scholarly journals Targeting USP7 Suppresses Tumor Development By Promoting Ubiquitination of PHF8 and Suppressing SNAI1/Wnt Signaling in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1156-1156
Author(s):  
Jie Zi ◽  
Chunhua Song ◽  
Zheng Ge
Keyword(s):  
Cell Proliferation ◽  
Wnt Signaling ◽  
Inflammatory Cell ◽  
Lymphoblastic Leukemia ◽  
Mrna Level ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Microarray Datasets ◽  
Marrow Cellularity

Abstract Introduction T-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy with a high unfavorable prognosis. Ubiquitin-specific-processing protease 7 (USP7) is one of the deubiquitinating enzymes attracting concentrated attention in current studies for cancers; it is also involved in regulation of oncogenic transcriptional program in T-ALL, and serves as a potential target to treat T-ALL. USP7 is interacted with PHD Finger protein 8 (PHF8), a histone lysine demethylase in T-ALL. However, the impact of the interaction in T-ALL development is undetermined. In this study, we explored the anti-tumor effect of targeting USP7 and its downstream substrate in T-ALL and underlying mechanism. Methods In vitro GST pull-down assay, co-immunoprecipitation assay, protein deubiquitination and stability assays were used to detect the interaction of USP7 with its direct substrate and the ubiquitination level. Gain-of-function(overexpression) and loss-of-function (lentiviral shRNA knockdown) approaches for USP7 were conducted for gene expression, CCK-8 cell proliferation assay, and apoptosis assay with annexin V + PI staining. ChIP-qPCR assay was used to explore the enrichment of histone markers in promoter region of target gene. In vivo human T-ALL xenograft mouse model was developed in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse by tail vein injection of 2.5 × 10 6 /mouse and the leukemia engraftment was evaluated with human CD45+ cells by flow cytometry. The qPCR and western blot were carried out for detection mRNA level and protein level of related genes. The mRNA level of USP7 and its substrate PHF8 was examined by qPCR in 43 newly-diagnosed T-ALL patients with an approval of the Ethics Committee. Microarray datasets of T-ALL patients from GEO database were used to determine the USP7 and its substrate -related genes with web-based platform MEM and Limma. Results Results showed that USP7 directly interacted with PHF8; and overexpressed USP7 deubiquitinated and stabilized PHF8, and enhanced cell proliferation of JURKAT T-ALL cells. These data indicates targeting USP7 promotes cell proliferation of T-ALL cells through its substrate PHF8. Moreover, SNAI1 was identified as one of key co-expressed genes with PHF8 in microarray datasets from T-ALL patients, and knock-down of PHF8 suppressed the SNAL1 expression and increased the recruitment of repressive histone marker, H3K9me2 but decreased the enrichment of H3K4me3 in SNAI1 promoter. SNAI1 knockdown significantly induced the cell proliferation arrest and apoptosis of T-ALL cells (Fig.1A & 1B). With in vivo human leukemia xenograft mouse model, we further demonstrated that SNAI1 knockdown significantly reduce the spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity and also the inflammatory cell infiltration compared to the scramble shRNA control (Fig.1C-1F). Moreover, Wnt signaling was identified as SNAI1 interaction partners by the pathway analysis in the SNAI1 interaction genes in the microarray data from T-ALL patients. SNAI1 knockdown significantly suppressed the expression of the Axin2 and Survivn, the key components of Wnt signaling. These data suggested oncogenic role of USP7/PHF8/SNAI1/Wnt signaling in T-ALL. Next, we explored the effect of SNAI1 on USP7 knockdown-induced anti-tumor effect. We found that USP7 knockdown induced cell proliferation arrest and apoptosis, and overexpression of SNAI1 could block the effect in vitro. Furthermore, the in vivo data showed that USP7 knockdown significantly suppressed spleen size and weight, the ratio of human CD45(+) cells, bone marrow cellularity, inflammatory cell infiltration, and the protein expression of PHF8, SNAI1, Axin2 and Survivin; and SNAI1 overexpression completely rescue the USP7 knockdown-mediated antitumor effect and restored the expression of Axin2 and Survivin in vivo (Fig.2). Conclusion: Our results demonstrated that targeting USP7 by shRNA induces the cell proliferation arrest and apoptosis by promoting ubiquitination of PHF8 and suppressing SNAI1/Wnt signaling in T-ALL. Our data also revealed the oncogenic roles of USP7/PHF8/SNAI1/Wnt signaling in T-ALL and suggested targeting the signaling pathway as potential therapy in T-ALL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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 The research significance of concomitant use of CAR-CD138-NK and CAR-CD19-NK to target multiple myelomas

2018 ◽  
Vol 16 ◽  
pp. 205873921878896 ◽  
Author(s):  
Songbo Zhao ◽  
Zhichao Han ◽  
Cheng Ji ◽  
Gangli An ◽  
Huimin Meng ◽  
...  
Keyword(s):  
Nk Cells ◽  
Lymphoblastic Leukemia ◽  
Small Subset ◽  
Novel Drugs ◽  
Prevalent Disease ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Multiple Myelomas ◽  
Made In

Multiple myeloma (MM) is a type of cancer characterized by abnormal proliferation of clonal cells; it is the very dangerous and highly prevalent disease. Although significant progress has been made in clinical research, especially with novel drugs such as bortezomib, lenalidomide, and carfilzomib, most of the patients with MM still suffer from often fetal relapses due to drug resistance. In this study, we aimed to develop immune cells that could specifically target and destroy MM cells. Chimeric antigen receptor–modified NK-92 (CAR-NK92) cells have been very effective against B-cell acute lymphoblastic leukemia (B-ALL); as MM shows high expression of CD138, we constructed CD138-directed CAR-NK-92MI cells (CAR-CD138). It 2is reported that there is a small subset of CD138–/CD19+ MM cells showing, to some extent, stem cell qualities. We therefore generated the CD19-directed CAR-NK-92MI cells (CAR-CD19) as well. These two CAR-NK cells showed strong in vitro biological activity in specifically killing target tumor cells. Thus, the concomitant use of these CAR-NK cells may achieve excellent results in vivo.

scholarly journals miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1726
Author(s):  
Valentina Saccomani ◽  
Angela Grassi ◽  
Erich Piovan ◽  
Deborah Bongiovanni ◽  
Ludovica Di Martino ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Target Genes ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Signaling Pathway

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.

scholarly journals Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood ◽  
2019 ◽  
Vol 133 (21) ◽  
pp. 2291-2304 ◽  
Author(s):  
Diego Sánchez-Martínez ◽  
Matteo L. Baroni ◽  
Francisco Gutierrez-Agüera ◽  
Heleia Roca-Ho ◽  
Oscar Blanch-Lombarte ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Antileukemic Activity ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

Efficacy of the CDK inhibitor dinaciclib in vitro and in vivo in T-cell acute lymphoblastic leukemia

2017 ◽  
Vol 405 ◽  
pp. 73-78 ◽  
Author(s):  
Sausan A. Moharram ◽  
Kinjal Shah ◽  
Fatima Khanum ◽  
Alissa Marhäll ◽  
Mohiuddin Gazi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Cdk Inhibitor ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals Application ofAntrodia camphorataPromotes Rat’s Wound HealingIn Vivoand Facilitates Fibroblast Cell ProliferationIn Vitro

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Zahra A. Amin ◽  
Hapipah M. Ali ◽  
Mohammed A. Alshawsh ◽  
Pouya H. Darvish ◽  
Mahmood A. Abdulla
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Granulation Tissue ◽  
Inflammatory Cell ◽  
Biological Activities ◽  
Fibroblast Cell ◽  
Parasitic Fungus ◽  
Antrodia Camphorata

Antrodia camphoratais a parasitic fungus from Taiwan, it has been documented to possess a variety of pharmacological and biological activities. The present study was undertaken to evaluate the potential ofAntrodia camphorataethanol extract to accelerate the rate of wound healing closure and histology of wound area in experimental rats. The safety ofAntrodia camphoratawas determinedin vivoby the acute toxicity test andin vitroby fibroblast cell proliferation assay. The scratch assay was used to evaluate thein vitrowound healing in fibroblast cells and the excision model of wound healing was testedin vivousing four groups of adultSprague Dawleyrats. Our results showed that wound treated withAntrodia camphorataextract and intrasite gel significantly accelerates the rate of wound healing closure than those treated with the vehicle. Wounds dressed withAntrodia camphorataextract showed remarkably less scar width at wound closure and granulation tissue contained less inflammatory cell and more fibroblast compared to wounds treated with the vehicle. Masson’s trichrom stain showed granulation tissue containing more collagen and less inflammatory cell inAntrodia camphoratatreated wounds. In conclusion,Antrodia camphorataextract significantly enhanced the rate of the wound enclosure in rats and promotes thein vitrohealing through fibroblast cell proliferation.

scholarly journals The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway

2008 ◽  
Vol 295 (6) ◽  
pp. G1150-G1158 ◽  
Author(s):  
Sharon DeMorrow ◽  
Heather Francis ◽  
Eugenio Gaudio ◽  
Julie Venter ◽  
Antonio Franchitto ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Calcium Release ◽  
Wnt Signaling Pathway ◽  
Xenograft Model ◽  
Orphan Receptor

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca2+-dependent pathway involving protein kinase C, or a Ca2+-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH2-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.

scholarly journals CXCR4 Blockade By BL-8040 in T Cell Acute Lymphoblastic Leukemia Decreases Mitochondrial Mass and Induces Non-Apoptotic Cell Death

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2745-2745
Author(s):  
Jun Xia ◽  
Stephanie Sun ◽  
Matthew RM Jotte ◽  
Geoffrey L. Uy ◽  
Osnat Bohana-Kashtan ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Lymphoblastic Leukemia ◽  
Apoptotic Cell Death ◽  
Employment Equity ◽  
Cxcr4 Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 10-15% of pediatric and 25% of adult ALL cases. CXCL12 is a CXC chemokine that is constitutively expressed at high levels in the bone marrow. CXCR4 is the major receptor for CXCL12 and is by far the most highly expressed chemokine receptor on T-ALL cells. Two groups recently showed that genetic loss of CXCR4 signaling in murine or human T-ALL cells markedly suppressed their growth in vivo. We previously reported that BL-8040, a potent new CXCR4 antagonist with sustained receptor occupancy, is active as monotherapy against T-ALL in mice. Indeed, a 2-week course of daily BL-8040 resulted in a median reduction in tumor burden of 32.1-fold (range 6.8 to 176) across 5 different T-ALL xenografts. Preliminary data from a clinical trial of BL-8040 plus nelarabine for relapsed T-ALL also suggest therapeutic activity, with a complete remission rate observed in 4/8 patients (50%), which compares favorably to published response rates of approximately 30% with single agent nelarabine. Here, we explore molecular mechanisms by which CXCR4 blockade induces T-ALL death. NOD-scid IL2Rgammanull (NSG) mice were injected with P12-Ichikawa cells, a T-ALL cell line modified to express click beetle red luciferase and GFP. Following T-ALL engraftment, mice were treated with a single dose of BL-8040, and then leukemic cells in the bone marrow harvested 24-48 hours later. Treatment with BL-8040 resulted in a marked suppression of Akt and Erk1/2 phosphorylation, suggesting that signaling through CXCR4 is the major source of PI3 kinase pathway activation in T-ALL cells. Surprisingly, treatment with BL-8040 did not affect cellular proliferation, as measured by Ki67/FxCycle Violet staining or by EdU labeling. Moreover, no increase in apoptosis, as measured by annexin V or activated caspase 3 expression, was observed. These data suggest that CXCR4 blockade induces a non-apoptotic cell death. To explore this possibility further, we performed transcriptome sequencing on T-ALL cells recovered from mice 24 hours after 1 dose of BL-8040. A total of 151 differentially expressed genes (FDR of < 0.05% and ≥ 2-fold change) were identified. Gene set enrichment analysis was strongly positive for alterations in oxidative phosphorylation, ribosome biogenesis, and carbohydrate metabolism. Ribosome function was assessed using O-propargyl-puromycin (OPP), which monitors global protein translation. No difference in global protein synthesis in T-ALL cells was observed after CXCR4 blockade in vivo. T-ALL cells are dependent on glutamine as a source of carbon, and PI3 kinase signaling positively regulates glutaminolysis. Thus, we hypothesized that CXCR4 blockade may induce T-ALL cell death by reducing glutamine metabolism. However, treatment of T-ALL cells in vitro with BL-8040 did not alter the cellular levels of glutamine or glutamate, as measured using a commercial bioluminescent assay. Confirmatory metabolic tracing studies using 13C-labeled glutamine and glucose are in progress. Finally, to explore the reduction in oxidative phosphorylation, we examined mitochondria function using Mitotracker Green. Treatment of T-ALL cells in vitro with BL-8040 for 24-48 hours induced a significant decrease in mitochondria number, suggesting induction of mitophagy. Collectively, these data suggest that T-ALL cells are addicted to CXCR4 signaling in vivo. CXCR4 blockade with BL-8040 induces a non-apoptotic cell death that is characterized by a loss of mitochondria. Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Bohana-Kashtan:BiolineRx: Employment, Equity Ownership. Sorani:BiolineRx: Employment, Equity Ownership. Vainstein:BiolineRx: Employment, Equity Ownership.

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