX5, genes critical for B cell development have also been identified in human ALL. To determine whether mutations in Ebf1 or Pax5 synergize with STAT5 activation to induce ALL, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice heterozygous for Ebf1 or Pax5. Haploinsufficiency of either Pax5 or Ebf1 synergized with Stat5b-CA to rapidly induce ALL in 100% of the mice. The leukemic cells displayed reduced expression of both Pax5 and Ebf1, but this had little effect on most EBF1 or PAX5 target genes. Only a subset of target genes was deregulated; this subset included a large percentage of potential tumor suppressor genes and oncogenes. Further, most of these genes appear to be jointly regulated by both EBF1 and PAX5. Our findings suggest a model whereby small perturbations in a self-reinforcing network of transcription factors critical for B cell development, specifically PAX5 and EBF1, cooperate with STAT5 activation to initiate ALL.

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Gradient of E2A Activity in B-Cell Development

Molecular and Cellular Biology ◽

10.1128/mcb.22.3.886-900.2002 ◽

2002 ◽

Vol 22 (3) ◽

pp. 886-900 ◽

Cited By ~ 61

Author(s):

Sabine Herblot ◽

Peter D. Aplan ◽

Trang Hoang

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Gene Dosage ◽

Lymphoblastic Leukemia ◽

Control Cell ◽

Cell Development ◽

B Cell Development ◽

Immunoglobulin M ◽

B Lineage

ABSTRACT The E2A locus is a frequent target of chromosomal translocations in B-cell acute lymphoblastic leukemia (B-ALL). E2A encodes two products, E12 and E47, that are part of the basic helix-loop-helix (bHLH) family of transcription factors and are central in B lineage differentiation. E2A haplo-insufficiency hinders progression through three major checkpoints in B-cell development: commitment into the B lineage, at the pro-B to pre-B transition, and in the induction of immunoglobulin M (IgM) expression required for a functional BCR. These observations underscore the importance of E2A gene dosage in B-cell development. Here we show that a higher proportion of pro-B cells in E2A+/− mice is in the cell cycle compared to that in wild-type littermates. This increase correlates with lower p21waf/cip1 levels, indicating that E2A has an antiproliferative function in B-cell progenitors. Ectopic expression in the B lineage of SCL/Tal1, a tissue-specific bHLH factor that inhibits E2A function, blocks commitment into the B lineage without affecting progression through later stages of differentiation. Furthermore, ectopic SCL expression exacerbates E2A haplo-insufficiency in B-cell differentiation, indicating that SCL genetically interacts with E2A. Taken together, these observations provide evidence for a gradient of E2A activity that increases from the pre-pro-B to the pre-B stage and suggest a model in which low levels of E2A (as in pro-B cells) are sufficient to control cell growth, while high levels (in pre-B cells) are required for cell differentiation. The antiproliferative function of E2A further suggests that in B-ALL associated with t(1;19) and t(17;19), the disruption of one E2A allele contributes to leukemogenesis, in addition to other anomalies induced by E2A fusion proteins.

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Regulation of Homeostatic and Malignant B Cell Development By the Tetraspanin CD53

Blood ◽

10.1182/blood-2018-99-111957 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3699-3699

Author(s):

Zev J. Greenberg ◽

Darlene A. Monlish ◽

Rachel L. Bartnett ◽

Laura G. Schuettpelz

Keyword(s):

B Cells ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Mapk Signaling ◽

Cell Development ◽

B Cell Development ◽

Malignant Cells ◽

Hematopoietic Stem ◽

Cell Counts ◽

B Lineage

Abstract Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, most commonly originating from the transformation of progenitor cells of the B cell lineage (B cell precursor-ALL; BCP-ALL). Treatment of patients with high-risk or relapsed disease is difficult and prognosis remains poor in pediatric patients, with an even worse survival rate for adult BCP-ALL. Previous studies have shown an association of enhanced CD53 expression with many B cell malignancies, suggesting upregulation of CD53 may be implicated in carcinogenesis or maintenance of malignant cells. CD53 is a member of the tetraspanin family of transmembrane proteins, classically involved in cell adhesion, proliferation, and survival, and expressed exclusively on hematopoietic cells. While several studies have implicated a role for CD53 in regulating mature B cell proliferation, its role in early B cell development is not yet known. To elucidate the contribution of CD53 to normal and malignant B cell development, we have generated a CD53 knockout mouse. In our CD53-/- mouse, we observe no differences in total white blood cell counts, yet the fraction of peripheral blood B cells is significantly reduced by 31% compared to wild-type (WT) controls (28.3% vs. 19.5%; p<0.005). During homeostatic B lymphopoiesis, CD53 increases through development, beginning at the pre-pro-B cell stage and reaching highest expression on mature B cells. Further investigation into the loss of B cells revealed that immature pre-B cells in the bone marrow and mature B cells in the spleen and lymph nodes are significantly diminished upon loss of CD53, resulting from increased apoptosis in CD53-/- mice. B cell differentiation of CD53-/- hematopoietic stem cells (HSCs) in vitro corroborates the dependence on CD53 for normal differentiation, as CD53-/- cultures have 26% fewer B cells than controls (p=0.033). Investigation into the signaling differences between WT and CD53-/- B cell progenitors by mass cytometry (CyTOF) suggests that decreased PI3K/Akt and MAPK signaling could be driving this loss. With the observed loss of both B cell progenitors and mature B cells in CD53-deficient mice, CD53-/- mice were recently crossed to Eμ-Myc transgenic mice, a model of B-lineage leukemia/lymphoma, to generate WT, CD53-/-, Eμ-Myc+;CD53+/+, and Eμ-Myc+;CD53-/- groups to assess whether loss of CD53 alters the pathology or survival of these mice. As observed in human patients, moribund Eμ-Myc+ mice significantly upregulate CD53 on malignant cells, suggesting a potential role for CD53 during pathogenesis. Ongoing experiments are aimed at elucidating the mechanism by which CD53 promotes homeostatic B cell development and determining the potential of CD53 as a therapeutic target for B lineage malignancies. Disclosures No relevant conflicts of interest to declare.

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NEK2 Regulates Immature B Cell Development and GC Formation in NEK2 Transgenic Mouse

Blood ◽

10.1182/blood.v120.21.257.257 ◽

2012 ◽

Vol 120 (21) ◽

pp. 257-257

Author(s):

Wen Zhou ◽

Xiao He ◽

He Wang ◽

Mohamed E Salama ◽

Xia Jiliang ◽

...

Keyword(s):

Drug Resistance ◽

B Cells ◽

B Cell ◽

Transgenic Mouse ◽

Cell Development ◽

B Cell Development ◽

Cell Surface Expression ◽

Brdu Incorporation ◽

Marginal Zone B Cells ◽

Immature B Cells

Abstract Abstract 257 Background: NEK2 is a cell cycle-regulated kinase of the never in mitosis A (NIMA) family that is highly enriched at the centrosome. We discovered that NEK2 expression was highly correlated to drug resistance, rapid relapse, and poor outcome in myeloma and multiple other cancers. Given that plasma cells in myeloma are originated from differentiated B cell lineage, we generated a B cell specific NEK2 transgenic mouse line, controlling by a B cell specific CD19 promoter, to explore the functions of NEK2 in B cell development. Materials and Methods: Floxed STOP-polyA -NEK2 (Nek2fl) mice generated in our laboratory were back crossed to C57BL/6 for six generations (F6). Since the expression of the NEK2 transgene was blocked by the floxed STOP-polyA until the STOP-polyA was removed by Cre, thus F6 was further crossed to the CD19-Cre knock in mice to generate B-cell-specific NEK2 transgenic mice (NEK2fl/fl CD19-cre heterozygous), termed here as NEK2+/CD19 mice. Single-cell suspensions were prepared from BM and spleen and cell phenotypes were evaluated on a BD FACSCanto II and FlowJo software. Spontaneous Cell proliferation was measured by BrdU incorporation in vivo (1mg per mouse, ip). Mouse spleens were fixed in 10% neutral formalin then processed and embedded in paraffin for sections and H&E stain. Gene expression profiling (GEP) comparison between B cells from NEK2+/CD19 mice and NEK2−/CD19 littermate controls (LMC) was performed using the Affymetrix mouse genome 430 2.0 array. Results: NEK2+/CD19 mice were healthy with no obvious abnormality in organogenesis. NEK2 transgene did not perturb early B cell development (up to the stage of pro/pre B cells in BM), but impaired B cell maturation after immature B cells egressed from the BM into the spleen. Analysis of B cell surface expression of CD21 and CD23, which discriminates immature B cells (CD21negCD23neg), follicular B cells (CD21intCD23high) and marginal-zone B cells (MZ B) (CD21highCD23high) cells in spleen, showed significantly decreased MZ B in the spleen of NEK2+/CD19 mice compared with that of LMC (7.2 ±1.3 versus 3.18±0.9, p<0.001, n=12). Germinal center (GC), the light zone in spleen, where GC centrocytes undergo specific changes in their transcriptional programs, committed to plasma or memory B cell differentiation. Eight of nine (88%) of unimmunized NEK2+/CD19 mice manifested numerous spontaneous GCs in the spleens, and only one spontaneous GC was observed in unimmunized LMC. BrdU uptake experiment showed that 3.69% of B cells in NEK2+/CD19 BM and 1.04% of B cells in NEK2+/CD19 spleen were proliferating, while no BrdU incorporation was detected in BM or spleen of LMC. Previous study suggested that PP1/AKT and Wnt pathways were involved in NEK2 inducing cancer cell drug resistance, proliferation and chromosomal instability. GEP performed on B cells from NEK2+/CD19 mice and LMC showed that BCAP, a B cell adaptor for PI3K that couples B-cell receptor signaling to PI3K/AKT activation as well as NF-kB signaling, was significantly up-regulated in the NEK2+/CD19 mice. Real-time PCR analysis confirmed the GEP result, showing that BCAP mRNA in NEK2+/CD19 mice was 7 folds higher than that of LMC. Conclusion: We conclude that NEK2 is required for the normal development of MZ B cells and may also contribute to the development of conventional B cells, particularly to the transition from immature to mature B cell. BCAP may play an important role in NEK2 signaling in regulating B cell development. Future studies will investigate B cells function in the NEK2+/CD19 mice. The new mouse model presented here suggests that specifically manipulating NEK2 expression in B cells may be of therapeutic value in combating myeloma. Disclosures: No relevant conflicts of interest to declare.

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