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.

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.

NRASmutDNMT3Amut Clone Identifies a Subset of de novo Cytogenetically Normal Acute Myeloid Leukemia with Adverse Prognosis.

OBJECTIVE: To investigate the molecular characteristics and clinical prognosis of the neuroblastoma RAS viral oncogene (NRAS) in patients with primary cytogenetically normal acute myeloid leukemia (AML). METHODS: A total of 171 adult patients with cytogenetically normal primary AML were collected, and 34 gene mutations in these patients were detected by targeted next-generation sequencing. RESULTS: Among 171 patients with cytogenetically normal AML(CN-AML), 17 (9.9%) patients had found NRAS mutations. Among the 17 NRAS mutant patients, 16 cases were associated with the concomitant gene, and NRAS mutation (NRASmut) was significantly positively correlated with DNMT3A mutation (DNMT3Amut) (P=0.011) and KRAS mutation (P=0.008) compared with the NRAS wild-type (NRASwt) group. The frequency of NRASmutDNMT3Amut clone was significantly higher in CN-AML patients with NRAS mutation (8/17, 47%). The total NRASmut group showed no significant differences on clinical characteristics, CR rate after induction therapy, OS, and RFS as compared with NRASwt group. However, patients with NRASmutDNMT3Amut provided a shorter effect on OS (median:7 vs 15 months; P=0.036) and RFS (median: 3 vs 12 months; P=0.003) than those with NRASwt, though no statistic differences on demographics, lab parameters, treatment and CR rate of patients receiving induction therapy. Multivariate analysis showed that NRASmutDNMT3Amut subtype could independently affect the RFS of CN-AML patients (HR:3.210, 95%CI:1.078-9.557, P=0.036). CONCLUSION: NRASmutDNMT3Amut clones have a high frequency of occurrence and show a poor survival prognosis. Our findings highlight potentially novel aspects of the underlying biology of NRASmutDNMT3Amut commutation in adult de novo CN-AML.

The impact of DNMT3A variant allele frequency and two different comutations on patients with de novo cytogenetically normal acute myeloid leukemia.

To refine the biological and prognostic significance of DNMT3A mutations in acute myeloid leukemia (AML), we assessed the impact of DNMT3A variant allele frequency (VAF) and its comutations in this study. Using targeted next-generation sequencing, we analyzed 171 adult patients with de novo cytogenetically normal AML for DNMT3A mutations and associated comutations. DNMT3Amut was detected in 35 patients. DNMT3Amut patients were divided into DNMT3AHigh and DNMT3ALow using a cut-off VAF value of 42%. We observed that DNMT3AHigh patients at diagnosis had increasing white blood cell (WBC) counts (p < 0.001) and a higher lactate dehydrogenase (LDH) level (p = 0.027), and were associated with lower complete remission (CR) rate (p = 0.015) and shorter overall survival (OS) (p = 0.032) than DNMT3ALow patients. We classified two different comutated genetypes, including DNMT3Amut NPM1mut FLT3-ITDmut and DNMT3Amut IDH1/IDH2mut . Patients with DNMT3Amut NPM1mut FLT3-ITDmut showed worse OS (p = 0.026) and relapse-free survival (RFS) (p = 0.003) than those with DNMT3Amut IDH1/IDH2mut , and showed a shorter OS (p = 0.027) than those with DNMT3Awt NPM1mut FLT3-ITDmut . We also observed that patients with DNMT3Amut IDH1/IDH2mut had higher platelet counts (p = 0.009) and a lower BM blast percentage (p = 0.040) than those with DNMT3Awt IDH1/IDH2mut . In multivariate analyses, DNMT3AHigh was independently associated with a lower CR rate (OR = 5.883; p = 0.004) and shorter OS (HR = 3.768; p < 0.001). DNMT3Amut NPM1mut FLT3-ITDmut independently affected worse OS (HR = 6.030; p < 0.001) and RFS (HR = 8.939; p < 0.001). Our findings might be potentially useful for predicting clinical outcomes.

A germline JAK2 exon12 mutation and a late somatic CALR mutation in a patient with essential thrombocythemia.

Background: It has been discovered that Janus kinase 2 (JAK2) exon12 mutations lead to the polycythemia vera (PV) phenotype, while somatic mutations of calreticulin (CALR) are associated with essential thrombocythemia (ET) or primary myelofibrosis. In this article, we report a case of ET with coexistence of JAK2 exon12 and CALR mutations. The objective of this study was to elucidate the pathogenicity mechanism of a JAK2 exon12 mutation (JAK2N533S) and the role of the coexistence of mutations on the hematological phenotype. Methods: We designed a colony analysis of tumor cells obtained from this patient, and attempted to identify mutant genes using DNA from hair follicles. Mutation impairment prediction and conservative analysis were conducted to predict the mutation impairment and structure of JAK2N533S. In addition, we conducted a functional analysis of JAK2N533S by constructing Ba/F3 cell models. Results: Three distinct tumor subclones, namely JAK2N533Shet+/CALRtype1het +, JAK2N533Shet+/CALR wt, and JAK2N533Shet+/CALRtype1hom +, were identified from the 17 selected erythroid and 21 selected granulocyte colonies. The analysis of hair follicles yielded positive results for JAK2N533S. According to the bioinformatics analysis, JAK2N533S may exert only a minor effect on protein function. Functional studies showed that JAK2N533S did not have a significant effect on the proliferation of Ba/F3 cells in the absence of interleukin-3 (IL-3), similar to wild-type JAK2. Notably, there were no increased phosphorylation levels of JAK2-downstream signaling proteins, including signal transducer and activator of transcription 3 (STAT3) and STAT5, in Ba/F3 cells harboring the JAK2N533S. Conclusion: Our study revealed that the JAK2N533Shet+/CALRtype1het+ subclone was linked to a significant expansion advantage in this patient, indicating that it may contribute to the development of the ET phenotype. We further demonstrated that JAK2N533S, as a noncanonical JAK2 exon12 mutation, is a germline mutation that may not exert an effect on cell proliferation and protein function. These results and the present body of available data imply that certain noncanonical JAK2 mutations are not gain-of-function mutations leading to the development of myeloproliferative neoplasms.

The characteristics and clinical prognosis analysis of ASXL1 mutations in Chinese adult patients with primary cytogenetically normal acute myeloid leukemia by next-generation sequencing.

We analyzed 156 adult patients with primary cytogenetically normal AML for ASXL1 mutations and co-mutations using targeted next-generation sequencing with a panel of 34 genes associated with myeloid neoplasms. ASXL1mut were identified in 15(10%) patients, more frequent at an older age (≥60years) (p = .014), and had significant associations with co-mutations in TET2, KIT, CBL and SRSF2, whereas inversely correlated to NPM1 and CEBPA mutations. ASXL1mut clustered in ELN2017 intermediate-risk group (p = .028). In the context of intermediate-risk, ASXL1mut had a worse overall survival(OS) (p = .038) and Relapse-free survival(RFS) (p = .016) than ASXL1wt. When coexisting DNMT3A or TET2 mutations, ASXL1mut/DNMT3Amut genetype revealed a superior OS than ASXL1mut/DNMT3Awt (p = .027), and ASXL1mut/TET2mut confered a worse RFS than ASXL1mut/TET2wt (p = .031). No significant prognosis impact of VAF (a cutoff value of 30%) and clone ranks of ASXL1mut were observed in this corhort. Our study provided a new understanding of characteristics of ASXL1mut AML.

The clinical characteristics and prognosis of Chinese acute myeloid leukemia patients with CSF3R mutations.

INTRODUCTION: The colony-stimulating factor 3 receptor (CSF3R) controls the proliferation of myeloid progenitors and differentiation into neutrophils. However, the clinical features and prognostic significance of CSF3R mutations in primary acute myeloid leukemia (AML) patients are still unclear. METHODS: 158 newly diagnosed AML patients were retrospectively evaluated in our study. Amplicon-based next-generation sequencing (NGS) and multiplex-nested reverse-transcription polymerase chain reaction (RT-PCR) were used to investigate the 34 genes and 43 fusion genes associated with leukemia. In addition, clinical features, mutation incidence, and survival outcomes were compared between patients with CSF3R mutation and patients with wild-type CSF3R. RESULTS: In our study, CSF3R mutations were found in 7.6% (12/158) cases. The membrane-proximal amino acid substitution T618I (58.3%) was the most frequent mutation. CSF3R mutations were associated with higher WBC counts (P = .035). CEBPA mutation, TET2 mutation, and RUNX1-RUNX1T1 translocation were the most common co-mutations of CSF3R. The CSF3R gene was mutually exclusive with signal transduction genes (P = .029), while positively associated with TET2 mutations (P = .014). CSF3R mutations had no effect on CR1 (P = .935), R (P = .625) and OS (P = .1172). Patients with CSF3R mutations had a worse DFS (P = .0352) than those with wild-type CSF3R. Multivariate survival analysis showed that CSF3R mutation was an independent risk factor for DFS of primary AML patients (HR=2.048, 95%CI: 1.006-4.170, P = .048). CONCLUSION: AML patients with CSF3R mutations had unique clinical features and gene co-mutation spectrum. CSF3R mutation was an independent risk factor for DFS and could be a potential prognostic marker and therapeutic target for Chinese primary AML patients.

GATA2 rs2335052 and GATA2 rs78245253 single-nucleotide polymorphisms in Chinese patients with acute myelocytic leukemia.

INTRODUCTION: GATA binding protein 2 (GATA2) gene, involved in progression of hematologic malignancies and various solid tumors, is a susceptibility gene for inherited acute myeloid leukemia (AML). However, the influence of its single-nucleotide polymorphisms (SNPs) on AML remains unknown. METHODS: We used allele-specific PCR to genotype GATA2 rs2335052 and rs78245253 in 159 newly diagnosed AML (non-M3) patients and 300 healthy volunteers, and all of participants came from China. And 34 common hematological tumor gene mutations in 159 AML patients were detected by next-generation sequencing. Kaplan-Meier survival analysis and Cox proportional hazard regression were used to analyze the association between the two SNPs and the prognosis of AML. RESULTS: We found GATA2 rs2335052 C/T genotype, rs2335052 T/T genotype and rs78245253 G/C genotype in 51.6%, 13.8% and 11.3% AML patients. Our results demonstrated that GATA2 rs2335052 and rs78245253 were associated with certain laboratory features in AML patients, which had no effect on the pathogeny, chemotherapy response and recurrence of patients. Nevertheless, Kaplan-Meier survival analysis showed that, compared with rs78245253 G/G genotype, rs78245253 G/C genotype was significantly related to a decrease in overall survival (OS) (P = .020). Additionally, multivariate cox regression analysis showed that GATA2 rs78245253 was an independent risk factor for OS of AML patients in China. CONCLUSION: GATA2 rs78245253 was an independent predictor for prognosis of AML patients in China and may be used as a potential indicator to predict the survival of AML patients in China. Further studies are needed to validate these findings and clarify the underlying mechanism.

The clinical characteristics and prognosis of cytogenetically normal AML with single mutations of CEBPA.

INTRODUCTION: CEBPA mutation is a common mutation in normal karyotype AML. CEBPAdm AML has been recognized as a separate entity, but there is still controversy to the prognosis of CEBPAsm patients. METHODS: A total of 151 newly diagnosed cytogenetically normal AML patients treated at the Second Hospital Center of Shanxi Medical University from February 2017 to December 2019 were the subjects of the study. According to the number of mutations in the CEBPA gene, the patients were divided into three groups, CEBPAsm, CEBPAdm, and CEBPAwt patients. The clinical characteristics, gene mutations, response, and prognosis were retrospectively compared among the three groups. RESULTS: CEBPAsm patients had lower hemoglobin values compared to CEBPAdm (P = .049). There was no statistical difference between the CEBPAsm cases and the CEBPAdm cases in the mutation types and the distribution of mutation regions (P > .050). Compared with CEBPAdm, cases with CEBPAsm were more likely associated with multiple other gene mutations (P = .023). Patients with CEBPAdm had a higher CR, ORR, and OS than those CEBPAwt (P < .050). CEBPAsm patients had a similar OS with CEBPAdm and CEBPAwt patients (P = .281). These CEBPAsm patients with VAF<30% had lower OS than the patients with VAF≥30%. FLT3-ITD mutations could reduce CEBPAsm patients' OS (P = .019). CONCLUSION: Our data first highlighted the impact of CEBPAsm VAF on OS, and the results showed the lower the VAF was, the shorter the OS tended to. The VAF of CEBPAsm could provide specific significance in some extent for the prognosis of patients.