Establishment of a Patient-Derived T-Cell Acute Lymphoblastic Leukemia Xenograft Model in Novel Immunodeficient NCG Mice / 中国实验血液学杂志

OBJECTIVE@#The leukemia cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) were inoculated into NCG mice to establish a stable human T-ALL leukemia animal model.@*METHODS@#Leukemia cells from bone marrow of newly diagnosed T-ALL patients were isolated, and the leukemia cells were inoculated into NCG mice via tail vein. The proportion of hCD45 positive cells in peripheral blood of the mice was detected regularly by flow cytometry, and the infiltration of leukemia cells in bone marrow, liver, spleen and other organs of the mice was detected by pathology and immunohistochemistry. After the first generation mice model was successfully established, the spleen cells from the first generation mice were inoculated into the second generation mice, and after the second generation mice model was successfully established, the spleen cells from the second generation mice were further inoculated into the third generation mice, and the growth of leukemia cells in peripheral blood of the mice in each group was monitored by regular flow cytometry to evaluate the stability of this T-ALL leukemia animal model.@*RESULTS@#On the 10th day after inoculation, hCD45+ leukemia cells could be successfully detected in the peripheral blood of the first generation mice, and the proportion of these cells was gradually increased. On average, the mice appeared listless 6 or 7 weeks after inoculation, and a large number of T lymphocyte leukemia cells were found in the peripheral blood and bone marrow smear of the mice. The spleen of the mice was obviously enlarged, and immunohistochemical examination showed that hCD3+ leukemia cells infiltrated into bone marrow, liver and spleen extensively. The second and third generation mice could stably develop leukemia, and the average survival time was 4-5 weeks.@*CONCLUSION@#Inoculating leukemia cells from bone marrow of patients with T-ALL into NCG mice via tail vein can successfully construct a patient-derived tumor xenografts (PDTX) model.

Study on the Regulation of Chidamide on CD8+T Cells in T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the regulatory effect of chidamide on CD8+ T cells in T-cell acute lymphoblastic leukemia.@*METHODS@#The expression levels of CXCL9 and CXCL3 mRNA in Jurkat cells, lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells were detected by fluorescence quantitative PCR. The proportion of CD8+ T cells in lymphocytes treated with chidamide and lymphocytes co-cultured with chidamide-treated Jurkat cells was determined by flow cytometry.@*RESULTS@#Chidamide upregulated CXCL9 mRNA expression in Jurkat cell line in a dose-dependent manner (r=0.950). The mRNA expression of CXCL9 in chidamide 5 μmol/L group was 164 times higher than that in control group. Chidamide upregulated CXCL9 mRNA expression in lymphocytes, but the up-regulated level was significantly lower than that in Jurkat cell line treated with the same concentration of chidamide. Co-culture with chidamide treated Jurkat cells upregulated the proportion of CD8+ T cells in lymphocytes.@*CONCLUSION@#In T-cell acute lymphoblastic leukemia, chidamide may increase the concentration of CXCL9 in the tumor microenvironment by up-regulating the expression of CXCL9 in tumor cells, leading to an increase in the number of CD8+ T cells.

The Effect of SP1 on the Progression of T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To study the transcriptional regulation of SP1 on the scaffold protein ARRB1 and its influence on the progression of T-cell acute lymphoblastic leukemia (T-ALL).@*METHODS@#pGL3-ARRB1-luc, pCDNA3.1-SP1 and other transcription factor plasmids that might be combined were constructed, and the binding of transcription factors to the promoter of ARRB1 was identified by dual luciferase reporter gene assay. Stable cell lines with over-expressed SP1 (JK-SP1) was constructed by lentiviral transfection, and the expression correlation of SP1 with ARRB1 was demonstrated by RT-PCR and Western blot. Further, the apoptosis, cell cycle and reactive oxygen species (ROS) were detected by flow cytometry. The effect of SP1 on propagation of leukemic cells was observed on NCG leukemic mice.@*RESULTS@#The expression of fluorescein were enhanced by co-transfection with pCDNA3.1-SP1 and pGL3-ARRB1-luc plasmids in HEK293T cell line (P<0.001), meanwhile, compared with the control group, the expression of ARRB1 mRNA and protein were increased in JK-SP1 cells (both P<0.01). Further in vitro experiments showed that, compared with the control group, the apoptosis rate was higher (x=22.78%) , the cell cycle was mostly blocked in G1 phase (63.00%), and the content of reactive oxygen species increased in JK-SP1 cells. And in vivo experiments showed that the mice injected with JK-SP1 cells through tail vein had a favorable overall survival time (average 33.8 days), less infiltration in liver and spleen tissue.@*CONCLUSION@#Transcription factor SP1 promotes the transcription and expression of ARRB1 by binding the the promoter of ARRB1 directly, thus delays the progress of T-ALL in vitro and in vivo. The study improves the pathogenesis of ARRB1 regulating the initiation and development of T-ALL, and provides theoretical basis for the development of new possible targeted drugs.

Chidamide inhibits the NOTCH1-MYC signaling axis in T-cell acute lymphoblastic leukemia / 医学前沿

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most dangerous hematological malignancies, with high tumor heterogeneity and poor prognosis. More than 60% of T-ALL patients carry NOTCH1 gene mutations, leading to abnormal expression of downstream target genes and aberrant activation of various signaling pathways. We found that chidamide, an HDAC inhibitor, exerts an antitumor effect on T-ALL cell lines and primary cells including an anti-NOTCH1 activity. In particular, chidamide inhibits the NOTCH1-MYC signaling axis by down-regulating the level of the intracellular form of NOTCH1 (NICD1) as well as MYC, partly through their ubiquitination and degradation by the proteasome pathway. We also report here the preliminary results of our clinical trial supporting that a treatment by chidamide reduces minimal residual disease (MRD) in patients and is well tolerated. Our results highlight the effectiveness and safety of chidamide in the treatment of T-ALL patients, including those with NOTCH1 mutations and open the way to a new therapeutic strategy for these patients.

Effect of Dimethyl Fumarate (DMF) on T-cell Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the effect and possible mechanism of dimethyl fumarate (DMF) on T-cell acute lymphoblastic leukemia (T-ALL), and provide experimental and theoretical basis for the clinical treatment of T-ALL.@*METHODS@#Jurkat cells were treated with different concentrations of DMF for 24 hours, and then the proportion and absolute count of Ki67-positive Jurkat cells were analyzed by flow cytometry. Meanwhile, the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and E3 ubiquitin ligase HACE1 in Jurkat cells treated with DMF for 24 hours were evaluated by Western blot. Nrf2 proteins were co-immunoprecipitated in Jurkat cells, and then HACE1 protein was assessed by Western blot. Plasmids of Flag-Nrf2 and different gradients of Flag-HACE1 were transfected into HEK293T cells, and the levels of Flag-Nrf2 were detected by Western blot after 48 hours.@*RESULTS@#DMF could significantly inhibit the proportion and absolute count of Ki67-positive Jurkat cells, and DMF inhibited the proliferation of Jurkat cells in a dose-dependent manner (r=0.9595, r=0.9054). DMF could significantly up-regulate the protein levels of Nrf2 and E3 ubiquitin ligase HACE1 in Jurkat cells (P<0.01, P<0.01). HACE1 physically interacted with Nrf2 in Jurkat cells. Overexpression of Flag-HACE1 significantly increased the protein level of Flag-Nrf2 in a dose-dependent manner (r=0.9771).@*CONCLUSION@#DMF inhibits the proliferation of T-cell acute lymphoblastic leukemia cell. The mechanism may be that, DMF significantly up-regulates the protein levels of Nrf2 and E3 ubiquitin ligase HACE1, and HACE1 interacts with Nrf2 and positively regulates Nrf2 protein level.

A Patient with Sequential Diseases of Langerhans Cell Sarcoma, Langerhans Cell Histiocytosis, and Acute Lymphoblastic Leukemia / 罕见病研究

Langerhans cell histiocytosis(LCH)and Langerhans cell sarcoma(LCS)are characterized by clone proliferation of Langerhans-type cells, which may occur concurrently or sequentially with T-cell acute lymphoblastic leukemia (T-ALL) and other Lymphoid neoplasms. A 15-year old female patient diagnosed with T-ALL developed LCH involving multiple systems during maintenance chemotherapy of T-AL. After treated with chemotherapy with improved result, the patient showed progression of the illness and refractory to the second-line treatment. We found c.G35A (p.G12D)mutation in the KRAS gene and used the targeted drug Trametinib for treatment. The treatment proved effective, leading to partial remission within a week. Three months after Trametinib treatment, the patient developed new lymphadenopathy. Biopsy revealed the existence of LCS. The disease progressed quickly, and the patient died 7 days after diagnosis of LCS. The case of patients with T-ALL then developing LCH and LCS sequentially is extraordinarily rare. The causes of the case is unclear and may be related to cell transdifferentiation, clonal evolution, and chemotherapy. Targeted drugs can contain this disease for a short time.

Effect of NACK knockdown on the proliferation and apoptosis of T-cell acute lymphoblastic leukemia Jurkat cells / 中华实用儿科临床杂志

Objective:To investigate the effect and mechanism of NACK knockdown on the proliferation and apoptosis of T-cell acute lymphoblastic leukemia (T-ALL) Jurkat cells. Methods:Lentivirus transfection technology was used to transfect Jurkat cells and knock down NACK gene.Real time fluorescent quantitative PCR and Western blot were used to detect the silencing efficiency of NACK gene.CCK-8 method and flow cytometry were used to detect the effects of NACK knockdown on the proliferation and apoptosis of Jurkat cells.The expressions of protein related with Notch1 pathway, such as Hes1 and c-Myc, were detected by Western blot. Results:After NACK-shRNA was successfully transfected into Jurkat cells by lentiviral vector, the expression of NACK mRNA and protein was reduced signi-ficantly ( P<0.05). Compared with the negative control group and the blank control group, the CCK-8 method showed that the cell proliferation in the experimental group was significantly inhibited [The inhibition rates of cell proliferation in the experimental group, negative control group and blank control group were (37.27±4.48)%, (4.25±2.10)% and (2.43±1.40)%, respectively]( F=132.640, P<0.05), and the flow cytometry test showed that the apoptosis in the experimental group increased significantly [The apoptosis rates of experimental group, negative control group and blank control group were (26.38±3.03)%, (6.07±2.61)% and (3.40±1.98)%, respectively]( F=90.534, P<0.05). Western blot results confirmed that the expression of Notch1 pathway-related proteins Hes1 and c-Myc was down-regulated compared with the negative control group and the blank control group, and the difference was statistically significant ( P<0.05). Conclusions:Targeting silent NACK can down-regulate the expression of Notch1 pathway-related proteins, which leads to the inhibition of Jurkat cell proliferation and increased apoptosis, thereby exerting its anti-T-ALL effect.

Childhood T-cell acute lymphoblastic leukemia in a single Latin American center: impact of improved treatment scheme and support therapy on survival

ABSTRACT Background and objective T-cell acute lymphoblastic leukemia (T-ALL) in children represents a high-risk disease. There is a lack of studies assessing the outcome of T-ALL in Hispanic populations, in which it is a rare malignancy. We report the characteristics and results of treatment for childhood T-cell ALL in children over 14 years at a Latin American reference center. Material and methods From January 2005 to December 2018, there occurred the analysis of twenty patients ≤ 16 years of age from a low-income open population diagnosed at a university hospital in Northeast Mexico. Clinical and laboratory characteristics, treatment regimens and outcomes were assessed by scrutinizing clinical records and electronic databases. Diagnosis was confirmed by flow cytometry, including positivity for CD-2, 5, 7 and surface/cytoplasmic CD3. Survival rates were assessed by the Kaplan-Meier method. Results There was a male preponderance (70 %), with a 2.3 male-to-female ratio (p= .074), the median age being 9.5 years. Leucocytes at diagnosis were ≥ 50 × 109/L in 13 (65 %) children, with CNS infiltration in 6 (30 %) and organomegaly in 10 (50 %). The five-year overall survival (OS) was 44.3 % (95 % CI 41.96-46.62), significantly lower in girls, at 20.8 % (95 % CI 17.32-24.51) vs. 53.1 % (95 % CI 50.30-55.82), (p= .035) in boys; there was no sex difference in the event-free survival (EFS) (p= .215). The survival was significantly higher after 2010 (p= .034). Conclusion The T-cell ALL was more frequent in boys, had a higher mortality in girls and the survival has increased over the last decade with improved chemotherapy and supportive care.

Expression of PD-1 in T-cell acute lymphoblastic leukemia cells and its clinical significance / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression and clinical significance of PD-1 molecule in tumor cells (T-ALL cells) derived from the patient with T-cell acute lymphoblastic leukemia (T-ALL). Methods: T-ALL cells from one patient and PBMCs from four healthy volunteers provided by the Department of Hematology in Jiangsu Provincial Hospital of Traditional Chinese Medicine in December 2015, and human 293T/PD-1 cells provided by Persongen Bio Therapeutics (Suzhou) Co., Ltd. were used for this study. The mouse T-ALL xenograft model was constructed by injecting T-ALL cells into tail vein of B-NDG mice, and flow cytometry was used to verify whether the cells obtained from the spleen of transplanted mice were mainly consisted of T-ALL cells. Flow cytometry was used to study the protein expression of PD-1 in T-ALL cells, and RT-PCR was applied to further verify the mRNA expression of PD-1 in T-ALL cells. The PD-1 gene in T-ALL cells was sequenced by SNP genotyping to detect whether the DNA sequence of PD-1 gene changed. PD-1 inhibitor was used in vitro to study their effects on proliferation, apoptosis, and the mRNA expression levels of related factors in T-ALL cells. Results: The mouse T-ALL xenograft model was successfully constructed and verified by flow cytometry as T-ALL. PD-1 was highly expressed at both mRNA and protein levels in T-ALL cells (all P<0.01). A C-to-T mutation was detected in the fifth exon of the PD-1 gene. PD-1 inhibitor had no significant effect on proliferation and apoptosis of T-ALL cells in vitro; PD-1 inhibitor up-regulated the mRNA expression of tumor-suppressor protein IGFBP3 and decreased the mRNA expression of oncoprotein SULT1A3 (all P<0.01). Conclusion: PD-1 is highly expressed in T-ALL cells, and PD-1 could be used as a target for clinical diagnosis and treatment for T-ALL.

Abnormal activation of Notch1 and T-cell acute lymphoblastic leukemia / 临床儿科杂志

Abnormal activation of Notch1 plays pivotal roles in the molecular pathogenesis of human T-cell acutelymphoblastic leukemia (T-ALL). Activating Notch1 mutations present in over 60% of the T-ALL patients. However, so far,there is no therapy with little side effects that specifically targets the abnormally activated Notch1 pathway-induced T-ALL. Thepresent study briefly reviewed the effects of abnormal activation of Notch1 in the pathogenesis of T-ALL, as well as the currentapproaches targeting Notch1 and its limitations, thus providing some guidance for the research and development of clinicaltherapies targeting T-ALL.

The characteristics and clinical signiifcance ofNOTCH1 mutations in childhood T-cell acute lymphoblastic leukemia / 临床儿科杂志

ObjectiveTo clarify the characteristics and clinical signiifcance of the NOTCH1 mutations in childhood T-cell acute lymphoblastic leukemia (T-ALL).MethodsAmplify and sequence the heterodimerization (HD) domain and the pro-line-glutamicacid-serine-threonine (PEST) domain of theNOTCH1 gene in 28 T-ALL children, in order to explore the frequency, position and type of the mutations as well as their reletions with prognosis.ResultsIn 28 children with T-ALL, 15 cases (51.57%) had been identiifed theNOTCH1 mutations, all of which were heterozygous mutations. The lymphoblast counts in peripher-al blood and bone marrow in theNOTCH1 mutant group at admission were signiifcantly higher than in the non-mutant group (P<0.05). The 1-year remission rate in the 28 children with T-ALL was 75% (21/28), including 80% (12/15) in mutant group in which 3 patients relapsed and all of them died (1-year mortality 20%) and 69.20% (9/13) in non-mutant group in which 4 patients relapsed but all survived (1-year mortality 0%).ConclusionsThe children with T-ALL had a high incidence of NOTCH1 mu-tations at various sites. In addition, the patients withNOTCH1 mutations had more severe disease at diagnosis, better short-term prognosis and poor outcome with salvage therapy after relapse.

Expression and clinical significance of Ikaros6 isoform in adult T-cell acute lymphoblastic leukemia patients / 白血病·淋巴瘤

Objective To investigate the expression and prognostic value of Ikaros6 in adult T-cell acute lymphoblastic leukemia(T-ALL) patients.Methods The RNA was extracted from mononuclear cells of bone marrow in 74 adult T-ALL patients.The expression of Ikaros6 was examined by reverse transcription-PCR,and the results were confirmed by sequencing.Clinical features and prognosis were analyzed based on clinical data.Results In 74 T-ALL patients,the incidence rate of Ikaros6 was 21.6 ％ (16/74).Extramedullary infiltrations were occurred often (x2 =4.084,P =0.043),had higher WBC (103.15 × 109/L vs 15.60×109/L,t =0.214,P =0.831),more severe anemia (75.95 g/L vs 107.00 g/L,t =1.504,P =0.142) and lower platelet count (26.0×109/L vs 67.0×109/L,t =1.421,P =0.164) in patients with Ikaros6 positive.Meanwhile Ikaros6-positive patients had inferior survival and were increased risk of relapse as compared with the Ikaros6-negative patients.Conclusions The incidence of Ikaros6 is high in adult T-ALL patients.Ikaros6-positive patients are more likely to have extramedullary infiltration and higher WBC,meanwhile they had inferior survival and increased risk of relapse.Thus Ikaros6 may be served as a valuable factor for risk stratification and prognosis evaluation in adult T-ALL patients.

Effect of 3-MA on arsenic trioxide induced apoptosis in Jurkat cells / 中国免疫学杂志

Objective:To discuss the effect and mechanism of autophagy inhibitor 3-MA on arsenic trioxide inducing apoptosis of acute T-cell leukemia cell line Jurkat cells.Methods:Proliferation inhibition of Jurkat cells treated with arsenic trioxide was detected by XTT.Morphological characteristics of Jurkat cells treated with different concentrations arsenic trioxide were observed by electron mi-croscope.Microtubule-associated protein 1 light chain 3B (LC-3B) protein expression was detected by Western blot and flow cytome-try.Apoptosis rates of Jurkat cells treated with 3-MA combining arsenic trioxide were detected by flow cytometry using AnnexinV-FITC/PI double staining.Results:Arsenic trioxide inhibited the growth of Jurkat cells in a dose and time dependence.We observed different morphological characteristics of autophagy , apoptosis and necrosis accompanying more autophagosomes in Jurkat cells which were treated with arsenic trioxide 2.5,5,10 μmol/L after 24 h.LC3B mean fluorescence intensity (MFI)relative multiples were(3.1±0.2) fold,(4.6±0.31)fold,(34.2±4.5)fold with 5 μmol/L arsenic trioxide treated Jurkat cells 0,24,48 h,and the P values between each of the two groups were less than 0.05,which increased depending time consistently with the growth inhibition rates.LC-3B protein expression gradually increased treated Jurkat cells with arsenic trioxide after 24 h,48 h.The growth inhibition rate (60.6±8.3)%was significantly different treated with arsenic trioxide combining 3-methyl adenine ( 3-MA ) while it was ( 33.4 ±9.1 )% treated with arsenic trioxide alone, however, LC-3B protein expression gradually decreased.Jurkat cell apoptosis rate ( 44.96 ±3.60 )% was significantly increased treated with arsenic trioxide combining autophagy inhibitor(3-MA) while it was (2.94±0.26)% treated with arsenic trioxide alone, and this difference was statistically significant.Conclusion: 3-MA increased apoptosis rates of Jurkat cells inducing by Arsenic trioxide and it may be related with inhibition of autophagy and induction of apoptosis.

Report on recent insight into molecular genetic alterations and target therapy in T-cell acute lymphoblastic leukemia in the 54th ASH annual meeting / 白血病·淋巴瘤

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy associated with poor prognosis.Increasing data regarding to alteration of gene expression signatures of oncogenes and tumor suppressors involved in the pathogenesis of T-ALL and the major mechanisms of T-cell transformation may contribute to define the biological markers for treatment response and prognosis,and has important clinical implications.In this review,advance knowledge concerning the characteristics of early T-cell precursor ALL,the alteration of TAL1 and NOTCH1 related genes and target inhibiton effects based on these alterations from 2012 the 54th ASH annual meeting ars summarized.

Resveratrol induces apoptosis and autophagy in T-cell acute lymphoblastic leukemia cells by inhibiting Akt/mTOR and activating p38-MAPK / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the effects of resveratrol-induced apoptosis and autophagy in T-cell acute lymphoblastic leukemia (T-ALL) cells and potential molecular mechanisms.</p><p><b>METHODS</b>The anti-proliferation effect of resveratrol-induced, apoptosis and autophagy on T-ALL cells were detected by using MTT test, immunofluorescence, electronic microscope, and flow cytometry, respectively. Western blotting was performed for detecting changes of apoptosis-associated proteins, cell cycle regulatory proteins and state of activation of Akt, mTOR, p70S6K, 4E-BP1, and p38-MAPK.</p><p><b>RESULTS</b>Resveratrol inhibited the proliferation and induced apoptosis and autophagy in T-ALL cells in a dose and time-dependent manner. It also induced cell cycle arrest at G0/G1 phase via up regulating cyclin-dependent kinase (CDK) inhibitors p21 and p27 and down regulating cyclin A and cyclin D1. Western blotting revealed that resveratrol significantly decreased the expression of antiapoptotic proteins (Mcl-1 and Bcl-2) and increased the expression of proapoptotic proteins (Bax, Bim, and Bad), and induced cleaved-caspase-3 in a time-dependent manner. Significant increase in ratio of LC3-II/LC3-I and Beclin 1 was also detected. Furthermore, resveratrol induced significant dephosphorylation of Akt, mTOR, p70S6K, and 4E-BP1, but enhanced specific phosphorylation of p38-MAPK which could be blocked by SB203580. When autophagy was suppressed by 3-MA, apoptosis in T-ALL cells induced by resveratrol was enhanced.</p><p><b>CONCLUSION</b>Our findings have suggested that resveratrol induces cell cycle arrest, apoptosis, and autophagy in T-ALL cells through inhibiting Akt/mTOR/p70S6K/4E-BP1 and activating p38-MAPK signaling pathways. Autophagy might play a role as a self-defense mechanism in T-ALL cells treated by resveratrol. Therefore, the reasonable inhibition of autophagy in T-ALL cells may serve as a promising strategy for resveratrol induced apoptosis and can be used as adjuvant chemotherapy for T-ALL.</p>

Aberrant signaling in T-cell acute lymphoblastic leukemia: biological and therapeutic implications

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous disease with respect to phenotype, gene expression profile and activation of particular intracellular signaling pathways. Despite very significant improvements, current therapeutic regimens still fail to cure a portion of the patients and frequently implicate the use of aggressive protocols with long-term side effects. In this review, we focused on how deregulation of critical signaling pathways, in particular Notch, PI3K/Akt, MAPK, Jak/STAT and TGF-ß, may contribute to T-ALL. Identifying the alterations that affect intracellular pathways that regulate cell cycle and apoptosis is essential to understanding the biology of this malignancy, to define more effective markers for the correct stratification of patients into appropriate therapeutic regimens and to identify novel targets for the development of specific, less detrimental therapies for T-ALL.