A Critical Role for the Bcl-2 Pathway in Notch-Mediated B Cell Apoptosis, Where Growth Arrest Is Independent of the E2A, IL-3 and p21Waf1/p27Kip1 Pathways.

Abstract The Notch receptor pathway regulates critical cell fate decisions in multiple developmental systems, including hematopoiesis. We have previously demonstrated that Notch signaling induces growth arrest and apoptosis in a wide range of human B cell malignancies and has potential as a B cell-specific therapeutic approach. In order to identify the mechanisms of growth arrest and apoptosis we analyzed an immortalized murine progenitor B cell line derived from Bax/Bak double knockout mice. These cells are unable to undergo apoptosis since they lack the pro-apoptotic effectors of the Bcl-2 pathway, and have been shown to be resistant to multiple apoptotic stimuli. Here we report that induction of Notch signaling through expression of several family members (Notch1, Notch4, Hes1) leads to rapid growth arrest, but not apoptosis, within 48 hours in these Bax-/Bak- progenitor B cells. These findings provide the first evidence for a critical role of the Bcl-2 pathway in Notch-mediated B cell apoptosis, and establish a mitochondrial-dependent mechanism for this effect. Importantly, the kinetics of growth arrest are accelerated with the expression of the Notch downstream target Hes1 as compared to the Notch receptors 1 and 4. These results extend our observation that Hes1 is sufficient to reproduce Notch-mediated B cell death, by demonstrating that Hes1 is more proximal to the critical growth inhibiting events, and may therefore provide a therapeutic target. In this model system we can isolate growth arrest from the effects on the apoptotic cascade. This provides a unique opportunity to explore the mechanism of Notch-mediated growth arrest. Prior studies have suggested that Notch signaling may induce growth arrest through inhibition of the E2A pathway, or through upregulation of the cell cycle regulators p21Waf1 and p27Kip1. In this model system, inhibition of the E2A pathway is not sufficient to induce growth arrest. Similarly, Hes1 does not upregulate either p21Waf1 or p27Kip1, suggesting that this is not the mechanism of growth arrest. To explore whether Notch/Hes1 induce growth arrest through inhibition of the IL-3 pathway, we compared phenotypic and functional aspects of Hes1 expression and IL-3 withdrawal. Although the timing and phenotypic effects (cell size, cell cycle and metabolic studies) were quite similar, Hes1 growth arrested cells lose their ability to migrate in response to the pan-B chemo-attractant SDF1a compared to IL-3 withdrawn cells. In summary, these results demonstrate that Notch/Hes1-mediated B cell apoptosis relies critically on pro-apoptotic members of the Bcl-2 pathway, Bax/Bak. Furthermore, growth arrest when isolated from apoptosis does not rely on inhibition of the E2A or IL-3 pathways, nor upregulation of p21Waf1/ p27Kip1. These findings provide the first insight into the mechanisms of Notch/Hes1-mediated B cell growth arrest and apoptosis and will help guide the development of Notch/Hes1 signaling as a cell-type specific therapeutic approach for B cell malignancies.

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Notch/HES1 Signaling Stabilizes p53 Expression Via a Novel PARP1-Chfr-PLK1-Mediated Mechanism in B-ALL

Blood ◽

10.1182/blood.v120.21.1292.1292 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1292-1292

Author(s):

Sankaranarayanan Kannan ◽

Patrick A Zweidler-McKay

Keyword(s):

Cell Cycle ◽

T Cell ◽

B Cell ◽

Notch Signaling ◽

Critical Role ◽

Growth Arrest ◽

Cyclin B ◽

Cell Type ◽

Cell Type Specific ◽

Notch Activation

Abstract Abstract 1292 Background: Notch signaling contributes to T cell leukemogenesis. However, we have found that activation of Notch signaling in human B-ALL promotes growth arrest and apoptosis. These contrasting effects of Notch in B versus T cell ALL, mirror effects seen in early lymphocyte development. As the Notch receptors are common between T and B cells, we hypothesized that these differences rely on the cell-type specific downstream mechanisms. We previously reported a critical role for Notch/HES1-mediated activation of Poly ADP-Ribose Polymerase 1 (PARP1) function in this B cell specific mechanism. Approach: To explore the cell-type specific downstream mechanisms of Notch activation in B-ALL, we used cell fractionation, westerns and immunoprecipitation to identify cell cycle regulators which were altered by Notch activation via HES1 expression in human B-ALL lines. Results: Notch activation in a panel of human B-ALL lines led to consistent growth arrest and apoptosis. Indeed, ligands, activated receptors and the Notch target gene HES1 all induced these leukemia lihibiting effects in B-ALL but not T-ALL lines. In this study we report a mechanism whereby HES1-mediated activation of PARP1 leads to PARylation of the E3 ligase Checkpoint with FHA and RING finger (CHFR) (Panel A) which results in targeting and ubiquitination of the cell cycle regulator Polo-Like Kinase 1 (PLK1) (Panel B). PLK1 is highly expressed in B vs. T-ALL and plays a critical role in B cell growth and survival. Following Notch activation, loss of ubiquitionated PLK1 through proteosomal degradation leads to cell cycle arrest through two mechanisms, namely cytoplasmic relocalization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at S20, which leads to decreased weakened p53-MDM2 interaction and accumulation of p53 (Panel C). siRNA to CHFR reveal that this mechanism is dependent on CHFR (Panel C). Importantly this mechanism is not seen in T-ALL cells as the activation of PARP1 by HES1 does not occur in T-ALL cells. Conclusions: Our findings reveal a novel molecular mechanism whereby Notch signaling induces disruption of the cell cycle in a cell type specific manner in B-ALL. Activation of PARP1, PARylation of CHFR, ubiquitination of PLK1 resulting in loss of nuclear cyclin B and accumulation of p53 demonstrates a series of events which can be initiated through activation of Notch in B-ALL. This mechanism reveals a potentially targetable approach to B-ALL. Disclosures: No relevant conflicts of interest to declare.

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Notch Agonists: Emerging as a Feasible Therapeutic Approach in AML.

Blood ◽

10.1182/blood.v108.11.1419.1419 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1419-1419 ◽

Cited By ~ 1

Author(s):

Robert M. Sutphin ◽

Wendy Fang ◽

Claudia Miller ◽

Patrick A. Zweidler-McKay

Keyword(s):

Cell Cycle ◽

Tumor Suppressor ◽

Notch Signaling ◽

Cell Fate ◽

Cell Lines ◽

Therapeutic Approach ◽

Caspase 3 ◽

Growth Arrest ◽

Notch Pathway ◽

Fold Increase

Abstract Introduction: The Notch pathway regulates critical cell-fate decisions affecting the growth and development of human hematopoietic cells. Although Notch1 is a known T cell oncogene, we have discovered that Notch signaling behaves as a tumor suppressor in acute myeloblastic leukemia (AML) inducing growth arrest and apoptosis in both cell lines and patient samples. To characterize the mechanism of this effect we have evaluated the influence of the Notch pathway on key effectors of differentiation, cell cycle, and apoptosis in human AML. Results: Notch signaling induces rapid growth arrest and apoptosis in a panel of human AML cell lines representing a range of AML FAB subtypes (M2–M6). Specifically, activated Notch1 expression caused a 70–95% reduction in AML cells compared to controls (p<0.001) (Figure 1). Notch-mediated growth arrest occurred in 24–48 hours with cells accumulating in G0/G1. Apoptosis was demonstrated by a 3.8-fold increase in AnnexinV binding (p<0.004) and a 3-fold upregulation of caspase 3 activity (p=0.0002) within 24 hours. The caspase 3 activity was abolished by the caspase 8 inhibitor IETD (p<0.0001) suggesting a potential role for the extrinsic death pathway. We also found that all four Notch receptors (1–4) are capable of inducing this effect, as is the Notch target gene HES1, suggesting a generalized Notch tumor suppressor effect in AML. Furthermore, Notch signaling through HES1 modulates the expression of key regulators of myeloid differentiation and cell cycle progression including downregulation of CEBPα 2.5-fold (p<0.02) and upregulation of p21WAF1 6-fold (p<0.004) suggesting potential mechanisms. As a novel therapeutic approach, we synthesized Notch agonists which effectively induce Notch signaling with a >18-fold increase in HES1 expression (p<.0001). Exposure of human AML cell lines and primary patient AML samples to this Notch agonist for 24 hours led to a 3 to 9-fold increase in apoptosis (p<0.017) compared to controls (Figure 2). Conclusions: We report here that Notch signaling is a novel tumor suppressor pathway in human AML. We demonstrate how Notch agonists can be used to induce growth arrest and apoptosis in human AML cell lines and patient AML samples. As a regulator of cell fate, proliferation and differentiation, Notch effectively disrupts multiple pathways in AML. We propose that Notch agonists represent a novel and feasible therapeutic approach in AML. Pre-clinical evaluation is underway. Figure.1 Effect of Notch on growth of AML cells Figure.1. Effect of Notch on growth of AML cells Figure.2 Notch Agonist induces apoptosis in AML Figure.2. Notch Agonist induces apoptosis in AML

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Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

Blood ◽

10.1182/blood-2005-01-0355 ◽

2005 ◽

Vol 106 (12) ◽

pp. 3898-3906 ◽

Cited By ~ 136

Author(s):

Patrick A. Zweidler-McKay ◽

Yiping He ◽

Lanwei Xu ◽

Carlos G. Rodriguez ◽

Fredrick G. Karnell ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Notch Signaling ◽

Cell Lines ◽

Growth Arrest ◽

B Cell Malignancies ◽

Notch Receptors ◽

Wide Range ◽

Notch Activation ◽

Human B Cell

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies.

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Dual Inhibition of Bruton’s Tyrosine Kinase and Phosphoinositide-3-Kinase p110δas a Therapeutic Approach to Treat Non-Hodgkin’s B Cell Malignancies

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.116.238022 ◽

2017 ◽

Vol 361 (2) ◽

pp. 312-321 ◽

Cited By ~ 3

Author(s):

Jennifer Alfaro ◽

Felipe Pérez de Arce ◽

Sebastián Belmar ◽

Glenda Fuentealba ◽

Patricio Avila ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Therapeutic Approach ◽

Bruton’S Tyrosine Kinase ◽

B Cell Malignancies ◽

Bruton's Tyrosine Kinase ◽

Dual Inhibition ◽

Phosphoinositide 3 Kinase

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The Combination of Romidepsin and Bortezomib Results in Synergistic Induction of Apoptosis in Human B-Lymphoma Cell Lines.

Blood ◽

10.1182/blood.v114.22.1689.1689 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1689-1689 ◽

Cited By ~ 1

Author(s):

Deshpande S. Deshpande ◽

Mary Jo Lechowicz ◽

Rajni Sinha ◽

Jonathan L. Kaufman ◽

Lawrence H. Boise ◽

...

Keyword(s):

Cell Cycle ◽

B Cell ◽

Cell Lines ◽

Mtt Assay ◽

Research Funding ◽

Annexin V ◽

Myeloma Cell ◽

Parp Cleavage ◽

Sequence Specificity ◽

B Cell Malignancies

Abstract Abstract 1689 Poster Board I-715 Introduction The use of the proteasome inhibitor bortezomib has demonstrated activity in multiple myeloma and lymphomas. The HDAC inhibitor romidepsin is being evaluated in CTCL and PTCL, though its activity in B-cell lymphomas is less clear. We hypothesized that the combination of bortezomib and romidepsin would result in synergistic apoptosis in different B-cell NHL cell lines based upon the observed activity of this combination in more mature B-cell malignancies such as myeloma. Experimental Design Daudi, HT, Ramos and SUDHL-4 cell lines were exposed to different concentrations of bortezomib and romidepsin, separately, concurrently, and sequentially. Cell viability was assessed using MTT-assay, induced apoptosis was evaluated using Annexin V and PI staining from 24-48 hours. Apoptosis was also evaluated using western blot analysis of caspases and PARP cleavage. LC3 and HDAC6 level expressions were performed to determine if the effect of the combination was a result of the aggresome or autophagy pathway. Cell cycle studies were also performed to study if there were any changes after treating cells with the combination. Results The combination of bortezomib and romidepsin resulted in synergistic B-cell apoptosis as measured by MTT-assay with combination indices of < 0.5. This was associated with increased caspases and PARP cleavage as early as 24 hours after exposure. Order of addition experiments demonstrated definite sequence specificity. When romidepsin was added first, and 6 hours later followed by bortezomib, apoptosis was enhanced, compared to both agents being given concurrently or when bortezomib was administered first. Cell cycle analysis studies demonstrated that pretreatment of cells with romidepsin for 6 hours followed by the addition of bortezomib arrested the cells in G2M phase. HDAC6 expression was significantly reduced following combination therapy, and LC3-I was cleaved to LC3-II in treated cells suggesting that the combination affected aggresome formation and autophagy. Conclusion The combination of romidepsin and bortezomib at low nanomolar concentrations suggests that this may be an important clinical combination to test in patients with relapsed or refractory B-cell malignancies. Sequence of administration data is currently being tested to determine if the effect is a result of autophagy inhibition as is seen in myeloma cell lines. Additional mechanistic studies will be presented with the goals of identifying predictors of response that can then be validated in prospective clinical trials. Disclosures Lechowicz: Gloucester: Consultancy. Kaufman:Millennium: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Gloucester: Research Funding; Novartis: Consultancy; BMS: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy. Flowers:Millennium: Research Funding.

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Notch Signaling In B-ALL Reveals PLK1 As A Potential Therapeutic Target In B-ALL: PLK1-Selective Inhibitor Poloxin Modulates Cyclin B and P53 Pathways, Leading To Growth Arrest and Apoptosis

Blood ◽

10.1182/blood.v122.21.2912.2912 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2912-2912 ◽

Cited By ~ 1

Author(s):

Sankaranarayanan Kannan ◽

Mandy A Hall ◽

Leonard S Golfman ◽

Patrick A Zweidler-McKay

Keyword(s):

Cell Cycle ◽

Notch Signaling ◽

Growth Arrest ◽

Selective Inhibitor ◽

Cyclin B ◽

Cell Type ◽

Specific Effects ◽

Cell Cycle Regulator ◽

Cell Type Specific ◽

Novel Therapeutic

Abstract Background Notch is a well-known oncogene in T-ALL, yet appears to have tumor suppressor effects in B-ALL. These cell type-specific effects of Notch signaling mirror consequences seen in early lymphocyte development and raises the question of how Notch leads to such divergent consequences in closely related cell types. In exploring these Notch mechanisms we discovered a B-ALL specific Notch-mediated reduction in the cell cycle regulator Polo-like kinase-1 (PLK1), revealing a novel targetable kinase in B-ALL. Approach To explore the consequences of Notch-mediated down regulation of cell cycle regulator kinase PLK1, we targeted PLK1 kinase function with the novel PLK1-selective inhibitor poloxin in human B-ALL lines. Results PLK1 is highly expressed in B-ALL verses normal tissues (panel A), correlates with cyclin B expression, is expressed >2-fold higher in B-ALL with t(1;19) than other B-ALL samples, and may predict response of ALL to methotrexate. In our panel of human B-ALL cell lines poloxin induced G2/M growth arrest and decreased cell number by >80% (panel B), and decreased survival in B-ALL cells (>75% AnnexinV+, panel C). PLK1 inhibition led to tumor suppressor p53 stabilization, revealing >5-fold increase in p53 protein levels following poloxin treatment in B-ALL (panel D). Mechanistically, PLK1 inhibition leads to both cytoplasmic re-localization of cyclin B, disrupting the CDC2-cyclinB complex, as well as phosphorylation of p53 at Ser20, which destabilizes p53-MDM2 interaction and thus accumulation of p53. Conclusions While exploring the mechanisms of cell type-specific effects of Notch signaling in ALL, we have found a novel therapeutic target, the cell cycle regulator PLK1. Our findings reveal a novel therapeutic approach whereby PLK1-selective inhibition via poloxin induces growth arrest and apoptosis in human B-ALL via consequences on cyclin B and p53 pathways. Disclosures: No relevant conflicts of interest to declare.

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Depletion of Normal and Malignant B cells with a CD37-specific SMIP molecule

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.3063 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 3063-3063

Author(s):

C. Cerveny ◽

L. Grosmaire ◽

E. Espling ◽

R. Bader ◽

C. Nilsson ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Target Cell ◽

Growth Arrest ◽

Xenograft Model ◽

B Cell Malignancies ◽

B Lymphoma ◽

Anti Tumor Activity

3063 Background: CD37 is a member of the tetraspanin family expressed at high levels by normal mature B cells and by most B cell malignancies. Previously, an antibody to CD37 has been labeled with 131I and tested in clinical trials for therapy of NHL. Treatment with 131I-MB-1, resulted in durable tumor remissions in patients lasting from 4 to 11 months (Press OW, Eary JF, Badger CC, et al. Treatment of refractory non-Hodgkin’s lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol. 1989;7:1027–1038). Here we assess the functional properties and therapeutic potential of a small modular immunopharmaceutical (SMIP) targeting CD37. Methods: Growth arrest and apoptosis of B lymphoma cell lines was assessed. ADCC activity was evaluated using BJAB targets and human peripheral blood mononuclear cells (PBMC) effectors. Drug-drug interactions were assessed by the Combination Index method. In vivo studies were performed utilizing established human B cell tumor xenografts in nude mice. Results: A CD37-directed SMIP drug candidate mediated growth arrest, apoptosis and ADCC, but not CDC, towards B lymphoma cell lines. The protein showed significant anti-tumor activity in a mouse xenograft model, and selectively depleted normal human B cells in short term cultures of PBMC. When combined with rituximab, the molecule increased apoptosis, C1q binding, and C’ dependent target cell death in vitro, and increased anti-tumor activity in vivo in a xenograft model. Conclusions: In vitro and in vivo characterization of the CD37-targeted SMIP drug suggest a potent capacity to eliminate target cells through combined effects of direct target cell signaling and effector cell recruitment. CD37-mediated growth was synergistic with standard chemotherapies in vitro and showed additive in vivo activity with CD20-targeted therapy. On the basis of these data CD37-directed SMIP therapy is being developed for clinical evaluation against B cell malignancies. No significant financial relationships to disclose.

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KLF4 suppresses transformation of pre-B cells by ABL oncogenes

Blood ◽

10.1182/blood-2006-03-011106 ◽

2006 ◽

Vol 109 (2) ◽

pp. 747-755 ◽

Cited By ~ 49

Author(s):

Michael G. Kharas ◽

Isharat Yusuf ◽

Vanessa M. Scarfone ◽

Vincent W. Yang ◽

Julia A. Segre ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

Tumor Suppressor ◽

B Cell ◽

Cell Activation ◽

Cell Transformation ◽

Cell Lineage ◽

Cell Types ◽

B Cell Malignancies

Abstract Genes that are strongly repressed after B-cell activation are candidates for being inactivated, mutated, or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4), a gene down-regulated in activated murine B cells, is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this, overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre–B-cell transformation by v-Abl and BCR-ABL, oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death, but not cell-cycle arrest, can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively, our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies.

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