S1P-S1PR3-RAS promotes the progression of S1PR3hi TAL1+ T-cell acute lymphoblastic leukemia that can be effectively inhibited by an S1PR3 antagonist.

TAL1+ T-cell acute lymphoblastic leukemia (T-ALL) is a distinct subtype of leukemia with poor outcomes. Through the cooperation of co-activators, including RUNX1, GATA3, and MYB, the TAL1 oncoprotein extends the immature thymocytes with autonomy and plays an important role in the development of T-ALL. However, this process is not yet well understood. Here, by investigating the transcriptome and prognosis of T-ALL from multiple cohorts, we found that S1PR3 was highly expressed in a subset of TAL1+ T-ALL (S1PR3hi TAL1+ T-ALL), which showed poor outcomes. Through pharmacological and genetic methods, we identified a specific survival-supporting role of S1P-S1PR3 in TAL1+ T-ALL cells. In T-ALL cells, TAL1-RUNX1 up-regulated the expression of S1PR3 by binding to the enhancer region of S1PR3 gene. With hyperactivated S1P-S1PR3, T-ALL cells grew rapidly, partly by activating the KRAS signal. Finally, we assessed S1PR3 inhibitor TY-52156 in T-ALL patient-derived xenografts (PDXs) mouse model. We found that TY-52156 attenuated leukemia progression efficiently and extended the lifespan of S1PR3hi TAL1+ T-ALL xenografts. Our findings demonstrate that S1PR3 plays an important oncogenic role in S1PR3hi TAL1+ T-ALL and may serve as a promising therapeutic target.

[The Effect of SP1 on the Progression of T-cell Acute Lymphoblastic Leukemia].

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.

Matrine induces autophagy in human neuroblastoma cells via blocking the AKT-mTOR pathway.

Neuroblastoma (NB) is one of the most common malignant solid tumors in children. Despite significant advances in the treatment strategy, the long-term survival rate of NB patients is only 50%. Developing new agents for NB patients deserves attention. Recent research indicates that matrine, a natural quinolizidine alkaloid component extracted from the traditional Chinese medicine Sophora root, is widely used for various diseases, including antitumor effects against a variety of cancers. However, the effect of matrine on NB is unknown. Herein, we found that matrine exerted antiproliferative activity in human NB cells in dose- and time-dependent manner. Matrine triggered autophagy in NB cells by blocking the AKT-mTOR signaling pathway and suppressing the phosphorylation of AKT and mTOR. 3-Methyladenine (3-MA), a PI3K inhibitor, protected against matrine-induced inhibition of cell proliferation, further supporting that the antitumor activity of matrine was at least partly autophagy-dependent. In vivo, matrine reduced tumor growth of SK-N-DZ cells in a dose-dependent manner. Matrine treatment significantly declined the phosphorylation of AKT and mTOR and enhanced the LC3 II/GAPDH ratio in NB xenografts. Altogether, our work uncovered the molecular mechanism underlying matrine-induced autophagy in NB and provided implications for matrine as a potential therapeutic agent against NB.

MiR-652-5p elevated glycolysis level by targeting TIGAR in T-cell acute lymphoblastic leukemia.

The effect of glycolysis remains largely elusive in acute T lymphoblastic leukemia (T-ALL). Increasing evidence has indicated that the dysregulation of miRNAs is involved in glycolysis, by targeting the genes coding glycolysis rate-limiting enzymes. In our previous studies, we found that overexpression of the ARRB1-derived miR-223 sponge repressed T-ALL progress and reduced the expression of miR-652-5p. However, little is known about miR-652-5p on T-ALL. Here, we showed that impaired miR-652-5p expression inhibited growth, promoted apoptosis of T-ALL cells in vitro and prolonged overall survival (OS) in vivo. Based on the GO enrichment of miR-652-5p target genes, we uncovered that impaired miR-652-5p decreased glycolysis, including reduced the lactate, pyruvate, ATP level and the total extracellular acidification rate (ECAR), elevated oxygen consumption rate (OCR) in T-ALL cell lines. Mechanically, miR-652-5p targeted the 3'UTR of Tigar mRNA and inhibited its expression. Furthermore, the alteration of glycosis level was attributed to Tigar overexpression, consistent with the effect of impaired miR-652-5p. Additionally, Tigar suppressed the expression of PFKFB3, a glycolysis rate-limiting enzyme, in vivo and in vitro. Taken together, our results demonstrate that impaired miR-652-5p/Tigar axis could repress glycolysis, thus to slow growth of T-ALL cells, which support miR-652-5p as a novel potential drug target for T-ALL therapeutics.

[ß-arrestin1 Promotes the Concentration of Mitochondrial Reactive Oxygen in T-ALL Cells via MiR-652-5p].

OBJECTIVE: To investigate the effect of ß-arrestin1 on the concentration of reactive oxygen species (ROS) in the mitochondria of acute T-lymphocytic leukemia (T-ALL) cells and its possible mechanisms. METHODS: The stable T-ALL cell line with knocked down ß-arrestin1 (Jurkat Siß1) was constructed. Flow cytometry and probe assays were used to detect ROS content in cell and mitochondrial, respectively. The relationship between ß-arrestin1 and microRNA was detected, analyzed and Q-PCR confirmed by microRNA microarray. The target genes of microRNA were predicated by miRbase software, identified by Western blot, and validated by Dual luciferase reporter gene. RESULTS: Jurkat Siß1 stable cell line was successfully constructed and it was found that ROS content was slightly reduced in Jurkat Siß1 at the whole cell level, and the ROS content was also significantly reduced in mitochondria. MicroRNA microarray analysis revealed that multiple T-ALL related microRNAs showed differentially expressed, in which the expression of miR-652-5p was significantly increased in Jurkat Siß1 (P<0.05 fold>2.0), and Q-PCR showed that miR-652-5p was nearly 5-fold up-regulated in Jurkat Siß1. miRbase predicted that the P62 gene was the target gene of miR-652-5p which could regulates mitochondrial function. P62 protein showed highly expressed in stably knocked down miR-652-5p in Jurkat cells. Dual luciferase reporter gene assay confirms that P62 was the target gene of miR-652-5p. CONCLUSION: ß-arrestin1 can decreases the expression of miR-652-5p and deregulates the translational inhibition of P62 mRNA, thus to increase ROS content in mitochondria of T-ALL cells.

[Induction of mouse T lymphocyte differentiation in vitro by thymic organoids].

Objective To induce the differentiation of hematopoietic stem progenitor cells (HSPCs) into T cell by creating thymic organoids and simulating the three-dimensional structure of thymus tissue in vitro. Methods The retroviral vector expressing the DLL1 and Green fluorescent protein (GFP) was constructed, and the OP9-DLL1 cell line was established in OP9 cells with the aid of retroviral infection. The mRNA and protein level of DLL1 in OP9-DLL1 cells was detected by quantitative real-time PCR and Western blot respectively. Immunofluorescence assay was used to detect the DLL1 protein expression and distribution in OP9-DLL1 cells. HSPCs were extracted from E13.5 fetal liver and bone marrow of C57BL/6 mouse, and mixed with OP9-DLL1 cells in an appropriate ratio respectively, then compacted by centrifuging and cultured at the air-liquid interface in medium. Fluorescence microscope was used to observe the growth of thymic organoids. Flow cytometry was used to detect the expression of T cell surface markers, including CD3, CD4, CD8, CD25, CD44, CD45, CD117 and TCRß. Immunofluorescence cytochemical staining was used to observe the distribution of hematopoietic cells in thymic organoids. Results The retroviral vector expressing DLL1 and GFP was successfully constructed. The OP9 cells were infected with the retrovirus constructed, and OP9-DLL1 cells were obtained by GFP screening. The mRNA and protein level of DLL1 in OP9-DLL1 cells significantly increased, and DLL1 was expressed in the membrane OP9-DLL1 cells. During the 40 days of culture, the thymic organoids remained in good condition and increased gradually in volume. The thymic organoids induced programmed differentiation of T cells, and differentiation of HSPCs into CD3+ T cells. Conclusion OP9-DLL1 cells can be used to construct thymic organoids and to induce differentiation of HSPCs into T cells in vitro.

[ß-arrestin1 overexpression suppresses progression of human T-cell acute lymphatic leukemia Molt-4 cell xenograft in mice].

OBJECTIVE: To investigate the effect of ß-arrestin1 overexpression on tumor progression in a NCG mouse model bearing T-cell acute lymphocytic leukemia (T-ALL) Molt-4 cell xenograft. METHODS: Molt-4 cells were tagged with firefly-luciferase (F-Luc) by lentiviral infection, and fluorescence intensity of the cells was detected using a luminescence detector. Molt-4 cell lines with ß-arrestin1 overexpression or knockdown were constructed by lentivirus infection and injected via the tail vein in sub-lethal irradiated NCG mice. Body weight changes and survival time of the xenografted mice were observed, and the progression of T-ALL in the mice was evaluated using an in vivo fluorescence imaging system. Sixteen days after xenografting, the mice were euthanatized and tumor cell infiltration was observed in the slices of the liver and spleen. RESULTS: We successfully tagged Molt-4 cells with F-Luc and overexpressed or knocked down ß-arrestin1 in the tagged cells. Bioluminescent imaging showed obvious luminescence catalyzed by F-Luc in Molt-4 cells. After injection of Molt-4-Luc cells into irradiated NCG mice, a gradual enhancement of luminescence in the xenografted mice was observed over time, while the body weight of the mice decreased. Compared with the control mice, the mice xenografted with ß-arrestin1-overexpressing Molt-4 cells had significantly prolonged survival time (P < 0.001), while the survival time of the mice xenografted with Molt-4 cells with ß- arrestin1 knockdown was significantly shortened (P < 0.001). Histological examination revealed fewer infiltrating tumor cells in the liver and spleen of the mice xenografted with ß-arrestin1-overexpressing Molt-4 cells in comparison with the mice bearing parental Molt-4 cell xenografts. CONCLUSIONS: ß-arrestin1 overexpression suppresses tumor progression in mice bearing Molt-4 cell xenograft.