Pharmacodynamic and Toxicity Studies of 6-Isopropyldithio-2'-guanosine Analogs in Acute T-Lymphoblastic Leukemia.

(1) Background: The research group has developed a new small molecule, 6-Isopropyldithio-2'-deoxyguanosine analogs-YLS004, which has been shown to be the most sensitive in acute T-lymphoblastic leukemia cells. Moreover, it was found that the structure of Nelarabine, a drug used to treat acute T-lymphoblastic leukemia, is highly similar to that of YLS004. Consequently, the structure of YLS004 was altered to produce a new small molecule inhibitor for this study, named YLS010. (2) Results: YLS010 has exhibited potent anti-tumor effects by inducing cell apoptosis and ferroptosis. A dose gradient was designed for in vivo experiments based on tentative estimates of the toxicity dose using acute toxicity in mice and long-term toxicity in rats. The study found that YLS010 at a dose of 8 mg/kg prolonged the survival of late-stage acute T-lymphoblastic leukemia mice in the mouse model study. (3) Conclusions: YLS010 has demonstrated specific killing effects against acute T-lymphoblastic leukemia both in vivo and in vitro. Preclinical studies of YLS010 offer a new opportunity for the treatment of patients with acute T-lymphoblastic leukemia in clinical settings.

[Potential Oncogenic Role of Genes in PLC Family on T-ALL Cell Lines by Suppression of Apoptosis].

OBJECTIVE: To explore the role of phospholipase C(PLC) family in the progression of acute T lymphoblastic leukemia (T-ALL). METHODS: The apoptosis of T-ALL cells was determined by Annexin V-PE/7-AAD staining after treatment of PLC inhibitor U73122 and Edelfosine. Cox regression and Kaplan-Meier were used to analyze the impact of PLC expressions on the event-free survival (EFS) of T-ALL patients. PLC expression in each subtype of T-ALL were analyzed by One-way ANOVA. The siRNA expression plasmids targeting the PLCß1, PLCγ1, PLCη1 gene were constructed, and T-ALL cells were infected with retrovirus packaging in HEK-293T cells. The mRNA and protein level were tested by RT-PCR and Western blot. RESULTS: P12-ICH and CCRF-CEM cell line were sensitive to U73122 and Edelfosine treatment, while Jurkat and MOLT4 were resistant to them. In the TARGET-ALL database, the prognosis of T-ALL patients with high expression of PLCß1, PLCγ1 and PLCη1 was poor, and PLCß1, PLCγ1, PLCη1 were unevenly distributed in T-ALL subtypes. PLCß1, PLCγ1 and PLCη1 maintained the survival of P12-ICH and CCRF-CEM cell lines, respectively, while they had no effect on the survival of MOLT4. CONCLUSION: PLCß1, PLCγ1 and PLCη1 can maintain the growth of T-ALL cell lines in vitro and promote the malignant progression of T-ALL, which are potential therapeutic targets.

[Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-ß Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia].

OBJECTIVE: To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-ß on T cell acute lymphoblastic leukemia (T-ALL) cell lines. METHODS: Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. RESULTS: Different concentrations of FA-2-b-ß significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-ß inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-ß reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/ß-catenin inhibitor (ICG001), the process was further intensified. CONCLUSION: Agaricus Blazei Extract FA-2-b-ß inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

The outcome of acute leukemia patients with SET-NUP214 fusion after allogeneic stem cell transplantation.

SET-NUP214 fusion gene, also known as TAF-1-CAN and SET-CAN, is observed in acute myeloid leukemia (AML) and T-cell lymphoblastic leukemia (T-ALL). SET-NUP214 fusion in T-cell lymphoblastic leukemia is associated with chemotherapy resistance, but the prognosis of patients with AML with SET-NUP214 has rarely been reported. In the present study, we retrospectively analyzed all patients with acute leukemia including AML and T-ALL patients with SET-NUP214 fusion who underwent allogeneic stem cell transplantation (alloHSCT) in our center from July 2017 to November 2022. Of the total 11 patients, 5 patients were diagnosed with AML and 6 patients were diagnosed with T-ALL de novo. All patients received myeloablative regimens in CR1, and there were three (60%) AML patients who relapsed post-alloHSCT and three T-ALL (50%) patients who relapsed post-alloHSCT. Only one patient with AML who relapsed post-alloHSCT responded to subsequent chemotherapy plus donor lymphocyte infusion and survived the last follow-up. The estimated 1-year overall survival and 3-year overall survival for all these 11 patients were 69.3% and 38.5%, respectively. The estimated 1-year leukemia-free survival and 3-year leukemia-free survival for all patients were 69.3% and 38.5%, respectively. The research shows a high incidence of relapse for patients with acute leukemia with the SET-NUP214 fusion gene, even after alloHSCT. More clinical trials or research with larger samples are urgently needed for this group of patients.

Imaging flow cytometry reveals a subset of TdT negative T-ALL blasts with very low forward scatter on conventional flow cytometry.

BACKGROUND: Studies in T-cell acute lymphoblastic leukemia (T-ALL) have shown that leukemic blast populations may display immunophenotypic heterogeneity. In the clinical setting, evaluation of measurable residual disease during treatment and follow-up is highly dependent on knowledge of the diversity of blast subsets. Here, we set out to evaluate whether variation in expression of the blast marker, TdT, in T-ALL blasts could correspond to differences in morphometric features. METHODS: We investigated diagnostic bone marrow samples from six individual T-ALL patients run in parallel on imaging flow cytometry (IFC) and conventional flow cytometry (CFC). RESULTS: Guided by the imagery available in IFC, we identified distinct TdTneg and TdTpos subpopulations with apparent differences in internal complexity. As TdTneg blasts predominantly displayed very low forward scatter (FSC) on CFC, these subsets were initially excluded from routine analysis as debris, elements of small diameter, apoptotic, and/or dead cells. However, IFC-based morphometric analyses demonstrated that cell size and shape of TdTneg blasts were comparable to the TdTpos cells and without morphometric apoptotic hallmarks, supporting that the TdTneg subpopulation corresponded to T-ALL blasts. Fluorescence in situ hybridization analyses substantiated the clinical relevance of TdTneg FSCvery-low cells by retrieving known diagnostic cytogenetic abnormalities at comparable frequencies in purified TdTneg FSCvery-low and TdTpos FSCint subsets. CONCLUSION: We highlight this finding as knowledge of phenotypic heterogeneity is of crucial importance in the clinical setting for delineation and quantification of blast subpopulations of potential biological relevance. We argue that the IFC imagery may allow for visual verification and improvement of applied gating strategies.

[Effect of Curcumin on Apoptosis of Acute T-Lymphoblastic Leukemia Cells induced by UMI-77 and Its Related Mechanism].

AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77. METHODS: T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 µmol/L curcumin treated cells), UMI-77 group (15 µmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 µmol/L curcumin and 15 µmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins. RESULTS: After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05). CONCLUSION: Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.

Case Report: A Case of Acute T Lymphoblastic Leukemia With Mixed Infection of Lethal Invasive Mucormycosis and Multi-Drug Resistant Bacteria.

Pulmonary mucormycosis (PM) is a rare and life-threatening fungal infection. Here, we report a case of an acute T lymphoblastic leukemia patient with mixed infections of lethal invasive Mucormycosis and multi-drug resistant (MDR) bacteria. After receiving anti-infection drugs to control the patient's fever, he was treated with induction chemotherapy. However, the malignant hematological disease was poorly controlled by the chemotherapy and the patient developed more symptoms of infection. Although the results of multiple ß-D-Glucan (G) and Galactomannan (GM) tests remained negative, several pathogens were detected using metagenomic next-generation sequencing (mNGS). In particular, mNGS identified Malassezia pachydermum, Mucor racemosus, and Lauteria mirabilis in the peripheral blood and local secretion samples. The Mucor and bacterial infections were further confirmed via multi-site and repeated fungal and bacterial cultures, respectively. Despite adjusting the anti-infection therapy according to the diagnostic results, the patient's blood disease and symptoms of infection were not alleviated. Additionally, the MDR Acinetobacter baumannii infection/colonization was not confirmed until the seventh culture of the peripheral venous catheter tip. Due to the patient's deteriorating conditions, his family decided to withdraw him from further treatment. Overall, mNGS can facilitate a diagnosis of Mucormycosis by providing clinical and therapeutic information to support conventional diagnostic approaches. For the early and timely diagnosis and treatment of PM, it is also necessary to consider the malignant hematological conditions and repeated tests through multiple detection methods.

Combination Effect of Notch1 and PI3K/AKT/mTOR Signaling Pathways Inhibitors on T-ALL Cell Lines.

Background: Acute T lymphoblastic Leukemia (T-ALL) is a highly aggressive hematologic malignancy. Chemotherapy resistance is one of the most important challenges in T-ALL treatment. Alterations in cellular signaling pathways such as Notch1 and PI3K/AKT/mTOR play a role in cell proliferation, survival, and resistance to chemotherapy. Combination of Notch1 and PI3K/AKT/mTOR inhibitors is an interesting and rational strategy in treatment of T-ALL. Interaction of AZD5363 as an inhibitor of PI3k/AKT/mTOR and Compound E as an inhibitor of Notch1 signaling pathway was investigated in a T-ALL pre-clinical model. Materials and Methods: T-ALL cell lines included Jurkat, Molt-4, and HPB- ALL cells were treated with AZD5363 and Compound E alone and in combination. Cell viability was determined by MTT assay. Flow cytometry was used to measure apoptosis. Interaction between AZD5363 and Compound E was assessed by Chou-Talalay method. Results: Combination treatment with AZD5363 and Compound E decreased cell viability with synergistic effect in all cell lines at 72 hours. Drug combination increased apoptosis even in Jurkat and HPB-ALL cells resistant to Compound E and AZD5363, respectively. Conclusion: Combination of AZD5363 with Compound E in T-ALL cell lines exhibited a synergistic effect. Cytotoxicity of drug combination increased in all T-ALL cell lines compared to each as a single drug. Simultaneous inhibition of Notch1 and PI3K/AKT signaling pathways as a possible treatment of T-ALL, provides a basis for future investigations.

[Erythrophagocytosis by blast cells and de novo T cell LAL without cytogenetic abnormalities in a Moroccan patient]. / Un cas marocain d'érythrophagocytose blastique et LAL T de novo sans anomalie cytogénétique.

Erythrophagocytosis by blast cells is due to hyperactivation of blast cells. Erythrophagocytosis is associated with T cell myeloid hemopathies (8;16). This study shows an exceptional case of erythrophagocytosis by blast cells in a patient with acute T-lymphoblastic leukemia without cytogenetic abnormalities. We here report the case of A.Z, aged 19 years presenting with febrile syndrome with dizziness and phosphenes, tumor syndrome with amygdala and gingival hypertrophy. Blood count revealed hyperleukocytosis (399.5 G/L), with aregenerative anemia (Hb: 9.3 g/dl) and thrombocytopenia (platelet count: 40 g/L). Myelogram showed 90% of blast cells (MPO-negative) with erythrophagocytosis by blast cells images. Immunophenotyping confirmed T-cell LAL. Cytogenetic analysis was normal. Erythrophagocytosis by blast cells in patients with T-cell LAL appears to be a separate entity, hence the importance of images on diagnosis, prognosis and treatment of T-cell LAL.

[Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells].

OBJECTIVE: To explore whether bortezomib and a Bcl-2 inhibitor exhibit synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells. METHODS: MTT assay was used to determine the cytotoxicity of bortezomib in the absence or presence of Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) in Jurkat cells. The effects of drug treatment on the expression of Bcl-2 family proteins, LC3B, p62, ubiquitin, BiP/Grp78, p-JNK, p-p38 and CHOP proteins were examined by Western blotting. Flow cytometry was used to determine the effects of bortezomib and Bcl-2 inhibitors (obatoclax, AT-101 and ABT-199) on cell apoptosis. Quantitative real-time PCR was used to measure the mRNA expression levels of the key regulatory factors of unfolded protein reaction (UPR). A zebrafish xenograft model was used to study the anti-tumor effect of bortezomib, obatoclax and their combination in vivo. RESULTS: Bortezomib or Bcl-2 inhibitors alone inhibited the cell viability of Jurkat cells, but only obatoclax and bortezomib showed synergistic cytotoxicity and pro-apoptotic effect. Obatoclax, rather than AT-101 and ABT- 199, blocked autophagic flux in the cells evidenced by concomitant accumulation of LC3B-Ⅱ and p62. Both bortezomib and obatoclax alone caused accumulation of polyubiquinated proteins, and their combination showed a synergistic effect, which was consistent with their synergistic cytotoxicity. The dual blockade of proteasome and autophagy by the combination of bortezomib and obatoclax triggered unfolded protein response followed by cell apoptosis. Preventing UPS dysfunction by tauroursodeoxycholic acid (TUDCA) significantly attenuated the cytotoxicity and pro-apoptotic effect of bortezomib in combination with obatoclax. In zebrafish xenograft models, bortezomib combined with obatoclax significantly decreased tumor foci formation. CONCLUSIONS: Bortezomib and obatoclax for dual blockade of protein degradation pathways show synergistic anti-tumor effect in human acute T lymphoblastic leukemia cells.

Extreme hyperleukocytosis in a pediatric T-ALL patient with a rare translocation, t(7;19)(q35;p13), and submicroscopic deletions at 4q25, 7q33 and 10q23.

Although childhood T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease the outcome can vary considerably. The varying outcomes suggest that unrecognized factors may contribute to disease progression. We report on a 2-year-old T-ALL patient presenting with a very short history of constipation and extreme hyperleukocytosis (WBC 882×10(9)/L). In her leukemic cells we detected the very rare translocation t(7;19)(q35;p13) and LYL1 overexpression. Additionally, we detected submicroscopic deletions at 4q25, 7q33 and 10q23 by oligo-aCGH analysis. We suggest that LYL1 overexpression contributed to the leukemic state and propose that the observed microdeletions may have influenced to the rapid disease progression.

Antitumor effects of a monoclonal antibody to human CCR9 in leukemia cell xenografts.

Tumor expression of certain chemokine receptors is associated with resistance to apoptosis, migration, invasiveness and metastasis. Because CCR9 chemokine receptor expression is very restricted in healthy tissue, whereas it is present in tumors of distinct origins including leukemias, melanomas, prostate and ovary carcinomas, it can be considered a suitable candidate for target-directed therapy. Here, we report the generation and characterization of 91R, a mouse anti-human CCR9 IgG2b monoclonal antibody that recognizes an epitope within the CCR9 N-terminal domain. This antibody inhibits the growth of subcutaneous xenografts from human acute T lymphoblastic leukemia MOLT-4 cells in immunodeficient Rag2(-/-) mice. Tumor size in 91R-treated mice was reduced by 85% compared with isotype-matched antibody-treated controls. Tumor reduction in 91R-treated mice was concomitant with an increase in the apoptotic cell fraction and tumor necrotic areas, as well as a decrease in the fraction of proliferating cells and in tumor vascularization. In the presence of complement or murine natural killer cells, 91R promoted in vitro lysis of MOLT-4 leukemia cells, indicating that this antibody might eliminate tumor cells via complement- and cell-dependent cytotoxicity. The results show the potential of the 91R monoclonal antibody as a therapeutic agent for treatment of CCR9-expressing tumors.

Protective effect of augmenter of liver regeneration on vincristine-induced cell death in Jurkat T leukemia cells.

Augmenter of liver regeneration (ALR) is a crucial factor in the process of proliferation of hepatocytes. Recently, it has been demonstrated that ALR plays an important role of anti-apoptosis in several cell lines, but the biological effects of ALR in acute T lymphoblastic leukemia have remained unclear. In this study, we investigated the effect of ALR on Jurkat T leukemia cell growth and survival. We found that ALR was up-regulated in Jurkat cells and could reduce the sensitivity of Jurkat cells to vincristine, but had a minimal effect on proliferation of Jurkat cells. Results from analysis of flow cytometry showed ALR attenuated apoptotic cells and inhibited G2/M-arrest in vincristine-treated Jurkat cells. Following incubation with ALR, an increase in pro-caspase8, pro-caspase3, pro-PARP and Bcl-2 levels was observed in vincristine-treated Jurkat cells. In summary, the results of this study demonstrate that ALR protects Jurkat T leukemia cells from vincristine-induced cell death via regulation of apoptotic signaling pathways and cell cycle.