Overexpression of RBM4 promotes acute myeloid leukemia cell differentiation by regulating alternative splicing of TFEB.

Alternative splicing (AS) is an efficient and ubiquitous transcriptional regulatory mechanism that expands the coding capacity of the genome and is associated with the occurrence and progression of cancer. The differentiation-promoting regimen is a potential therapeutic approach in cancer treatment. In this study, we screened NPMc-positive and NPMc-negative AML samples from the Cancer Genome Atlas (TCGA), focusing on the splicing factor RBM4 and its splicing mechanism on the target gene TFEB, which are most relevant to the prognosis of AML. We also investigated the impact of the TFEB-dominant spliceosome on autophagy and differentiation of THP-1 and K562 cells. The results showed that RBM4 recognized the CU-rich sequence in intron 8 of TFEB, increasing the production of the TFEB-L spliceosome, which promoted autophagy. Overexpression of RBM4 increased autophagy and promoted cell differentiation. The combination of TFEB-L with the therapeutic drug rapamycin further promoted the differentiation of leukemia cell lines and primary leukemia cells in AML patients. This study suggested that overexpression of RBM4 could promote cell differentiation by promoting the production of the TFEB-dominant spliceosome, demonstrating the potential of the TFEB-dominant spliceosome combined with chemotherapy drugs to promote leukemia cell differentiation and improve patient prognosis.

Synergistic effect and molecular mechanism of PVA and UM171 in ex vivo expansion of primitive hematopoietic stem cells.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) used for transplantation; the number of cells in a single UCB is too small to quickly establish bone marrow (BM) implantation, and ex vivo expansion of HSCs has the potential to overcome this limitation. The purpose of this study is to explore the culture conditions conducive to the maintenance and expansion of hematopoietic stem and progenitor cells (HSPCs) and long-term hematopoietic stem cells (LT-HSCs) derived from human umbilical cord blood, compare the different effects of albumin (HSA) and polyvinyl alcohol (PVA), optimize the culture system using UM171 and investigate the molecular mechanism of PVA and UM171 promoting the expansion of primitive hematopoietic stem cells. CD34+ cells were purified from UCB using MacsCD34 beads, and then cultured in serum-free medium supplemented with cytokines for 12 days, with PVA or UM171 added according to experimental requirements; the relative percentage of different HSCs subsets after culture were detected by flow cytometry; CFU Assay Setup for detecting the multilineage differentiation potential of HSCs; RT-PCR detection of gene expression levels; reactive oxygen detection assessment of intracellular ROS levels. (1) The conditions of 20 ng/mlSCF, 100 ng/mlTPO, and 5% oxygen concentration are conducive to the maintenance of LT-HSCs. (2) Compared with HSA, PVA significantly increased the proportion of HSPCs and LT-HSCs, as well as dramatically promoted the expression of antioxidant enzymes and reduced the production of reactive oxygen species (ROS). (3) After adding UM171 to PVA-based medium, the proportion of HSPCs and LT-HSCs further increased, and downstream genes of Notch and Wnt pathways were selectively activated. (1) PVA may inhibit ROS production by upregulating the expression of antioxidant enzymes, which is beneficial for maintaining stemness and inhibiting differentiation of HSCs. (2) The antioxidant properties of PVA can delay differentiation, while UM171 can promote self-renewal by regulating the stem cell pathway, and the combination of them is beneficial for the maintenance and expansion of HSCs in vitro.

Advances in hematopoietic stem cells ex vivo expansion associated with bone marrow niche.

Hematopoietic stem cells (HSCs) are an ideal source for the treatment of many hematological diseases and malignancies, as well as diseases of other systems, because of their two important features, self-renewal and multipotential differentiation, which have the ability to rebuild the blood system and immune system of the body. However, so far, the insufficient number of available HSCs, whether from bone marrow (BM), mobilized peripheral blood or umbilical cord blood, is still the main restricting factor for the clinical application. Therefore, strategies to expand HSCs numbers and maintain HSCs functions through ex vivo culture are urgently required. In this review, we outline the basic biology characteristics of HSCs, and focus on the regulatory factors in BM niche affecting the functions of HSCs. Then, we introduce several representative strategies used for HSCs from these three sources ex vivo expansion associated with BM niche. These findings have deepened our understanding of the mechanisms by which HSCs balance self-renewal and differentiation and provided a theoretical basis for the efficient clinical HSCs expansion.

Baicalein attenuates oxidative damage in mice haematopoietic cells through regulation of PDGFRß.

Platelet-derived growth factor receptor ß (PDGFRß) plays a crucial role in murine haematopoiesis. Baicalein (BAI), a naturally occurring flavonoid, can alleviate disease damage through anti-oxidative, anti-apoptotic, and anti-inflammatory mechanisms. However, whether BAI attenuates oxidative damage in murine haematopoietic cells by PDGFRß remains unexplored. In this study, we utilized a tert-butyl hydroperoxide (TBHP)-induced BaF3 cell injury model and an ionising radiation (IR)-induced mice injury model to investigate the impact of the presence or absence of PDGFRß on the pharmacological effects of BAI. In addition, the BAI-PDGFRß interaction was characterized by molecular docking and dynamics simulations. The results show that a specific concentration of BAI led to increased cell viability, reduced reactive oxygen species (ROS) content, upregulated nuclear factor erythroid 2-related factor 2 (NRF2) expression, and its downstream target genes heme oxygenase 1 (HO-1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), and activated protein kinase B (AKT) pathway in cells expressing PDGFRß plasmid and experiencing damage. Similarly, BAI elevated lineage-Sca1+cKIT+ (LSK) cell proportion, promoted haematopoietic restoration, enhanced NRF2-mediated antioxidant response in PDGFRß+/+ mice. However, despite BAI usage, PDGFRß knockout mice (PDGFRß-/-) showed lower LSK proportion and less antioxidant capacity than the total body irradiation (TBI) group. Furthermore, we demonstrated an interaction between BAI and PDGFRß at the molecular level. Collectively, our results indicate that BAI attenuates oxidative stress injury and helps promote haematopoietic cell recovery through regulation of PDGFRß.

The Study of SRSF1 Regulates Abnormal Alternative Splicing of BCL2L11 and the Role in Refractory Acute Myeloid Leukemia.

INTRODUCTION: Abnormalities in splicing factors, such as mutations or deregulated expression, can lead to aberrant splicing of target genes, potentially contributing to the pathogenesis of acute myeloid leukemia (AML). Despite this, the precise mechanism underlying the abnormal alternative splicing (AS) induced by SRSF1, a splicing factor associated with poor AML prognosis, remains elusive. METHODS: Using strict splicing criteria, we globally screened for AS events in NPMc-positive and NPMc-negative AML samples from TCGA. An AS network associated with AML prognosis was then established. Functional assays, including CCK-8, flow cytometry, and Western blot, were conducted on K562 and THP-1 cells overexpressing SRSF1. Cell viability following 72-h Omipalisib treatment was also assessed. To explore the mechanism of SRSF1-induced AS, we created a BCL2L11 miniGene with a site-specific mutation at its branch point. The AS patterns of both wild-type and mutant miniGenes were analyzed following SRSF1 overexpression in HEK-293T, along with the subcellular localization of different spliceosomes. RESULTS: SRSF1 was significantly associated with AML prognosis. Notably, its expression was markedly upregulated in refractory AML patients compared to those with a favorable chemotherapy response. Overexpression of SRSF1 promoted THP-1 cell proliferation, suppressed apoptosis, and reduced sensitivity to Omipalisib. Mechanistically, SRSF1 recognized an aberrant branch point within the BCL2L11 intron, promoting the inclusion of a cryptic exon 3, which in turn led to apoptosis arrest. CONCLUSION: Overexpression of SRSF1 and the resulting abnormal splicing of BCL2L11 are associated with drug resistance and poor prognosis in AML.

Expansion strategies for umbilical cord blood haematopoietic stem cells in vitro.

Haematopoietic stem cell transplantation (HSCT) is considered an effective treatment for some haematopoietic malignancies, haematopoietic failure and immunodeficiency. Compared with bone marrow and mobilized peripheral blood, cord blood has the advantages of easy access, being harmless to donors and low requirement for HLA matching. In addition, umbilical cord blood transplantation (UCBT) has achieved remarkable clinical success in the past 30 years due to the low recurrence rate of malignancies treated by UCBT, mild degree of chronic graft-versus-host disease (GVHD) and good quality of life for patients after transplantation. However, the number of cells in a single cord blood is too small for rapid bone marrow implantation. We summarize the various factors involved that need to be considered in the expansion of haematopoietic stem cells (HSCs) in vitro, which all avoid complex operations, such as vector construction and virus transfection. We also found it necessary to identify a new molecule as the carrier of HSCs cultured in vitro, which not only would provide a three-dimensional structure conducive to the self-renewal of HSCs but also prevent their differentiation.

A new pathway for the transformation of AML in MDS: APA mechanism regulated by NUDT21 and RUNX1.

We have identified that NUDT21 plays a vital role in MDS transformations, while the transcription factor RUNX1 is essential for normal hematopoiesis, which is a high expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and we aim to explore the linkage between the two genes and new pathways for MDS transformation to AML. Prediction of RUNX1 expression levels and its relationship with NUDT21 in AML and MDS patients was performed using bioinformatics techniques and validated in patients. Using lentiviral packaging technology, NUDT21 knockdown and overexpression models were developed in AML and MDS cell lines. These models were validated using quantitative polymerase chain reaction (qPCR) and western blotting. The cell cycle, apoptosis, differentiation, and cytokines were examined by flow cytometry, CCK-8 analyzed proliferation, and the intracellular localization of NUDT21 and RUNX1 was examined by immunofluorescence. mRNA transcriptome sequencing was performed on THP-1, MUTZ-1, and Dapars analyzed SKM-1 cell lines and the sequencing data to observe the knockdown effect of NUDT21 on RUNX1. qPCR and western blot revealed a positive correlation between NUDT21 and RUNX1; both were located in the nucleus. Overexpression of NUDT21 reduced apoptosis, promoted cell proliferation, and possibly increased the invasive ability of cells. It also altered the APA site in the RUNX1 3'-UTRs region. NUDT21 regulates RUNX1 gene expression and promotes AML transformation in MDS through an APA mechanism.

BCR/ABL1ΔE7-8-9 isoform contributes to tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

In chronic myeloid leukemia (CML) patients, the involvement of the BCR/ABL1 isoform in tyrosine kinase inhibitors (TKIs) resistance has attracted lots of attention. In this work, a novel isoform that encoded truncated protein due to the deletion of ABL1 exon7, 8, and 9 was reported and named BCR/ABL1ΔE7-8-9 here. This isoform was detected only in 10.2% of CML patients with inadequate responses to TKIs. BCR/ABL1Δexon7-8-9 isoform promoted S phase cell proliferation and reduced the expression of fusion gene and ABL1 phosphorylation level more slowly than that of control cells after TKIs treatment. The novel isoform has the qualities of a functional tyrosine kinase, localized in the cytoplasm, and could not be imported into the nucleus by TKIs. These results indicated that BCR/ABL1Δexon7-8-9 showed poorer sensitivity to imatinib and nilotinib than wild-type BCR/ABL1. According to molecular docking studies, nilotinib and imatinib present different binding sites and have a lower binding capacity with BCR/ABL1ΔE7-8-9 protein than the wild type. Our findings suggested that the novel isoform BCR/ABL1ΔE7-8-9 may contribute to TKIs resistance in CML due to its weakened TKIs binding ability. It enriched the mechanism of spliceosome involved in TKIs resistance. Monitoring the expression of BCR/ABL1ΔE7-8-9 helps guide the treatment of CML patients in the clinic.

Early platelet elevation after complete remission as a prognostic marker of favourable outcomes in favourable- and intermediate-risk acute myeloid leukaemia: A retrospective study.

OBJECTIVES: Platelet (PLT) recovery after chemotherapy is associated with the prognosis of patients with acute myeloid leukaemia (AML). This study aimed to explore the prognostic significance of early high PLT values in patients with de novo non-M3 AML who achieved first complete remission (CR). METHODS: A total of 206 patients with de novo non-M3 AML were analysed in this retrospective study. A receiver operating characteristic (ROC) curve was used to determine the optimal PLT cut-off. The overall survival (OS) and relapse-free survival (RFS) were assessed using Kaplan-Meier and Cox regression analyses. RESULTS: 312×109 /L was confined as the cut-off of the PLT count. The estimated 3-year OS of patients with high PLT was higher than that of their counterparts (72.3% vs. 34.6%, p = 0.001). In subgroup analysis, patients with high PLT had better OS in the favourable- and intermediate-risk (non-adverse-risk) AML (p = 0.001). The estimated 3-year RFS for the high and low PLT groups was 75.1% and 45.7% respectively (p = 0.078). Multivariate analyses revealed that high PLT count was an independent favourable variable for OS (HR = 0.264, p < 0.001) and RFS (HR = 0.375, p = 0.011) in the non-adverse-risk group. CONCLUSION: Our results showed that early high PLT count recovery at first CR in non-adverse-risk AML patients is a positive prognostic marker for survival outcomes.

Coagulopathy in cytogenetically and molecularly distinct acute leukemias at diagnosis: Comprehensive study.

We analyzed the characteristics of coagulopathy in cytogenetically and molecularly distinct acute leukemias. We retrospectively analyzed 211 adult patients with de novo non-acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML), and 105 newly diagnosed patients with B-cell acute lymphoblastic leukemia (B-ALL). Disseminated intravascular coagulation (DIC) occurrence was assessed according the International Society of Thrombosis and Haemostasis (ISTH) criteria. Further, we analyzed the associations of the cytogenetics and mutations with DIC development and coagulation profile. Significant differences were observed between APL and non-APL AML (P = 0.001), APL and B-ALL (P = 0.002), and non-APL AML and B-ALL (P = 0.009) in the distribution of ISTH DIC scores of the acute leukemia patients that met the criteria for diagnosis of DIC. Except for the elevated leukocyte count, a normal karyotype with NPM1 mutations or/and FLT3-ITD mutations was independently associated with the development of DIC in non-APL AML, characterized by significant PT prolongation and significantly elevated D-Dimers. The P210BCR-ABL1 transcript strongly predicted hypofibrinogenemia in B-ALL in the final multivariate model, but Philadelphia chromosome negatively affected elevated D-dimers. In conclusion, DIC occurrence and the coagulation profile were associated with the cytogenetics and mutations in acute leukemia.

TET2 rs2454206, TET2 rs12498609 and ASXL1 rs3746609 single nucleotide polymorphisms in patients with myelodysplastic syndromes.

To study the association between TET2rs2454206, TET2rs12498609 and ASXL1rs3746609 and Myelodysplastic syndromes (MDS), a total of 90 MDS patients and 143 healthy volunteers were included. The clinical data, bone marrow samples of patients and peripheral blood samples of volunteers were obtained. We found TET2rs2454206 G/A genotype, TET2rs12498609 G/C genotype and ASXL1rs3746609 A/G genotype in 13.3%, 11.1%, 10.1% MDS patients and in 42.7%, 22.4%, 23.8% healthy volunteers (P < 0.001; P = 0.029; P = 0.009, respectively). TET2 rs2454206 G/A genotype was associated with higher serum LDH level in MDS (P = 0.025). Patients with TET2rs12498609 G/C genotype were characterized with higher frequency of mutated SRSF2 gene (P = 0.042) and lower occurrence rate of anemia (P = 0.026) than those with C/C genotype. ASXL1rs3746609 A/G genotype linked with higher thrombocyte counts (P = 0.02) and percent of total T lymphocyte (P = 0.029), whereas with lower percent of NK cell (P = 0.032) and B lymphocyte (P = 0.007). None of these three SNPs had impact on the overall survival and disease progression to AML. We concluded that People with TET rs2454206 G/A genotype, TET2rs12498609 G/C genotype or ASXL1rs3746609 A/G genotype were related to lower prevalence of MDS. All of the three SNPs were associated with certain laboratory features in MDS patients.

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression.

OBJECTIVE: This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS: Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS: At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2  ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. CONCLUSION: In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.

Detection of PML/RARα Fusion Gene in Bone Marrow of Patients with Functionalized Graphene Oxide.

BACKGROUND: Current diagnostic methods of acute promyelocytic leukemia (APL) have some inevitable shortcomings; therefore, further studies are needed to develop more effective diagnostic methods. We used functionalized graphene oxide (GO) to detect the promyelocytic leukemia/retinoic acid receptor, α fusion gene (PML/RARα fusion gene) in bone marrow of APL patients. This method was more convenient and time-saving, and we can obtain the detection results in 1 hour. METHODS: Our group consists of 36 cases, among them are 16 cases which are PML-RARα positive, 20 cases which are PML-RARα negative. Firstly, samples were fixed, drilled, and incubated with antibody CD45. Secondly, GO, fluorescent probes, and buffer liquid were added. One hour later, samples were washed with PBS (1 x) buffer, centrifuged, and fluorescent signals were detected with flow cytometry. RESULTS: Using functional GO to carry the fluorescent probe we ascertained whether bone marrow samples contain the L type PML/RARα fusion gene. Using the probe, only cells which contain L type PML/RARα fusion gene will have fluorescent signals compared to no signals (p < 0.05). The GO detection method was accurate and has clinical diagnostic values (p < 0.05). CONCLUSIONS: The GO detection method has the advantages of accurate, time-saving, energy-saving simple operation, and no pollution.

Quantitative analysis of Plasmodium ookinete motion in three dimensions suggests a critical role for cell shape in the biomechanics of malaria parasite gliding motility.

Motility is a fundamental part of cellular life and survival, including for Plasmodium parasites--single-celled protozoan pathogens responsible for human malaria. The motile life cycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. Although gliding is based on the well-studied system of actin and myosin, its core biomechanics are not completely understood. Currently accepted models suggest it results from a specifically organized cellular motor that produces a rearward directional force. When linked to surface-bound adhesins, this force is passaged to the cell posterior, propelling the parasite forwards. Gliding motility is observed in all three life cycle stages of Plasmodium: sporozoites, merozoites and ookinetes. However, it is only the ookinetes--formed inside the midgut of infected mosquitoes--that display continuous gliding without the necessity of host cell entry. This makes them ideal candidates for invasion-free biomechanical analysis. Here we apply a plate-based imaging approach to study ookinete motion in three-dimensional (3D) space to understand Plasmodium cell motility and how movement facilitates midgut colonization. Using single-cell tracking and numerical analysis of parasite motion in 3D, our analysis demonstrates that ookinetes move with a conserved left-handed helical trajectory. Investigation of cell morphology suggests this trajectory may be based on the ookinete subpellicular cytoskeleton, with complementary whole and subcellular electron microscopy showing that, like their motion paths, ookinetes share a conserved left-handed corkscrew shape and underlying twisted microtubular architecture. Through comparisons of 3D movement between wild-type ookinetes and a cytoskeleton-knockout mutant we demonstrate that perturbation of cell shape changes motion from helical to broadly linear. Therefore, while the precise linkages between cellular architecture and actomyosin motor organization remain unknown, our analysis suggests that the molecular basis of cell shape may, in addition to motor force, be a key adaptive strategy for malaria parasite dissemination and, as such, transmission.

Glycan imaging in intact rat hearts and glycoproteomic analysis reveal the upregulation of sialylation during cardiac hypertrophy.

In the heart, glycosylation is involved in a variety of physiological and pathological processes. Cardiac glycosylation is dynamically regulated, which remains challenging to monitor in vivo. Here we describe a chemical approach for analyzing the dynamic cardiac glycome by metabolically labeling the cardiac glycans with azidosugars in living rats. The azides, serving as a chemical reporter, are chemoselectively conjugated with fluorophores using copper-free click chemistry for glycan imaging; derivatizing azides with affinity tags allows enrichment and proteomic identification of glycosylated cardiac proteins. We demonstrated this methodology by visualization of the cardiac sialylated glycans in intact hearts and identification of more than 200 cardiac proteins modified with sialic acids. We further applied this methodology to investigate the sialylation in hypertrophic hearts. The imaging results revealed an increase of sialic acid biosynthesis upon the induction of cardiac hypertrophy. Quantitative proteomic analysis identified multiple sialylated proteins including neural cell adhesion molecule 1, T-kininogens, and α2-macroglobulin that were upregulated during hypertrophy. The methodology may be further extended to other types of glycosylation, as exemplified by the mucin-type O-linked glycosylation. Our results highlight the applications of metabolic glycan labeling coupled with bioorthogonal chemistry in probing the biosynthesis and function of cardiac glycome during pathophysiological responses.

Bifunctional unnatural sialic acids for dual metabolic labeling of cell-surface sialylated glycans.

Sialic acid analogues containing a unique chemical functionality or chemical reporter have been metabolically incorporated into sialylated glycans. This process, termed metabolic glycan labeling, has emerged as a powerful tool for studying sialylation as well as other types of glycosylation. Currently, this technique can install only a single functionality. Here we describe a strategy for dual labeling of sialylated glycans using a new class of bifunctional sialic acid analogues containing two distinct chemical reporters at the N-acyl and C9 positions. These bifunctional unnatural sialic acids were metabolically incorporated into cellular glycans, where the two chemical reporters exerted their distinct functions. This approach expands the capability of metabolic glycan labeling to probe sialylation and glycan-protein interactions.

The short isoform of the long-type PML-RARA fusion gene in acute promyelocytic leukaemia lacks sensitivity to all-trans-retinoic acid.

Alternative splicing is associated with human disease. In acute promyelocytic leukaemia (APL) patients with the long (L)-type promyelocytic leukaemia-retinoic acid receptor α fusion gene (PML-RARA), three alternative splicing isoforms can be detected: E5(+)E6(+), E5(-)E6(+), and E5(-)E6(-). This study is the first to demonstrate that alternative splicing of L-type PML-RARA is associated with time to achieve complete remission (CR) in APL. Higher expression of the E5(-)E6(-) isoform, the short isoform, was related to longer time to achieve CR. Each isoform was constructed into recombinant lentiviral vector and transfected into U937 cells. Compared with the E5(-)E6(+) and E5(+)E6(+) groups, the U937 cells with E5(-)E6(-) showed lower sensitivity to all-trans-retinoic acid treatment.

Detection of promyelocytic leukemia/retinoic acid receptor α (PML/RARα) fusion gene with functionalized graphene oxide.

An attempt was made to use functionalized graphene oxide (GO) to detect the Promyelocytic leukemia/Retinoic acid receptor α fusion gene (PML/RARα fusion gene), a marker gene of acute promyelocytic leukemia. The functionalized GO was prepared by chemical exfoliation method, followed by a polyethylene glycol grafting. It is found that the functionalized GO can selectively adsorb the fluorescein isothiocyanate (FITC)-labeled single-stranded DNA probe and quench its fluorescence. The probe can be displaced by the PML/RARα fusion gene to restore the fluorescence, which can be detected by laser confocal microscopy and flow cytometry. These can be used to detect the presence of the PML/RARα fusion gene. This detection method is verified to be fast, simple and reliable.

Acute myeloid leukemia cells and MSC-derived exosomes inhibiting transformation in myelodysplastic syndrome.

AIMS: To investigate the mechanism of exosomes' role in the transformation of MDS to AML. METHODS: Exosomes in culture supernatants of MDS and AML cell lines, were extracted by ultrafiltration and identified in three ways: morphology, size, and exosome protein surface markers. Exosomes from AML cell lines were then co-cultured with MDS cell lines and their impacts on MDS cell microenvironment, proliferation, differentiation, cell cycle, and apoptosis were analyzed by CCK-8 assay and flow cytometry. Furthermore, exosomes from MSC were extracted for further authentication. RESULTS: The transmission electron microscopy, nanoparticle tracking analysis, Western blotting, and flow cytometry methods all verify that ultrafiltration is a reliable method to extract exosomes in the culture medium. Exosomes from AML cell lines inhibit the proliferation of MDS cell lines, block cell cycle progression, and promote apoptosis and cell differentiation. It also leads to increased secretion of tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS) in MDS cell lines. In addition, MSC-derived exosomes were found to inhibit the proliferation of MDS cell lines, arrest cell cycle progression, promote apoptosis, and inhibit differentiation. CONCLUSION: Ultrafiltration is a proper methodology in extracting exosomes. The exosomes of AML origin and MSC origin may play a role in MDS leukemia transformation via targeting TNF-α/ROS-Caspase3 pathway.

[Impact of CSF3R Mutation on Treatment Response and Survival of Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the expression of CSF3R mutation in acute myeloid leukemia (AML) and analyze its clinical characteristics and prognosis. METHODS: A retrospective study was conducted in 212 patients with AML who were newly diagnosed in the Second Hospital of Shanxi Medical University from January 1th 2018 to June 30th 2021, including 22 patients with CSF3R mutations as mutation group and 190 patients with CSF3R wild type ï¼»66 cases of them were screened by propensity score matching (PSM), as control groupï¼½. The early efficacy and survival between the two groups were compared. RESULTS: The median age of patients in the mutation group was 50(17-73) years old, and the ratio of male to female was 1.2:1 The main types were AML with maturation (11 cases) and acute myelomonocytic leukemia (9 cases). Prognostic stratification was carried out according to the risk stratification system of the European leukemia network in 2017, with 16 cases (72.73%) in the middle and high-risk group. At the initial diagnosis, the median count of white blood cell (WBC) was 44.75(1.30-368.71)×109/L, among which 15 cases (68.18%) were >10×109/L, and the median count of platelet (PLT) was 24(4-55)×109/L. CSF3R T618I (68.18%) was a common mutation site, which had concomitant gene mutations, in which CEBPA mutation was the most common (10 cases, 45.45%), but only existed in CSF3R T618I mutation. The CR/CRi rate was 68.18% and 71.21% in the mutant group and the control group (P >0.05), the median over all survival time was 15 months and 9 months (P >0.05), and the median disease-free survival time was 8 months and 4 months (P >0.05), respectively. CONCLUSION: Most AML patients with CSF3R mutation are middle-aged patients, the main types are AML with maturation and acute myelomonocytic leukemia, and most of them have middle and high-risk prognosis. CSF3R mutation may not be an independent prognostic marker for newly diagnosed AML patients.