A Novel DLL1-Anti-CD19 Chimeric Protein Effectively Targets Notch Activation to B-Cell Acute Lymphoblastic Leukemia and Induces Cell Death

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most prevalent form of cancer in children, and those who relapse continue to have poor survival. Therefore, the development of improved and specifically targeted treatment options is vital. The Notch pathway has been shown to act as a tumor suppressor in B-ALL via cell type specific induction of growth-inhibiting and pro-apoptotic pathways. In this study, we aim to therapeutically activate Notch signaling in B-ALL via targeting the Notch ligand DLL1 to B cells using an anti-CD19 scFv chimeric protein. Methods: A soluble chimeric protein composed of the extracellular domain of the Notch ligand DLL1 linked to a validated anti-CD19 scFv was isolated from HEK-293 producer cells. Human B-ALL (SB, Nalm6) and T-ALL (Jurkat) lines were treated with DLL1-anti-CD19scFv chimeric protein, and expression of the Notch target gene HES1 and effects on cell growth and survival were measured. Results: Both B- and T-ALL lines express Notch1 and Notch2 receptors on the cell surface. Exposure of B-ALL cells to DLL1-anti-CD19scFv chimeric protein led to an increase of Notch signaling, measured via 3-14 fold increase in HES1 mRNA expression. As expected, surface expression of the Notch receptors decreased upon chimera exposure, as Notch receptors are cleaved and destroyed upon activation. Importantly, exposure of the B-ALL lines to the chimeric protein led to a maximum 60% decrease in cell counts over 3-4 days, in contrast to T-ALL, where exposure did not effect growth significantly. Conclusions: This study demonstrates that Notch signaling can be feasibly activated in human B-ALL cells through a soluble DLL1-anti-CD19 scFv chimeric protein. Activation of Notch signaling via this method leads to growth inhibition and cell death in B-ALL, but not T-ALL cells. Based on our findings, we suggest that this soluble DLL1-anti-CD19 chimera may be a potential therapeutic approach for B-ALL, and further in vivo testing is warranted. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Select γ-Secretase Inhibitors Induce Apoptosis in Pre-B ALL Cells and Disrupt the Balance Between Constitutive Notch Signaling and Repression.

Blood ◽

10.1182/blood.v112.11.1917.1917 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1917-1917

Author(s):

Bridget S. Wilson ◽

Xiangbing Meng ◽

Tomas Mazel ◽

Cheryl L. Willman ◽

Susan Atlas ◽

...

Keyword(s):

Cell Death ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Caspase Activation ◽

Notch 1 ◽

Activating Mutations ◽

Important Group ◽

Notch Receptors ◽

Activation Status

Abstract Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI’s tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.

Download Full-text

Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Blood ◽

10.1182/blood.v116.21.1809.1809 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1809-1809

Author(s):

Hisashi Harada ◽

Nastaran Heidari ◽

Mark Hicks

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Small Molecule ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Annexin V ◽

Glucocorticoid Resistance ◽

Down Regulation

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Notch Expression Expands the Pre-Malignant Pool of T-Cell Acute Lymphoblastic Leukemia Clones without Affecting Self-Renewal

Blood ◽

10.1182/blood.v116.21.3161.3161 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3161-3161

Author(s):

Jessica S Blackburn ◽

Sali Liu ◽

David M. Langenau

Keyword(s):

T Cell ◽

Notch Signaling ◽

Primary Tumor ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Leukemic Cells ◽

Distinct Advantage ◽

Rodent Models ◽

Self Renewal

Abstract Abstract 3161 60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, making it the most commonly mutated oncogene in T-ALL. Notch signaling is critical for T cell development, and activating Notch mutations are found in all subtypes of T-ALL, suggesting that Notch deregulation may be a dominant initiating event in human disease. In human and rodent models of T-ALL, Notch directly induces cMyc expression. However, cMyc over expression cannot completely rescue Notch inhibition, suggesting that Notch may have other important roles in T-ALL progression. Classic viral insertion screens in mice have indentified that insertional activation of Notch1 is common in Myc induced T-cell malignancies, suggesting that Notch imparts a distinct advantage to leukemic clones independent of cMyc. Notch-induced transgenic zebrafish models of T-ALLs are unique in that Notch signaling does not induce cMyc expression, allowing new opportunities to determine the function of Notch which are independent of cMyc. As with rodent models, the co-expression of Notch and cMyc in zebrafish T cells significantly enhanced T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with Myc did not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to Myc alone expressing T-ALLs. Moreover, clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch collaborates with Myc to increase the number of T-ALL clones contained within the primary tumor by 2–4 fold when compared to single transgenic animals that express only cMyc. Following serial transplantation, a large portion of T-ALL clones present in primary Notch/Myc leukemias are not found in transplanted animals. This starkly contrasts to results seen in Myc-alone expressing T-ALLs where all primary clones are capable of engraftment and reinitiation of leukemia. Paradoxically, transplant animals developing T-ALL from leukemias that coexpress Notch and Myc have similar numbers of clones as found in primary Myc-induced leukemias. Primary Myc-induced T-ALLs express high levels of endogenous scl and lmo2, recapitulating the most common and treatment resistant subtype in human T-ALL. By contrast, T-ALLs that co-express Notch and Myc fail to upregulate any of the T-ALL oncogenes; however, following transplantation into recipient animals, double transgenic Notch/Myc leukemias now express high levels of scl and lmo2. Finally, large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that Notch does not collaborate with Myc to increase self-renewal of leukemia initiating cells (LICs). Primary T-ALLs expressing both Notch and Myc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, these Notch/Myc leukemias exhibit similar leukemia-initiating frequency as Myc-induced T-ALLs. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, Myc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells accounting for the longer latency likely reflecting acquisition of additional genetic changes in clones. Our data may explain why a subset of relapse human T-ALLs develop disease from an underrepresented clone found in the primary leukemia. Primary human T-ALLs likely have a large pool of premalignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

High Mir-221 Expression Is Associated with Poorer Treatment Outcome of Patients with T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.1439.1439 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1439-1439 ◽

Cited By ~ 1

Author(s):

Hamilton L. Gimenes-Teixeira ◽

Guilherme A. dos Santos ◽

Dalila L. Zanette ◽

Priscila S Scheucher ◽

Luciana Correa Oliveira de Oliveira ◽

...

Keyword(s):

Treatment Outcome ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Mirna Expression ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Disease Free Survival ◽

Cell Counts ◽

Cell Acute Lymphoblastic Leukemia ◽

The Impact

Abstract Abstract 1439 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells that accounts about 15% of pediatric and 25% of adult ALL cases. In the last years, several clinical and laboratory features have been described as prognostic markers; nevertheless, with intensification of therapy most of them have lost their predictive value. MicroRNA (miRNA) expression analysis has proved to be an useful tool for identifying specific subsets of cancer patients with relevant cytogenetic, laboratorial and clinical features. The aim of the present study was to determine if miRNAs may be useful markers in T-ALL. First, we performed a supervised analysis comparing the miRNA expression profile of T-ALL blasts from 36 T-ALL/CD56− and 12 T-ALL/CD56+. We selected CD56 as prognostic marker based on our previous report showing that the disease-free survival (DFS) of T-ALL/CD56+ patients was of 28.5 months compared to 69.8 in the CD56− group. Also patients tended to be older and to present normal platelet counts in the T-LLA/CD56+ group. We used the Taqman MicroRNA Assay Human Panel (Applied Biosystems) to perform a screening of 164 knowledge mature miRNA sequences using specific primers and probes according to manufacturer instructions. Total RNA input was normalized based on the geometric means of Ct values obtained from four endogenous RNAs. All reactions were run in duplicate and a coefficient of variation greater than 5% was used as an exclusion factor (seven miRNAs were excluded). The fold change was calculated using comparative 2−δCt method. We have identified a set of 14 miRNAs differentially expressed, of which miR-374 and miR-221 best distinguished T-ALL/CD56+ from T-ALL/CD56− blasts. Based on this profile, we selected miR-221 and miR-374 as potential markers and quantified their expression in the same samples using RQ-PCR. Patients were stratified as high and low expression using the median value as cut off. We detected a significant association between the miR-221 high expression and poorer treatment outcome. On the contrary, miR-374 expression levels were not associated with treatment outcome. We evaluate the impact of age, white blood cell counts, CD56 and miR221 expression on overall survival (OS). Age and miR-221 were the only ones found to be significant. The estimate 5-year OS (mean and confidence interval 95%) was of 67.0 ± 10.3% in the group of patients expressing miR-221 below the cut-off value, whereas this value was of 28.5 ± 14.5% in the alternative group. Even among T-ALL/CD56− patients, the higher expression of miR-221 was significantly associated with poorer outcome. Our data suggest that miR-221 play an important role in T-ALL and its regulation may represent a potential therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

EGFL7 Antagonizes NOTCH Signaling, Stimulates Blast Proliferation and Confers Poor Prognosis in Cytogenetically-Normal Acute Myeloid Leukemia (CN-AML)

Blood ◽

10.1182/blood.v128.22.2689.2689 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2689-2689

Author(s):

Changxian Shen ◽

Dimitrios Papaioannou ◽

Betina Mcneil ◽

Deedra Nicolet ◽

Malith Karunasiri ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Notch Signaling ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Notch Pathway ◽

Western Blots ◽

Notch Receptors ◽

Subsequent Activation ◽

Expression Status ◽

Acute Myeloid

Abstract Epidermal growth factor-like domain 7 (EGFL7) is a secreted protein and plays an important role in angiogenesis by regulating the growth, proliferation and migration of endothelial cells. Recent studies in solid tumors have shown that EGFL7 is overexpressed and is associated with a more aggressive disease phenotype. Whether EGFL7 plays a similar role in blood cancers such as acute myeloid leukemia (AML) however, has not been previously reported. To investigate the association of EGFL7 expression with outcome in AML, we measured EGFL7 mRNA expression in newly diagnosed older (≥60 years, n=126) CN-AML patients. In these patients, those with high EGFL7 expression were less likely to achieve CR (52% v 76%, P=.009). Patients with high EGFL7 expression status had shorter event-free survival (5-year rates: 6% v 13%, P=.03) and overall survival (5-year rates: 10% v 16%, P=.009) than patients with low EGFL7 expression status. To validate our clinical data we measured EGFL7 mRNA in primary AML blasts (n=11) compared to normal bone marrow (NBM) (n=5) using RT-PCR, and found a ~2.4 fold-increase in the AML samples, P<0.05. These results were consistent with the protein levels, measured using western blots. Using both ELISA and western blotting, we observed significant increased levels of EGFL7 protein in the medium of cultured primary AML cells (n=3, 31-443 pg/mL) compared to media alone. Importantly, detection of serum EGFL7 levels by ELISA revealed variable but increased levels of EGFL7 in the sera from 3 of 5 (P<0.05) AML patients tested compared with sera from normal healthy controls (n=7). To investigate the functional consequences of EGFL7 stimulation on blast growth we treated primary AML blast cells from three patients with recombinant EGFL7 (rEGFL7) protein and performed colony forming unit (CFU) assays. Treatment with rEGFL7 led to an increase in the number of CFUs (P<0.05) and was associated with increased phosphorylation of AKT. In addition, under starvation conditions, recombinant EGFL7 protein alone promoted the survival and proliferation of MLL-PTD AML cells (n=3) but not normal BM cells (n=3) (P<0.05). To explore the underlying molecular mechanism(s) by which EGFL7 maintains the survival and proliferation of AML cells, we screened for proteins that interact with EGFL7 by using a protein antibody array on human primary AML blasts. This screen revealed that EGFL7 binds to multiple components of key signal transduction pathways important for leukemia, including NOTCH. Previous studies by Lobry C et al., (JExpMed 2013) demonstrated that although NOTCH2 and to a lesser extent, NOTCH1, mRNA was relatively abundant, NOTCH signaling was inhibited in AML blasts. Re-activation of the canonical NOTCH pathway, released a block in differentiation resulting in eradication of the leukemic blasts. Schmidt MH et al., (Nat Cell Biol. 2009) demonstrated that EGFL7 was capable of binding NOTCH receptors and blocking their subsequent activation. Therefore, we hypothesized that one mechanism by which the NOTCH pathway is kept inactive in AML is through autocrine binding of EGFL7 protein to NOTCH receptors. Co-immunoprecipitation assays confirmed binding of EGFL7 to NOTCH2 in both human primary AML cells (n=3, P<0.05) and mouse MLL-PTD AML cells (n=3, P<0.05). Moreover, stimulation of AML blasts with rEGFL7 protein suppressed the cleavage and subsequent activation of NOTCH2, resulting in decreased transcription of NOTCH downstream target genes, HES1 and NRARP (n=3, P<0.05). In summary, we show here that high EGFL7 is associated with worse prognosis in patients with CN-AML, and that EGFL7 is expressed and secreted by the AML cells in an autocrine fashion, promoting their growth in part through antagonizing NOTCH2 activation. Targeting EGFL7 with antibodies to reduce NOTCH binding might represent a novel therapeutic approach to reactivate NOTCH signaling, allowing for blast differentiation and elimination of AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Direct Inhibition of the Notch Transactivation Complex with Synthetic Constrained Peptides in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v110.11.2819.2819 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2819-2819 ◽

Cited By ~ 2

Author(s):

Raymond Moellering ◽

Melanie Cornejo ◽

Jennifer Rocknik ◽

Michael Hancock ◽

Christina DelBianco ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Notch Signaling ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Stem Cell Differentiation ◽

Gamma Secretase ◽

Clinical Resistance ◽

Cell Acute Lymphoblastic Leukemia

Abstract Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

Download Full-text

The Notch Signaling Pathway as a Suppressor of Myeloid Transformation

Blood ◽

10.1182/blood.v118.21.sci-13.sci-13 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-13-SCI-13

Author(s):

Iannis Aifantis

Keyword(s):

Bone Marrow ◽

T Cell ◽

Notch Signaling ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Stem Cell Differentiation ◽

Lineage Tracing ◽

Specific Gene ◽

Hematopoietic Stem

Abstract Abstract SCI-13 Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types. Its activity is controlled by the multi-subunit γ-secretase complex (γSE) complex. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations. We identified somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) results in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis reveals that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. These studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue. These observations also suggest that Notch activity is not simply a promoter of the T cell lineage in the thymus but that Notch signaling thresholds could regulate commitment and/or survival of distinct hematopoietic lineages in the bone marrow. To address these issues in vivo, we have generated Notch receptor lineage tracing and activity reporter genetic tools. Analysis of these animal models identified unique novel functions for the Notch pathway during early bone marrow hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

PIM2 Upregulation Leads to Physiological Buffering of Metformin-Induced Cell Death Mediated by ER Stress/UPR in Acute Lymphoblastic Leukemia,

Blood ◽

10.1182/blood.v118.21.3496.3496 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3496-3496

Author(s):

Gilles M. Leclerc ◽

Guy J. Leclerc ◽

Jeffim N. Kuznetsov ◽

Julio C. Barredo

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Cell Growth ◽

Growth Inhibition ◽

Er Stress ◽

Conflicts Of Interest ◽

Synthetic Lethality ◽

Lymphoblastic Leukemia ◽

Protein Translation ◽

Western Blots

Abstract Abstract 3496 Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and adolescents. Despite significant overall improvements in cure rates, outcome remains dismal for patients with resistant phenotypes or after relapse. Therefore, novel treatment strategies are warranted. Recently, we identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells, as a potential target for ALL therapy due to its effects on cell growth, proliferation, and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We showed that activation of AMPK using metformin (1-5 mM) induced significant cell growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cell line models. Western blot analysis revealed that metformin led to activation of p-AMPK (Thr172) and its downstream target p-ACC (Ser79), the cell proliferation regulator p-Akt (Ser473), and the protein translation regulator p-4EBP1 (Thr70), suggesting that protein translation may be an important determinant in the mechanism of metformin-induced cell death. Indeed, we demonstrated that blocking protein translation with the mTOR inhibitor rapamycin (1 μg/ml) rescued ALL cells from metformin-induced cell death (p < 0.01). In addition, knockdown of AMPK1α expression using shRNAs (shAMPK) abrogated metformin-induced growth inhibition and apoptosis in ALL cells as compared to control cells expressing scramble shRNAs (shCTRL), indicating that AMPK mediates metformin's cytotoxicity in our models. Western blots demonstrated that ALL cells expressing shAMPK exhibit decreased expression of total AMPK, p-p38MAPK (Thr180), p-mTOR (Ser2448), and p-4EBP1 (Thr70) compared to shCTRL cells, implicating regulation of protein translation in the mechanism of cell death induced by metformin. In addition, metformin-induced p-Akt (Ser473) activation observed in shCTRL cells is blocked in shAMPK expressing cells, suggesting that the contextual crosstalk between AMPK and Akt is relevant for metformin's cytotoxicity. Indeed, experiments co-targeting Akt and AMPK using perifosine (6 μM) or the Akt inhibitor X (AIX, 5 μM) plus metformin (5 mM) for 72 h induced synergistic cell death in NALM6 cells (Combination Index (CI) values of 0.21 for perifosine + metformin, and 0.19 for AIX + metformin). Our studies uncovered that apoptotic death in NALM6 and CCRF-CEM cells treated with metformin correlated with metformin's induction of ER stress/UPR in ALL cells, as demonstrated by increased expression of the UPR markers IRE1α and CHOP. More important, rapamycin rescued metformin-treated ALL cells by relieving ER stress/UPR as demonstrated by decreased IRE1α and CHOP. These observations support our previous findings that ER stress/UPR mediates cell death in ALL cells under metabolic stress, and is tightly coupled to regulation of protein translation (Mol Cancer Ther 10:437, 2011). To further investigate the relationship between protein translation and ER stress/UPR, we examined the role of PIM1/2 kinases, particularly PIM2 known to regulate CAP protein translation, in metformin-induced ALL cell death. Our results indicate that expression of PIM2 is significantly increased in NALM6 cells treated with metformin (5–10 mM) for 72 h. We also observed concomitant decrease in the expression of the UPR markers IRE1α, ATF6, and CHOP, raising the possibility that PIM2 upregulation may be a compensatory survival mechanism to regulate protein translation and suppress metformin-induced ER stress/UPR. To test this hypothesis, we co-treated NALM6 cells with the small molecule PIM1/2 kinase inhibitor V (80 μM) and metformin (5 mM) and found that inhibition of PIM2 in metformin-treated NALM6 cells induced synergistic cell death (CI = 0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in metformin treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies that exploit synthetic lethality by combining activators of AMPK such as metformin and compounds that target regulation of protein translation or protein degradation as suitable for clinical translation in patients with ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Incidence and Outcome In Adults With Acute Lymphoblastic Leukemia With Primary Central Nervous System Involvement

Blood ◽

10.1182/blood.v122.21.2639.2639 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2639-2639

Author(s):

Muhamed Baljevic ◽

Hagop M Kantarjian ◽

Deborah Thomas ◽

Michael Rytting ◽

Jyothsna Dasarathula ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Central Nervous System Involvement ◽

Cns Involvement ◽

Cytologic Examination ◽

Cell Counts ◽

Remission Duration

Abstract Background Presence of primary central nervous system (pCNS) involvement at the time of diagnosis of acute lymphoblastic leukemia (ALL) in adults is a poor prognostic feature. Few reports have systematically analyzed the outcomes of adult patients (pts) diagnosed with CNS involvement at diagnosis of ALL. This report provides analysis of single institution experience outcomes in adult pts diagnosed and treated for ALL with pCNS involvement with the hyperfractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone (HCVAD) or augmented Berlin-Frankfurt-Munster (AUG-BFM) based induction regimens, with or without the frontline use of Rituximab and tyrosine kinase inhibitors (TKIs). Methods The records of 623 consecutive pts with newly diagnosed ALL treated at the M. D. Anderson Cancer Center between January 2001 and June 2013 were reviewed. Those who had CNS involvement at diagnosis were treated with intrathecal (IT) chemotherapy twice weekly until at least 2 consecutive cerebrospinal fluid (CSF) cell counts normalized and cytologic examination was negative for evidence of malignant cells. IT therapy was subsequently administered weekly for at least 6-8 weeks, then according to the prophylactic schedule (2 intrathecals per course) for the remaining courses of intensive chemotherapy. Results A total of 68 (11%) pts had pCNS involvement by analysis of CSF; 5 (7%) had additional brain, leptomeningeal, base of skull or spine evidence of involvement. They were treated with either HCVAD (n=52) or AUG-BFM (n=16). HCVAD with Rituximab was used in 22 (32%), and HCVAD with Dasatinib or Ponatinib in 17 (25%). Median age at diagnosis was 38 (range 13-80); 45 (66%) were male; median white blood cell count 9.9 (vs. 6.7 for those with no pCNS involvement; p=0.007); peripheral blood blast 46% (vs. 18; p=0.0008); lactate dehydrogenase 1266 (vs. 1013; p=0.03); albumin 3.3 (vs. 3.55; p=0.03); platelets 54; hemoglobin 9.3; bone marrow blast 83%. Philadelphia chromosome (Ph+) was seen in 18 (26%) vs. 140 (25%) of pts with no pCNS involvement. Among pts with pCNS, 46% expressed CD20 vs. 47% of pts with no pCNS involvement. Complete response (CR) was achieved in 61 (90%) pts compared to 513 (92%) among those with no pCNS involvement (p=0.465). Of those with CNS disease who achieved CR, 21 (34%) had a relapse, compared to 138 (27%) among those with no CNS involvement. Median complete remission duration has not been reached; Kaplan-Meier estimates for remission duration at 18 months are 66% vs. 81% for pts with with or without pCNS, respectively (p=0.147). Overall, 6 (10%) of pts with pCNS disease who had a recurrence had an isolated CNS relapse; 3 (50%) of them had a baseline Ph+. They were treated with combination therapy including HCVAD and IT cytarabine and/or methotrexate with or without craniospinal radiation and allogeneic stem cell transplantation. Five (24%) reached the second CR. The median overall survival (OS) was 28 mo for pts with pCNS involvement vs. 86 mo for those without CNS involvement at presentation (p=0.036). Of those who were evaluable in the CNS cohort, 48 (74%) pts were alive at 1 year, and 24 (41%) were alive at 4 years. Conclusion The incidence of pCNS involvement in adults with ALL has remained virtually the same over the last 20 years; 10% for HCVAD treated cohort (Cortes et al. Blood. 1995). Despite effective and wider therapeutic arsenal for ALL including Rituximab and advanced generation TKIs since year 2000, adults with ALL who present with pCNS involvement have an inferior outcome, with shorter median OS compared to pts who do not present with pCNS disease. However, pCNS is still compatible with cure if properly treated. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Clinical Significance of High C-MYC and Low MYCBP2 Expression and Their Association with Ikaros Dysfunction in Adult Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3713.3713 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3713-3713

Author(s):

Zheng Ge ◽

Xing Guo ◽

Jianyong Li ◽

Melanie Hartman ◽

Yuka Imamura Kawasawa ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Zinc Finger Protein ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Luciferase Assay ◽

Lower Percentage ◽

Dependent Manner ◽

Cell Counts ◽

Low Expression ◽

Ck2 Inhibitor

Abstract Objective: The Myc proteins are transcription factors with essential roles in cell growth and proliferation through their ability to regulate gene expression. MYC binding protein 2(MYCBP2) is probable E3 ubiquitin-protein ligase and its function in leukemia is undetermined. IKZF1 encodes a kruppel-like zinc finger protein Ikaros that is essential for normal hematopoiesis and acts as a tumor suppressor in acute lymphoblastic leukemia(ALL). IKZF1 deletion is associated with the development of ALL and poor clinical outcome. This study aimed to explore the expression of c-MYC and MYCBP2 and their correlation with clinical features in adult ALL, as well as the mechanism by which Ikaros directly regulates c-MYC/MYCBP2 expression in ALL. Methods: Quantitative PCR (qPCR) was performed to explore the expression of c-MYC and MYCBP2 in 151 newly diagnosed adult patients with ALL. The correlations of c-MYC/MYCBP2 expression with clinical parameters and survival status were analyzed. In addition, luciferase assay, quantitative Chromatin Immunoprecipitation (qChIP) and Ikaros shRNA knockdown were performed to further explore the mechanism underlying regulation of c-MYC/MYCBP2 expression. Results: Expression of c-MYC is significantly higher and MYCBP2 is significantly lower in both B-ALL and T-ALL patients compared with that in normal controls. C-MYC expression is also negatively co-related with the MYCBP2 in ALL cohorts. The patients with c-MYC high and MYCBP2 low expression (c-MYChigh +MYCBP2low) showed higher median white blood cell counts (WBC) (101.5×109/L vs 29.4×109/L, P =0.007), incidence of splenomegaly and liver infiltration (75.0% vs 33.3%, P =0.004;75.0% vs19.4%, P =0.000), percentage of CD34(+) and CD33(+) cells (90.0% vs 61.3%, P =0.025; 80.0% vs 25.8%, P =0.000) and a lower percentage of complete remission (CR) rate (60.0% vs 92.0%,P =0.027) compared with that of patients with c-MYC low and MYCBP2 high expression (c-MYClow +MYCBP2high). Notably, our Ikaros ChIP-seq data showed strong Ikaros binding peaks in the promoter region of both c-MYC and MYCBP2. The qChIP assay showed that Ikaros significantly binds to c-MYC and MYCBP2 promoter regions in both Nalm6 B-ALL and Molt4 T-ALL cells. Moreover, expression of Ikaros suppressed c-MYC but increased MYCBP2 expression in both Nalm6 and CEM T-ALL cells. Conversely, Ikaros knockdown induced the increase of c-MYC but decrease of MYCBP2 in Nalm6 and CEM cells. Ikaros activator,Ck2 inhibitor TBB suppress c-MYC and increase MYCBP2 expression in a dose-dependent manner in Nalm6 and CEM cells. Ikaros knockdown with shRNA could block the TBB-induced suppression of c-MYC and increase of MYCBP2 expression. These data indicated that both c-MYC and MYCBP2 are direct Ikaros targets in ALL and Ikaros regulates their expression. Importantly, we also observed Ikaros binding to c-MYC and MYCBP2 promoters in primary B-All and T-ALL. The expression of c-MYC significantly increased and MYCBP2 decreased in patients with Ikaros deletion compared to that of Ikaros wild type. These data indicated Ikaros regulatory effect on c-MYC and MYCBP2 in ALL patients and Ikaros deletion is one of the reasons for expression change of c-MYC and MYCBP2 in the patients. Conclusion: We observed the expression of c-MYC significantly increased and MYCBP2 decreased in adult ALL patients. C-MYC high and MYCBP2 low expression is correlated with high-risk leukemia. Ikaros dysfunction is one of the reasons underlying c-MYC high and MYCBP2 low expression in the patients. Our data revealed the oncogenic effect of Ikaros/MYCBP2/c-MYC on oncogenesis in adult ALL, also suggested CK2 inhibitor exert its anti-leukemia effect through Ikaros-mediated regulation on c-MYC and MYCBP2 expression in leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text