scholarly journals Pharmacological Inhibition of MAP2K7 Induces Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3889-3889
Author(s):  
Taylor J Chen ◽  
Ye Shen ◽  
Cory Seth Bridges ◽  
Chun Shik Park ◽  
Jacob J. Junco ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Pharmacological Inhibition ◽  
Cycle Arrest ◽  
Caspase 7 ◽  
Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2488-2488 ◽  
Author(s):  
Yana Pikman ◽  
Andrew Furman ◽  
Emily S. Lee ◽  
Andrew E. Place ◽  
Gabriela Alexe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cyclin D3 ◽  
Cycle Arrest ◽  
Cell Acute Lymphoblastic Leukemia

Abstract While significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), approximately 10-20% of newly diagnosed patients will experience either induction failure or relapse. Additionally, fewer than 50% of T-ALL patients who experience a relapse are long-term survivors. New targeted therapies are needed for the treatment of this disease. Multiple lines of evidence point to Cyclin D3/CDK4/6 as a potential therapeutic target in T-ALL. Cyclin D3 (CCND3), a direct target of activated NOTCH1, is upregulated in T-ALL, and CCND3 null animals are refractory to NOTCH1 driven T-ALL. CCND3 binds and activates CDK4/6, and the CCND3-CDK complex phosphorylates the tumor suppressor RB leading to cell cycle progression. Previous studies have demonstrated that CDK4/6 small-molecule inhibition is an effective therapeutic strategy for the treatment of NOTCH1-driven T-ALL mouse models. Using the publicly available Genomics of Drug Sensitivity in Cancer data set, we identified NOTCH1 mutations as a biomarker of response and RB mutations as a biomarker of resistance to the CDK4/6 inhibitor palbociclib. We validated that RB null status predicts resistance to the Novartis CDK4/6 inhibitor LEE011 in a panel of T-ALL cell lines. Interestingly, we identified both NOTCH1 mutant, as well as NOTCH1 wildtype, T-ALL cell lines that were sensitive to LEE011 treatment. Mining of publicly available data revealed that CDK6 is consistently marked by a super-enhancer in T-ALL cell lines, both NOTCH1 mutant and wildtype, suggesting another potential reason for sensitivity to CDK4/6 inhibition in this lineage. Treatment with LEE011 also led to a dose-dependent cell cycle arrest and cell death in T-ALL cells, including MOLT4 (NOTCH1 mutant) and MOLT16 (NOTCH1 wildtype). Combinations of drugs with CDK4/6 inhibitors will likely be critical for the successful translation of this drug class because they generally do not induce cell death. Combinations with cytotoxic chemotherapy are predicted to be antagonistic, however, as most of these drugs rely on rapidly proliferating cells, and CDK4/6 inhibition induces cell cycle arrest. To discover effective, and immediately translatable combination therapies with LEE011 in T-ALL, we performed combination studies of LEE011 with agents standardly used for T-ALL treatment, including corticosteroids, methotrexate, mercaptopurine, asparaginase, vincristine and doxorubicin. Combinations of LEE011 with methotrexate, mercaptopurine, vincristine or asparaginase were antagonistic in T-ALL cell lines while the combination with doxorubicin was additive. Combination treatment of LEE011 with corticosteroids had a synergistic effect on cell viability in MOLT4 and MOLT16 cell lines as measured by excess over Bliss additive and isobologram analyses. This combination also decreased phospho RB signaling, increased cell cycle arrest and induced cell death to a greater degree than either drug alone. LEE011 treatment increased CCND3 protein levels, an effect mitigated by glucocorticoid treatment, one possible mechanism contributing to the observed synergy. Additionally, the combination of LEE011 with everolimus, an mTOR inhibitor, was synergistic in these cell lines. We next extended testing to in vivo models of T-ALL. In a MOLT16 orthotopic mouse model, the combination of LEE011 and everolimus significantly prolonged mouse survival compared to treatment with each individual drug alone. The combination of LEE011 with dexamethasone did not extend survival over treatment with LEE011 alone and dexamethasone was inactive in vivo. Both LEE011 and everolimus had on-target activity in the treated mice as measured by inhibition of peripheral blood phospho-RB and phospho-4EBP1. We then tested the combination of LEE011 with dexamethasone in a second mouse model, a MOLT4 orthotopic model. Here, the combination of LEE011 with dexamethasone was more effective in prolonging survival compared to each treatment alone, supporting a heterogeneous response to the combination of LEE011 with dexamethasone in vivo. We conclude that LEE011 is active in T-ALL, and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation in the clinical setting. Disclosures Kim: Novartis Pharmaceuticals: Employment. Stegmaier:Novartis Pharmaceuticals: Consultancy.

Ligand Activation of FLT3 Induces Cell Cycle Arrest in Acute Lymphoblastic Leukemia with 11q23 Translocation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1888-1888
Author(s):  
Yoshiyuki Furuichi ◽  
Kanji Sugita ◽  
Takeshi Inukai ◽  
Kumiko Goi ◽  
Kazuya Takahashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Cycle Arrest

Abstract A fms-like kinase (FLT3) is widely known to be involved in proliferation of normal hematopoietic stem cells and precursors. Acute lymphoblastic leukemia (ALL) with 11q23 translocation, often found in infantile leukemia with very poor prognosis, is thought to be derived from a population of cells at the developmental stage very close to hematopoietic stem cells, and was recently shown that they express FLT3 at high levels compared with other types of leukemia. In the present study, we examined the effects of FLT3 ligand (FL) on leukemia cells with or without 11q23 translocation to evaluate a biological implication of the FLT3/FL interaction in ALL. Three of 8 leukemic cell lines without 11q23 translocation showed a proliferative response to FL in the 3H-thymidine uptake assays. However, five of 7 B-precursor leukemic cell lines with 11q23 translocation, unexpectedly, showed an inhibitory response (23-69% inhibition) to FL in a dose-dependent manner (1–20ng/ml), although the special cell line with D835 mutation in FLT3 (KOCL-33) was not affected by the addition of FL. This inhibitory effect was almost abrogated in the presence of a FLT-3 kinase inhibitor PKC412. Inhibition of 3H-thymidine uptakes were not due to induction of apoptosis but due to induction of the Go/G1 arrest. This cell cycle arrest was mediated, at least in part, by a marked up-regulation of p27 due to suppression of its degradation, and promoted resistance of cell lines to radiation-induced apoptosis. Of interest, the addition of FL induced a complete disappearance of constitutive phosphorylation of STAT5 but upregulated phosphorylation of MAPK and Akt. These results suggest that the FLT3/FL interaction in ALL with 11q23 translocation transmits the inhibitory signal specifically to the JAK/STAT pathway via the kinase activity of FLT3, in the process of which the JAK/STAT-specific inhibitory molecules such as SOCS-2 and CIS-1 may be implicated.

Hydroalcoholic leaves extract of Vaccinium ashei Reade promotes cell cycle arrest and apoptosis on T-cell acute lymphoblastic leukemia

2021 ◽  
pp. 1-5
Author(s):  
Dalila Meneghetti ◽  
Verciane Schneider Cezarotto ◽  
Natália Paiva do Nascimento ◽  
Natacha Azussa Migita ◽  
Juliana Ronchi Corrêa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Cycle Arrest ◽  
Lymphoblastic Leukemia ◽  
Vaccinium Ashei ◽  
Cycle Arrest ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals Synergistic activity of rapamycin and dexamethasone in vitro and in vivo in acute lymphoblastic leukemia via cell-cycle arrest and apoptosis

2012 ◽  
Vol 36 (3) ◽  
pp. 342-349 ◽  
Author(s):  
Chong Zhang ◽  
Yong-Ku Ryu ◽  
Taylor Z. Chen ◽  
Connor P. Hall ◽  
Daniel R. Webster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Cycle Arrest ◽  
Lymphoblastic Leukemia ◽  
Synergistic Activity ◽  
Cycle Arrest

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2372-2372
Author(s):  
Kam Tong Leung ◽  
Karen Kwai Har Li ◽  
Samuel Sai Ming Sun ◽  
Paul Kay Sheung Chan ◽  
Yum Shing Wong ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Jnk Pathway ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induced Apoptosis

Abstract Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

Epigenetic Control Of Cell Cycle-Promoting Genes By Ikaros and Casein Kinase II In B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3734-3734
Author(s):  
Sinisa Dovat ◽  
Chunhua Song ◽  
Xiaokang Pan ◽  
Yali Ding ◽  
Chandrika S. Gowda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Regulatory Elements ◽  
Preclinical Model ◽  
Cycle Arrest ◽  
Cell Acute Lymphoblastic Leukemia

Abstract IKZF1 (Ikaros) encodes a kruppel-like zinc finger protein that is essential for normal hematopoiesis and acts as a tumor suppressor in acute lymphoblastic leukemia (ALL). The deletion and/or mutation of Ikaros is associated with the development of human T-cell and B-cell acute lymphoblastic leukemia (B-ALL) with poor outcome. In vivo, Ikaros binds DNA and regulates gene expression by chromatin remodeling. Since there is a paucity of known genes that are regulated by Ikaros, the molecular mechanisms through which Ikaros exerts its tumor suppressor function remain unknown. Here we describe studies that identify the targets and mechanisms of Ikaros-mediated epigenetic regulation in human B-ALL. We used chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) to identify target genes that are bound by Ikaros in vivo in human B-ALL, and to define epigenetic patterns associated with Ikaros binding. ChIP-seq revealed a large set of Ikaros target genes that contain a characteristic Ikaros binding motif. The largest group of genes that are direct Ikaros targets included genes that are essential for cell cycle progression. These included CDC2, CDC7, CDK2 and CDK6 genes whose deregulation is associated with malignant transformation. The strong binding of ikaros to the promoters of cell cycle-promoting genes was confirmed by quantitative immunoprecipitation in primary leukemia cells. To establish whether Ikaros directly regulates transcription of the cell cycle-promoting genes, their expression was measured in B-ALL cells that were transduced with either a retroviral vector that contains Ikaros, or a control vector. Target gene expression was monitored by qRT-PCR. Ikaros strongly repressed transcription of the cell cycle-promoting genes, which resulted in cell cycle arrest. Global epigenetic profiling using ChIP-seq suggested that Ikaros represses cell cycle-promoting genes by inducing epigenetic changes that are consistent with repressive chromatin. High-resolution epigenetic profiling of the upstream regulatory elements of the cell cycle-promoting genes targeted by Ikaros showed that increased Ikaros expression results in the formation of heterochromatin, which is characterized by the presence of the H3K9me3 histone modification and associated transcriptional repression. Functional analysis revealed that phosphorylation of Ikaros by the oncogenic protein. Casein kinase II (CK2), impairs its function as a transcriptional repressor of the cell cycle-regulating genes. Inhibition of CK2 by specific inhibitors enhances Ikaros-mediated repression of the cell cycle-regulating genes resulting in cessation of cellular proliferation and cell cycle arrest in vitro and in vivo in a B-cell ALL preclinical model. This was associated with increased Ikaros binding and the formation of heterochromatin at upstream regulatory elements of the cell cycle-promoting genes. Our results provide evidence that Ikaros functions as a repressor of cell cycle-promoting genes in B-ALL by directly binding their promoters and inducing the formation of heterochromatin with characteristic H3K9me3 histone modifications Ikaros repressor function is negatively regulated by CK2 kinase in B-cell ALL. Inhibition of CK2 enhances Ikaros mediated-repression of cell cycle-promoting genes resulting in an anti-leukemia effect in a preclinical model of B-cell ALL. Presented data identified the mechanism of action of CK2 inhibitors and demonstrated their efficacy in B-cell ALL preclinical model. Results support the use of CK2 inhibitors in Phase I clinical trial. Supported by National Institutes of Health R01 HL095120 and a St. Baldrick’s Foundation Career Development Award (to S.D.). Disclosures: No relevant conflicts of interest to declare.

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