scholarly journals CARMA1 Is Required for Notch1-Induced NF-κb Activation in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1260-1260 ◽  
Author(s):  
Rong Wang ◽  
Ninghan Zhang ◽  
Yuan Feng ◽  
Tingting Shao ◽  
Huihui Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
T Cell ◽  
Cell Lines ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Accession Number ◽  
Nsg Mice ◽  
Control Shrna ◽  
Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm originated from early T cell progenitors. Constitutive activation of the Notch1-signaling pathway plays an important role in T-ALL. After three shears, Notch1 generates intracellular fragment of Notch1 (Notch1-IC), which translocates to the necleus and exerts its biological functions. NF-κB pathway is an important regulator of cell survival and a major downstream target of Nothc1 in T-ALL. However, the molecular mechanism of Notch1 activating NF-κB pathway is still unknown. CARMA1 forms a complex with MALT1 and BCL10 (CBM complex), which plays a role in activation of NF-κB pathway in leukomonocyte. Therefore, we asked whether CARMA1 mediates Notch1-induced NF-κB pathway. Methods Microarray gene expression data were obtained from GEO database (accession number GSE26713) to assess expression of CARMA1, BCL10, MALT1 in T-ALL patients. In addition, we apply microarray dataset (GEO accession number GSE42328) to examine the relationship between gene expression and patients outcome. Western blot was used to analyze CRAMA1 expression in different cell lines. CCK-8 assay, EdU assay, cell cycle and colony formation experiments were performed in CARMA1-shRNA and control-shRNA T-ALL cell lines to investigate the effect of CARMA1 in T-ALL. We intravenously injected the Control-shRNA and CARMA1-shRNA MOLT-4 cells into NOD-SCID/IL2Rgnull (NSG) mice to evaluate the influence of CARMA1 knockdown on overall survival. Dual luciferase reporter assay was applied to detect transcriptional activity of NF-κB. Results We found that CARMA1 was highly expressed in T-ALL cell lines, but not observed in B-ALL and AML cell lines. The T-ALL patients with high CARMA1 expression had better outcome compared with the low expression group. However, we failed to observe any significant differences in the expression of BCL10 and MALT1 between primary normal and T-ALL cells or relationship of survival with MALT1 and BCL10 in T-ALL. A previous finding showed that Notch1 mutations are correlated with an improved long-term prognosis (J Clin Oncol 2013;31:4333). The above results suggested both high expression of CARMA1 and activating Notch1 mutaions relate to favorable prognosis, indicating the possible relevance between CARMA1 expression and Notch1 activation. To elucidate the effect of CARMA1 on cell proliferation, we performed CARMA1 knockdown experiment with shRNAs in T-ALL cells. CARMA1 knockdown in MOLT-4 cells significantly decrease the quantity and size of cell colonies. MOLT-4 and Jurkat cell lines showed significant reductions in cell viability through CARMA1 silencing compared with control-shRNA cells. The EdU proliferation assay demonstrated that knockdown of CARMA1 in MOLT-4 and Jurkat cell lines markedly reduced the number of EdU-positive cells. Interestingly, CARMA1 had no significant effect on cell proliferation in CCRF-CEM cells. This finding is consistent with a recent report, which demonstrated that CARMA1 knockdown showed no difference in proliferation of CCRF-CEM cells, but only one cell line was used (Leukemia. 2017;31:255). CCRF-CEM is a SIL-TAL1 fusion gene positive cell line and its survival depends on the activation of TAL1 transcriptional complex. Therefore, the proliferation of SIL-TAL1 fusion gene positive cell lines may be independent of CARMA1 expression. We next explored the significance of CARMA1 in cell cycle and in vivo tumorigenic ability. Compared to the control-shRNA cells, CARMA1 knockdown arrested T-ALL cell lines in G1 phase. CARMA1 knockdown remarkably led to the downregulation of the percentage of CD45 positive cells and prolonged overall survival of NSG mice. To further determine the role of CARMA1 in Notch1-induced NF-Κb activation, we transfected Notch1-IC into control-shRNA and CARMA1-shRNA cells, respectively. We found that Notch1-IC significantly upregulated the activity of NF-κB in control-shRNA cells. However, the Notch1-induced NF-κB was abolished in CARMA1 knockdown T-ALL cells. The results demonstrate that CARMA1 is involved in Notch1-mediated NF-κB activation. Conclusion Taken together, CARMA1 markedly contributes to cell survival in SIL-TAL1 negative T-ALL cells and may play a crucial role in Notch1-induced activation of NF-κB pathway. Furthermore, CRAMA1 might be a prognostic marker and a prospective therapeutic target for T-ALL. Disclosures No relevant conflicts of interest to declare.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2440-2440
Author(s):  
Tian Yuan ◽  
Yaling Yang ◽  
Jeffrey You ◽  
Daniel Lin ◽  
Kefeng Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Rt Pcr ◽  
Altered Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

TYK2-STAT1 Pathway Positively Regulates BCL2 Gene Expression in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1470-1470
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Jason M Glover ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Protein ◽  
Bcl2 Expression

Abstract Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals BRD4 Proteolysis Targeting Chimera (PROTAC) ARV-825 Targets Both NOTCH1-MYC Regulatory Circuit and Leukemia-Microenvironment in T-ALL

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 716-716
Author(s):  
Sujan Piya ◽  
Hong Mu ◽  
Seemana Bhattacharya ◽  
Teresa McQueen ◽  
Richard E Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
New Haven ◽  
Down Regulation ◽  
Regulatory Circuit ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Background: Salvage options for patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) are limited, with less than 25% of these patients achieving second remission 1, 2. 70% of T-ALL cases have activating mutations of the NOTCH1 pathway, which transcriptionally activates MYC by binding to its `superenhancer' region 3, 4. Other deregulated oncogenic pathways in T-ALL include PI3K/Akt, the anti-apoptotic Bcl-2 family, and CDKN2A/2B cell cycle regulators 5, 6. The NOTCH1-MYC regulatory circuit is an attractive therapeutic target, but clinical development of gamma-secretase inhibitors (GSI) to target NOTCH1 has been limited by 'on target' toxicities. A better target may be BRD4, a critical component of superenhancer complexes that binds to acetylated histone (3 and 4) and drives NOTCH1 mediated MYC transcription7. ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that has 3 components: a thienodiazepine-based BRD4 ligand, a linker arm, and a cereblon-binding ligand. ARV-825 recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4, resulting in down-regulation of MYC. Methods: We investigated the effectiveness of ARV-825 against T-ALL cell lines, including GSI-resistant lines. Since microenvironmental signals are critical for the survival of T-ALL, we specifically tested the impact of BRD4 degradation on CD44/CD44v, which integrates cell-extrinsic microenvironmental signals and is part of cysteine transporter that maintains low intra-cellular reactive oxygen species (ROS), necessary for T-ALL survival and the persistence of disease. We also examined the anti-leukemic effect of ARV-825 in a T-ALL patient-derived xenograft (PDX) mouse model of disseminated leukemia with a constitutively active NOTCH1 mutation. Results: The IC50s for all tested T-ALL cell lines at 72 hours were in the low nanomolar range (< 50 nM). ARV-825 leads to sustained degradation of BRD4 and down-regulation of its transcriptional targets MYC, Bcl-2 and Bcl-XL and inhibits cell proliferation and induces apoptosis in GSI-sensitive (HPB-ALL, KOPT1) and GSI-resistant (MOLT4, SUPT1) cell lines. Mass cytometry based proteomic analysis (CyTOF) and immunoblotting showed that ARV-825 down-regulated cell intrinsic oncogenic molecules: transcription factors Myc and NFkB, cell cycle regulator CDK6, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins. In addition ARV-825 down regulated two key molecules involved in leukemia-stroma interaction; CD44 (Fig. 1), and CD98, a component of amino acid transporters xCT, LAT1 and 2, both essential in regulation of oxidative stress. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs . untreated). As a functional correlate of down-regulation of CD98/CD44/CD44v, flow cytometry confirmed increased intracellular ROS generation (Fig. 2). Finally, in a PDX mouse model of human T-ALL, ARV-825 treatment resulted in lower leukemia burden (confirmed by flow cytometry for human CD45+ cells in bone marrow) and better survival compared to vehicle-treated control mice (p=0.002) (Fig.3). Reference: 1. Marks DI, Rowntree C. Management of adults with T-cell lymphoblastic leukemia. Blood 2017; 129(9): 1134-1142. 2. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood 2015; 126(7): 833-41. 3. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood 2017; 129(9): 1124-1133. 4. Demarest RM, Ratti F, Capobianco AJ. It's T-ALL about Notch. Oncogene 2008; 27(38): 5082-91. 5. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics and molecular biology of T-ALL. Blood 2017; 129(9): 1113-1123. 6. Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC et al. Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 2009; 113(8): 1689-98. 7. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153(2): 320-34. Disclosures Qian: 4Arvinas, LLC. New Haven, CT: Employment. Raina: 4Arvinas, LLC. New Haven, CT: Employment. McKay: 6 ImmunoGen, Inc.Waltham, MA: Employment. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Andreeff: Daiichi Sankyo: Consultancy.

scholarly journals Therapeutic Stratification of PTEN-MYC Axis Appears to be Promising for Molecular Targeted Therapies in Refractory and Relapsed T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3883-3883
Author(s):  
Heinz Ahlert ◽  
Sanil Bhatia ◽  
Marc Remke ◽  
Melf Sönnichsen ◽  
Niklas Dienstbier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Drug Combinations ◽  
Killing Effect ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Lines

Therapy refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL) is still one of the most common causes for cancer related death in children. T-ALL is a highly heterogeneous disease with several established biomarkers, which promote its progression. These biomarkers are promising tools for patient stratification and can be used for targeted therapeutic approaches. For instance, PTEN is one of the most displayed mutations in pediatric T-ALL and plays an important role in proteolysis of the oncoprotein MYC. Therefore, it is not surprising that PTEN activity in T-ALL patients correlates with higher MYC protein levels. Importantly, PTEN mutations have recently been associated with poor overall survival in T-ALL patients, underscoring its importance for future treatment options using molecular targeted therapies. In this study, we validated the importance of PTEN as a MYC regulator in T-ALL and elucidated PTEN mutational status as a stratification marker for T-ALL patients. Comparative quantification of PTEN, phosphorylated AKT and MYC in leukemic T-cell lines (n=10) was analyzed at protein level, relative to healthy T-cells. PTEN loss or deceased expression was observed in half of the screened leukemic T-cell lines with concomitant high MYC expression and increased AKT phosphorylation. To identify the drug sensitivity of PTEN mutant (PTENm) and PTEN wild type (PTENwt) cell lines, high-throughput drug screening was performed with 180 inhibitors, including among others kinase-, BET- and gamma secretase inhibitors, as well as conventional chemotherapeutics. PTENm cell lines showed remarkable sensitivity in nanomolar ranges against PI3K inhibitors compared to PTENwt (P=0.001). Anticipating that tumor cells eventually escape the killing effect of single pathway targeted drugs, the need for molecular identification and characterization of synergistic drug combinations is evident. Therefore, diverse compound combinations were tested in an 11 by 11 matrix of different concentrations in PTENm and PTENwt cell lines. Synergism was predicted with Combenefit software using Loewe model to assess putative additive versus synergistic effects. We observed that simultaneous use of copanlisib (PI3K inhibitor) with JQ1 (BET inhibitor) had a strong killing effect in PTENm cell lines. To assess target downregulation after 24 hours of drug treatment, lysates were taken and visualized using western blot. A strong decrease of MYC expression was observed already with a combination of low concentrations of copanlisib and JQ1 with subsequent higher apoptosis induction as measured by Annexin V/PI staining. A second drug combination that showed synergy in PTENm was observed with copanlisib and alisertib, an Aurora kinase-A (AURKA) inhibitor, expecting changes in cell cycle and reduced MYC stability. Indeed, results of cell cycle analysis have revealed an inhibition during mitosis and cellular MYC levels were decreased, suggesting that this combination affects highly proliferative MYC-dependent tumor cells. Contrary to PTENm, in PTENwt cell lines, PI3K inhibition alone showed no cytotoxic effect, but synergy was detected in combination with the MEK inhibitor cobimetinib. Moreover, in this synergistic combination MYC was effectively downregulated on protein level accompanied with changes in regulatory pathways including AKT and MEK. Interestingly, phosphorylation of MYC changed from the stabilizing site (S62) to the degradative site (T58), proposing the progress of MYC degradation. In summary, we observed the importance of PTEN as a regulator for MYC proteolysis and related pathways including AKT and MEK. The loss of PTEN is accompanied with increased MYC. Synergy studies have observed effective drug combinations for PTENm and PTENwt with the aim to downregulate MYC and induce apoptosis in tumor cells. Based on these findings, PTEN-MYC axis seems suitable as a potential stratification marker for future therapy options in refractory and relapsed T-ALL. In the next step, promising drug combinations will be tested in a patient derived xenograft mouse model to validate these findings. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia

Leukemia ◽  
2008 ◽  
Vol 23 (1) ◽  
pp. 125-133 ◽  
Author(s):  
C Graux ◽  
◽  
M Stevens-Kroef ◽  
M Lafage ◽  
N Dastugue ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals Clinical characterization and prognosis of T cell acute lymphoblastic leukemia with high CRLF2 gene expression in children

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0224652
Author(s):  
Mingmin Wang ◽  
Jinquan Wen ◽  
Yuxia Guo ◽  
Yali Shen ◽  
Xizhou An ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia

Interleukin-7 promotes survival and cell cycle progression of T-cell acute lymphoblastic leukemia cells by down-regulating the cyclin-dependent kinase inhibitor p27kip1

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1524-1531 ◽  
Author(s):  
Joao T. Barata ◽  
Angelo A. Cardoso ◽  
Lee M. Nadler ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Cycle Progression ◽  
Lymphoblastic Leukemia ◽  
Cyclin Dependent Kinase ◽  
Malignant Cells ◽  
Cycle Progression ◽  
Interleukin 7 ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Immature Thymocytes

In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27kip1 was mandatory for IL-7–mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7–mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7–mediated proliferation and p27kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.

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