BACKGROUND: T-cell lymphoblastic lymphoma/acute lymphoblastic leukemia (T-LBL/ALL) is an aggressive form of hematological malignancy associated with poor prognosis in adult patients. Histone deacetylases (HDACs) are aberrantly expressed in T-LBL/ALL and are considered potential therapeutic targets. Here, we investigated the antitumor effect of a novel HDAC inhibitor, chidamide, on T-LBL/ALL. METHODS: HDAC1, HDAC2 and HDAC3 levels in T-LBL/ALL cell lines and patient samples were compared with those in normal controls. Flow cytometry, transmission electron microscopy, and lactate dehydrogenase release assays were conducted in Jurkat and MOLT-4 cells to assess apoptosis and pyroptosis. A specific forkhead box O1 (FOXO1) inhibitor was used to rescue pyroptosis and upregulated gasdermin E (GSDME) expression caused by chidamide treatment. The role of the FOXO1 transcription factor was evaluated by dual-luciferase reporter and chromatin immunoprecipitation assays. The efficacy of chidamide in vivo was evaluated in a xenograft mouse. RESULTS: The expression of HDAC1, HDAC2 and HDAC3 was significantly upregulated in T-LBL/ALL. Cell viability was obviously inhibited after chidamide treatment. Pyroptosis, characterized by cell swelling, pore formation on the plasma membrane and lactate dehydrogenase leakage, was identified as a new mechanism of chidamide treatment. Chidamide triggered pyroptosis through caspase 3 activation and GSDME transcriptional upregulation. Chromatin immunoprecipitation assays confirmed that chidamide led to the increased transcription of GSDME through a more relaxed chromatin structure at the promoter and the upregulation of FOXO1 expression. Moreover, we identified the therapeutic effect of chidamide in vivo . CONCLUSIONS This study suggested that chidamide exerts an antitumor effect on T-LBL/ALL and promotes a more inflammatory form of cell death via the FOXO1/GSDME axis, which provides a novel choice of targeted therapy for patients with T-LBL/ALL.
Humans ; Pyroptosis/drug effects* ; Forkhead Box Protein O1/genetics* ; Aminopyridines/pharmacology* ; Animals ; Mice ; Benzamides/pharmacology* ; Cell Line, Tumor ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy* ; Phosphate-Binding Proteins/metabolism* ; Histone Deacetylase Inhibitors/pharmacology* ; Jurkat Cells ; Histone Deacetylases/metabolism* ; Apoptosis/drug effects* ; Gasdermins

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

OBJECTIVE: To study the expression of SPOP in patients with acute myeloid leukemia (AML) and its effect on proliferation, apoptosis and cycle of AML cells. METHODS: RT-qPCR was used to detect the expression of SPOP mRNA in bone marrow samples of patients with newly diagnosed AML and normal controls. The stable overexpression of SPOP in AML cell lines THP-1 and U937 were constructed by liposome transfection. The effect of SPOP on cell proliferation was detected by CCK-8, and the effect of SPOP on apoptosis and cell cycle was detected by flow cytometry. The expressions of anti-apoptotic protein Bcl-2 and apoptotic protein Bax, Caspase3 were detected by Western blot. RESULTS: The median expression level of SPOP mRNA in normal control group was 0.993 1(0.6303, 1.433), while that in AML group was 0.522 1(0.242 2, 0.723 7). The expression level of SPOP in AML group was significantly lower than that in normal control group ( P < 0.001). After the overexpression of SPOP, the proportion of apoptotic cells in the U937 overexpression group and THP-1 overexpression group was 10.9%±0.3% and 4.6%±015%, which were higher than 8.9%±0.3% and 3.0%±0.30% in the Empty Vector group, respectively (both P < 0.05). The expression of Caspase3 in U937 overexpression group and THP-1 overexpression group was 1.154±0.086 and 1.2±0.077, which were higher than 1 in Empty Vector group, respectively (both P < 0.05). The ratio of Bax/Bcl-2 in U937 overexpression group and THP-1 overexpression group was 1.328±0.057 and 1.669±0.15, which were higher than 1 in Empty Vector group, respectively (both P < 0.05). In the cell proliferation experiment, the number of cells in the U937 overexpression group and THP-1 overexpression group were both slightly lower than those in the Empty Vector group, but the differences were not statistically significant (P >0.05). In the cell cycle experiment, the proportion of G₁ cells in the U937 overexpression group and THP-1 overexpression group were both slightly higher than those in the Empty Vector group, but the differences were not statistically significant (P >0.05). CONCLUSION SPOP can promote the apoptosis of leukemic cells, and its mechanism may be related to down-regulation of Bcl-2 expression and up-regulation of Bax and Caspase3 expression.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Apoptosis ; Repressor Proteins/genetics* ; Cell Proliferation ; Nuclear Proteins/genetics* ; Cell Cycle ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; U937 Cells ; Cell Line, Tumor ; RNA, Messenger/genetics*

OBJECTIVE: To investigate the role of nucleolin (NCL) in acute myeloid leukemia (AML) Kasumi-1 cells and its underlying mechanism. METHODS: The Kasumi-1 cells were infected with lentivirus carrying shRNA to downregulate NCL expression. Cell proliferation was detected by CCK-8 assay, and cell apoptosis and cell cycle were determined by flow cytometry. Transcriptome next-generation sequencing (NGS) was performed to predict associated signaling pathways, the expression levels of related genes were measured by RT-PCR. RESULTS: Down-regulation of NCL expression significantly inhibited the proliferation of Kasumi-1 cells (P <0.01) and markedly increased the apoptosis rate (P <0.001). Cell cycle analysis showed significant changes in the distribution of cells in the G₁ and S phases after NCL knockdown (P <0.05), while no significant difference was observed in the G₂ phase (P >0.05). Transcriptome sequencing analysis demonstrated that differentially expressed genes in Kasumi-1 cells with low expression of NCL were primarily enriched in key signaling pathways, including ribosome, spliceosome, RNA transport, cell cycle, and amino acid biosynthesis. qPCR validation showed that the expression of BAX, CASP3, CYCS, PMAIP1, TP53 , and CDKN1A was significantly upregulated after NCL downregulation (P <0.05), with CDKN1A exhibiting the most pronounced difference. CONCLUSION NCL plays a critical role in regulating the proliferation, apoptosis, and cell cycle progression of Kasumi-1 cells. The mechanism likely involves suppressing cell cycle progression through activation of the TP53-CDKN1A pathway and promoting apoptosis by upregulating apoptosis-related genes.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Down-Regulation ; Cell Proliferation ; Apoptosis ; RNA-Binding Proteins/genetics* ; Nucleolin ; Cell Line, Tumor ; Phosphoproteins/metabolism* ; Cell Cycle ; Signal Transduction ; RNA, Small Interfering

Glutamine provides carbon and nitrogen to support the proliferation of cancer cells. However, the precise reason why cancer cells are particularly dependent on glutamine remains unclear. In this study, we report that glutamine modulates the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) to promote cancer cell proliferation and survival. Specifically, lysine 604 (K604) in the sixth of the 7 substrate-recruiting WD repeats of FBW7 undergoes glutaminylation (Gln-K604) by glutaminyl tRNA synthetase. Gln-K604 inhibits SCFFBW7-mediated degradation of c-Myc and Mcl-1, enhances glutamine utilization, and stimulates nucleotide and DNA biosynthesis through the activation of c-Myc. Additionally, Gln-K604 promotes resistance to apoptosis by activating Mcl-1. In contrast, SIRT1 deglutaminylates Gln-K604, thereby reversing its effects. Cancer cells lacking Gln-K604 exhibit overexpression of c-Myc and Mcl-1 and display resistance to chemotherapy-induced apoptosis. Silencing both c-MYC and MCL-1 in these cells sensitizes them to chemotherapy. These findings indicate that the glutamine-mediated signal via Gln-K604 is a key driver of cancer progression and suggest potential strategies for targeted cancer therapies based on varying Gln-K604 status.
Glutamine/metabolism* ; Myeloid Cell Leukemia Sequence 1 Protein/genetics* ; Humans ; Proto-Oncogene Proteins c-myc/genetics* ; Cell Proliferation ; Signal Transduction ; Neoplasms/pathology* ; F-Box-WD Repeat-Containing Protein 7/genetics* ; Cell Survival ; Cell Line, Tumor ; Apoptosis

The objective of the present study was to investigate the role and mechanism of bone marrow microenvironmental cells in regulating the mitochondrial mass of leukemia cells, and to uncover the mechanism of leukemia progression at the metabolic level. A mouse model of acute myeloid leukemia (AML) induced by the overexpression of the MLL-AF9 (MA9) fusion protein was established, and the bone marrow cells of AML mice were transplanted into mitochondrial fluorescence reporter mice expressing the Dendra2 protein (mito-Dendra2 mice). The proportion of Dendra2⁺ cells in bone marrow leukemia cells at different stages of AML was quantified by flow cytometry. The effects of transferred mitochondria on leukemia cells were studied by fluorescence-activated cell sorting (FACS), followed by functional experiments and bulk RNA sequencing. Finally, components within the bone marrow niche, such as mesenchymal stromal cells (MSCs) and endothelial cells (ECs), were co-cultured with leukemia cells in vitro. The proportion of leukemia cells that underwent mitochondrial transfer and the apoptosis level of leukemia cells were then detected by flow cytometry. The results showed that mitochondria from bone marrow cells were transferred to leukemia cells in the AML mouse model, and the proportion of mitochondrial transfer decreased with AML progression. The proportion of mitochondria transferred to leukemia stem cells (LSCs) was lower than that of mature AML cells. In AML cells receiving Dendra2⁺ mitochondria, there was a significant increase in the levels of intracellular reactive oxygen species (ROS) and apoptosis, while the levels of protein translation and their colony-forming capacities were decreased. The transplantation of Dendra2⁺ AML cells resulted in an extension of the survival of mice. RNA sequencing analysis demonstrated a significant downregulation of pathways related to translation, aerobic respiration and mitochondrial organization in AML cells that had received mitochondria. In vitro co-culture experiments indicated that MSCs within the bone marrow niche tended to transfer their mitochondria to leukemia cells and promoted the apoptosis of leukemia cells. These results indicate that in the MA9-induced AML mouse model, bone marrow niche cells can transfer mitochondria to leukemia cells, resulting in a reduction in the overall survival and function of the leukemia cells. Mitochondrial transfer in the bone marrow microenvironment may serve as a self-defensive mechanism of the host bone marrow niche cells, inhibiting the progression of AML.
Animals ; Mice ; Mitochondria/metabolism* ; Disease Progression ; Leukemia, Myeloid, Acute/genetics* ; Apoptosis ; Mesenchymal Stem Cells/metabolism* ; Bone Marrow Cells/metabolism* ; Humans ; Tumor Microenvironment ; Mice, Inbred C57BL

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.
Humans ; Atorvastatin/pharmacology* ; PTEN Phosphohydrolase/pharmacology* ; Sincalide/metabolism* ; Drug Resistance, Neoplasm/genetics* ; TOR Serine-Threonine Kinases/metabolism* ; Leukemia, Promyelocytic, Acute/drug therapy* ; Doxorubicin/pharmacology* ; Apoptosis ; RNA, Small Interfering/pharmacology* ; Glycolysis ; Glucose/therapeutic use* ; Cell Proliferation

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.
Humans ; Animals ; Mice ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; HEK293 Cells ; Reactive Oxygen Species ; Transcription Factors ; T-Lymphocytes ; Cell Line, Tumor ; Sp1 Transcription Factor/metabolism*

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.
Humans ; Apoptosis/genetics* ; bcl-2-Associated X Protein ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Leukemia, Myeloid, Acute/pathology* ; Luciferases ; MicroRNAs/metabolism* ; PAX9 Transcription Factor/genetics*

OBJECTIVE: To investigate the effects of long non-coding RNA (lncRNA) GATA3 antisense RNA 1 (GATA3-AS1) targeting miR-515-5p on the proliferation and apoptosis of childhood acute lymphoblastic leukemia (ALL) cells. METHODS: RT-qPCR was used to determine the expression of GATA3-AS1 and miR-515-5p in the plasma of controls and ALL children. Human ALL cells Jurkat were divided into si-GATA3-AS1, si-NC, miR-NC, miR-515-5p, si-GATA3-AS1+anti-miR-NC and si-GATA3-AS1+anti-miR-515-5p groups. CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the cell apoptosis. The targeting relationship between GATA3-AS1 and miR-515-5p was determined by dual-luciferase reporter assay. RESULTS: The expression level of GATA3-AS1 in the plasma of ALL children was significantly higher than that of controls (P <0.001), while the expression level of miR-515-5p was significantly lower than that of controls (P <0.001). Compared with the si-NC group, the cell inhibition rate, apoptosis rate, and miR-515-5p expression level in si-GATA3-AS1 group were significantly increased (P <0.001). Compared with the miR-NC group, the cell inhibition rate and apoptosis rate in miR-515-5p group were significantly increased (P <0.001). GATA3-AS1 could directly and specifically bind to miR-515-5p. Compared with the si-GATA3-AS1+anti-miR-NC group, the cell inhibition rate and apoptosis rate in si-GATA3-AS1+anti-miR-515-5p group were significantly decreased (P <0.001). CONCLUSION Down-regulation of GATA3-AS1 can inhibit proliferation and induce apoptosis of childhood ALL cells by targeting up-regulation of miR-515-5p expression.
Child ; Humans ; MicroRNAs/metabolism* ; RNA, Long Noncoding/metabolism* ; Antagomirs/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Apoptosis ; Gene Expression Regulation, Neoplastic ; GATA3 Transcription Factor/metabolism*