[Study on the inhibitory and pro-apoptotic effects of different concentrations of total tanshinone alone and in combination with tyrosine kinase inhibitors on human myeloid leukemia cell lines].

Objective: To explore the effect and investigate the molecular mechanism of different concentrations of total tanshinones alone and in combination with tyrosine kinase inhibitors (TKIs) on the proliferation inhibition and apoptosis of human myeloid leukemia cell lines. Methods: K562 and Kasumi-1 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the TKIs-resistant strain K562/T315I cell line was constructed in Molecular Medicine Research Center, Beijing Lu Daopei Institute of Hematology. Logarithmic growth phase cells were taken and divided into intervention groups with total tanshinone of 0, 2.19, 4.38, 8.75, 17.50 and 35.00 µg/ml intervention groups, which were inoculated in 96-well plates at a density of 1×104 cells/well and exposed to the drug for 24 h, and a control group treated with dimethyl sulfoxide was also set up simultaneously. All experiments were repeated independently 3-5 times. The proliferative activity of the cells was assessed using the CCK-8 assay, the apoptotic rates were measured by flow cytometry, and the expression levels of apoptosis-regulating proteins Bcl-2 and Bax were analyzed by Western blotting. The cell lines treated and untreated with total tanshinone were subjected to transcriptome sequencing and gene set enrichment analysis to identify differentially expressed genes. Results: The half-inhibitory concentration (IC50) values of 8.75 µg/ml total tanshinone at 24 h for K562, K562/T315I and Kasumi-1 cells were (4.11±0.02), (4.95±0.04) and (3.98±0.01) µg/ml, respectively. When combined with 0.25 µmol/L imatinib, 8.75 µg/ml total tanshinone could enhance the induction of apoptosis effects on K562 and K562/T315I cell lines. After being treated with 4.38, 8.75, and 17.50 µg/ml of total tanshinone for 24 h, compared with the control group, total tanshinone upregulated the expression level of Bax protein, downregulated the expression level of Bcl-2 protein, and decreased the Bcl-2/Bax ratio (all P<0.05). Total tanshinone inhibited the proliferation-related signaling pathway and DNA damage repair pathway of myeloid leukemia cell lines, and activated the signaling pathway that induces apoptosis in leukemia cells. Conclusion: Different concentrations of total tanshinoneinhibites proliferation and promote apoptosis in K562, Kasumi-1 and TKIs-resistant K562/T315I cell lines, and further enhance the anti-leukemic effect when combined with TKIs.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

FL118 Is a Potent Therapeutic Agent against Chronic Myeloid Leukemia Resistant to BCR-ABL Inhibitors through Targeting RNA Helicase DDX5.

Chronic myeloid leukemia (CML) is induced by the expression of the fused tyrosine kinase BCR-ABL, which is caused by a chromosomal translocation. BCR-ABL inhibitors have been used to treat CML; however, the acquisition of resistance by CML cells during treatment is a serious issue. We herein demonstrated that BCR-ABL induced the expression of the RNA helicase DDX5 in K562 cells derived from CML patients in a manner that was dependent on its kinase activity, which resulted in cell proliferation and survival. The knockout of DDX5 decreased the expression of BIRC5 (survivin) and activated caspase 3, leading to apoptosis in K562 cells. Similar results were obtained in cells treated with FL118, an inhibitor of DDX5 and a derivative compound of camptothecin (CPT). Furthermore, FL118 potently induced apoptosis not only in Ba/F3 cells expressing BCR-ABL, but also in those expressing the BCR-ABL T315I mutant, which is resistant to BCR-ABL inhibitors. Collectively, these results revealed that DDX5 is a critical therapeutic target in CML and that FL118 is an effective candidate compound for the treatment of BCR-ABL inhibitor-resistant CML.

Gene expression profiling on CML patients with Philadelphia translocation.

OBJECTIVE: The use of tyrosine kinase inhibitors (TKIs) and other targeted therapeutics plays a pivotal role in treatment management for individuals diagnosed with chronic myeloid leukemia (CML). However, some patients may experience fewer favorable outcomes and treatment resistance. Our work aims to use whole transcriptome sequencing to evaluate the variations in gene expression patterns among individuals with CML based on their response to TKI therapy. PATIENTS AND METHODS: Ten blood samples were obtained from two groups of patients diagnosed with CML: those at the initial diagnosis stage and those at the recurrence stage. RNA extraction was performed on all samples and used for next-generation sequencing. The data analysis was performed using the DESeq2 R program. RESULTS: In total, 499 genes were identified as having statistically significant differences in expression levels between the two groups. Of these, 122 genes exhibited upregulation, and 377 genes exhibited downregulation. We observed a notable dysregulation in the expression levels of NTRK2 (with a fold change more significant than +5). A significant proportion of the genes that were expressed demonstrated involvement in several biological processes, including the cell cycle, PI3K-AKT signaling system, cellular senescence, oxidative phosphorylation, microRNA in cancer, FOXO signaling pathway, P53 signaling pathway, and other related pathways. CONCLUSIONS: The results demonstrate a correlation between signaling pathways and the development of treatment resistance in patients with CML. These pathways exhibited enhanced efficacy in transmitting signals downstream of the TKI target, BCR-ABL. Several target genes require additional validation in a more extensive cohort study to verify their correlation with TKI resistance. The present research highlights that many BCR-ABL-independent pathways may be correlated with resistance, thus enhancing the prospective therapy options for patients with CML.

Inhibition of mitochondrial folate metabolism drives differentiation through mTORC1 mediated purine sensing.

Supporting cell proliferation through nucleotide biosynthesis is an essential requirement for cancer cells. Hence, inhibition of folate-mediated one carbon (1C) metabolism, which is required for nucleotide synthesis, has been successfully exploited in anti-cancer therapy. Here, we reveal that mitochondrial folate metabolism is upregulated in patient-derived leukaemic stem cells (LSCs). We demonstrate that inhibition of mitochondrial 1C metabolism through impairment of de novo purine synthesis has a cytostatic effect on chronic myeloid leukaemia (CML) cells. Consequently, changes in purine nucleotide levels lead to activation of AMPK signalling and suppression of mTORC1 activity. Notably, suppression of mitochondrial 1C metabolism increases expression of erythroid differentiation markers. Moreover, we find that increased differentiation occurs independently of AMPK signalling and can be reversed through reconstitution of purine levels and reactivation of mTORC1. Of clinical relevance, we identify that combination of 1C metabolism inhibition with imatinib, a frontline treatment for CML patients, decreases the number of therapy-resistant CML LSCs in a patient-derived xenograft model. Our results highlight a role for folate metabolism and purine sensing in stem cell fate decisions and leukaemogenesis.

[Optimal search conditions for identification of patients with chronic myeloid leukemia from Japanese National Health Insurance data].

This study investigated which conditions could be used to identify patients with chronic myeloid leukemia (CML) from a National Health Insurance claims dataset. During April 2012 and September 2018, 1,789,462 employees were enrolled in the dataset for Shizuoka Prefecture residents. The number of patients with the ICD-10 code for CML was 761. Among them, 246 who had been prescribed a tyrosine kinase inhibitor were considered as having true CML. The positive predictive value was calculated as 32.3% when CML was identified by ICD-10 code alone. Combination of ICD-10 code with prescribed drugs was required to accurately identify patients with CML from the insurance database.

BCR/ABL-Positive Chronic Myeloid Leukemia in Children: Current Treatment Approach.

PURPOSE OF REVIEW: The purpose of this review is to summarize the most updated treatment recommendations for pediatric CML, and to discuss current areas of investigation. RECENT FINDINGS: There is new phase 1 data to support the safety of the non-ATP competitive tyrosine kinase inhibitor (TKI) asciminib in the pediatric cohort. Ongoing studies are investigating the role of treatment-free remission in children. Chronic phase CML in children is managed with lifelong TKI therapy; however, evidence of deeper remissions sustained with second-generation TKIs may permit shorter treatment courses. Use of more specific TKIs may mitigate some of the side effects specific to the pediatric cohort. Children with advanced phase CML should achieve a complete hematologic remission with use of a second-generation TKI prior to transplant to achieve the best outcome.

Leukaemia exposure alters the transcriptional profile and function of BCR::ABL1 negative macrophages in the bone marrow niche.

Macrophages are fundamental cells of the innate immune system that support normal haematopoiesis and play roles in both anti-cancer immunity and tumour progression. Here we use a chimeric mouse model of chronic myeloid leukaemia (CML) and human bone marrow (BM) derived macrophages to study the impact of the dysregulated BM microenvironment on bystander macrophages. Utilising single-cell RNA sequencing (scRNA-seq) of Philadelphia chromosome (Ph) negative macrophages we reveal unique subpopulations of immature macrophages residing in the CML BM microenvironment. CML exposed macrophages separate from their normal counterparts by reduced expression of the surface marker CD36, which significantly reduces clearance of apoptotic cells. We uncover aberrant production of CML-secreted factors, including the immune modulatory protein lactotransferrin (LTF), that suppresses efferocytosis, phagocytosis, and CD36 surface expression in BM macrophages, indicating that the elevated secretion of LTF is, at least partially responsible for the supressed clearance function of Ph- macrophages.

miR-1202 acts as anti-oncomiR in myeloid leukaemia by down-modulating GATA-1S expression.

Transient abnormal myelopoiesis (TAM) is a Down syndrome-related pre-leukaemic condition characterized by somatic mutations in the haematopoietic transcription factor GATA-1 that result in exclusive production of its shorter isoform (GATA-1S). Given the common hallmark of altered miRNA expression profiles in haematological malignancies and the pro-leukaemic role of GATA-1S, we aimed to search for miRNAs potentially able to modulate the expression of GATA-1 isoforms. Starting from an in silico prediction of miRNA binding sites in the GATA-1 transcript, miR-1202 came into our sight as potential regulator of GATA-1 expression. Expression studies in K562 cells revealed that miR-1202 directly targets GATA-1, negatively regulates its expression, impairs GATA-1S production, reduces cell proliferation, and increases apoptosis sensitivity. Furthermore, data from TAM and myeloid leukaemia patients provided substantial support to our study by showing that miR-1202 down-modulation is accompanied by increased GATA-1 levels, with more marked effects on GATA-1S. These findings indicate that miR-1202 acts as an anti-oncomiR in myeloid cells and may impact leukaemogenesis at least in part by down-modulating GATA-1S levels.

[Effect of TRIM59 Expression Interference on Daunorubicin Chemosensitivity of Chronic Myeloid Leukemia K562 Cells and Its Mechanism].

OBJECTIVE: To investigate the effect of tripipartite motif 59 (TRIM59) expression interference on the chemosensitivity of daunorubicin (DNR) in chronic myeloid leukemia (CML) K562 cells and the related molecular mechanism. METHODS: The expressions of TRIM59 mRNA in bone marrow tissues of patients with CML and K562 cells were detected by RT-qPCR. Liposome-based transfection technology was used to transfect TRIM59-specific siRNA (si-TRIM59) into K562 cells which then were treated with DNR. The proliferation and apoptosis of cells were detected by CCK-8 assay and flow cytometry, respectively, and the expressions of apoptosis-related protein and Wnt/ß-catenin signaling pathway-related protein were detected by Western blot. RESULTS: Compared with the bone marrow tissue of CML patients at the time of initial treatment, the expression of TRIM59 mRNA in bone marrow tissue of CML patients at the time of chemotherapy resistance was significantly increased (P <0.05). Compared with control group, the cell proliferation inhibition rate and apoptosis rate in si-TRIM59 group and DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased (P <0.05), while the expressions of Bcl-2, Wnt3α, GSK-3ß protein and the ratio of p-ß-catenin/ß-catenin were significantly decreased (P <0.05). Compared with si-TRIM59 group and DNR group, the proliferation inhibition rate and apoptosis rate of si-TRIM59+DNR group were significantly increased (P <0.05), the expression of Bax, Caspase3 and Cleaved-Caspase3 protein were significantly increased, while the expression of Bcl-2, Wnt3α, GSK-3ß protein and the ratio of p-ß-catenin/ß-catenin were significantly decreased (P <0.05). CONCLUSION: TRIM59 expression interference may enhance the chemosensitivity of K562 cells to DNR, and its mechanism may be related to the regulation of Wnt/ß-catenin signaling pathway.

Myeloid leukemia-derived galectin-1 downregulates CAR expression to hinder cytotoxicity of CAR T cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown significant activity in B-lineage malignancies. However, their efficacy in myeloid leukemia has not been successful due to unclear molecular mechanisms. METHODS: We conducted in vitro and in vivo experiments to investigate whether myeloid leukemia cells directly induce CAR down-regulation. Furthermore, we designed a CD33 CARKR in which all lysines in the cytoplasmic domain of CAR were mutated to arginine and verified through in vitro experiments that it could reduce the down-regulation of surface CARs and enhance the killing ability. Transcriptome sequencing was performed on various AML and ALL cell lines and primary samples, and the galectin-1-specific inhibitory peptide (anginex) successfully rescued the killing defect and T-cell activation in in vitro assays. RESULTS: CAR down-regulation induced by myeloid leukemia cells under conditions of low effector-to-tumor ratio, which in turn impairs the cytotoxicity of CAR T cells. In contrast, lysosomal degradation or actin polymerization inhibitors can effectively alleviate CAR down-regulation and restore CAR T cell-mediated anti-tumor functions. In addition, this study identified galectin-1 as a critical factor used by myeloid leukemia cells to induce CAR down-regulation, resulting in impaired T-cell activation. CONCLUSION: The discovery of the role of galectin-1 in cell surface CAR down-regulation provides important insights for developing strategies to restore anti-tumor functions.

Prognostic significance of multiparametric flow cytometry minimal residual disease at two time points after induction in pediatric acute myeloid leukemia.

BACKGROUND: Prompt response to induction chemotherapy is a prognostic factor in pediatric acute myeloid leukemia. In this study, we aimed to evaluate the prognostic significance of multiparametric flow cytometry-minimal residual disease (MFC-MRD), assessed at the end of the first and second induction courses. METHODS: MFC-MRD was performed at the end of the first induction (TP1) in 524 patients and second induction (TP2) in 467 patients who were treated according to the modified Medical Research Council (UK) acute myeloid leukemia 15 protocol. RESULTS: Using a 0.1% cutoff level, patients with MFC-MRD at the two time points had lower event-free survival and overall survival. Only the TP2 MFC-MRD level could predict the outcome in a separate analysis of high and intermediate risks based on European LeukemiaNet risk stratification and KMT2A rearrangement. The TP2 MFC-MRD level could further differentiate the prognosis of patients into complete remission or non-complete remission based on morphological evaluation. Multivariate analysis indicated the TP2 MFC-MRD level as an independent adverse prognostic factor for event-free survival and overall survival. When comparing patients with MFC-MRD ≥ 0.1%, those who underwent hematopoietic stem cell transplant during the first complete remission had significantly higher 5-year event-free survival and overall survival and lower cumulative incidence of relapse than those who only received consolidation chemotherapy. CONCLUSIONS: The TP2 MFC-MRD level can predict the outcomes in pediatric patients with acute myeloid leukemia and help stratify post-remission treatment.