Pedigree investigation, clinical characteristics, and prognosis analysis of haematological disease patients with germline TET2 mutation.

BACKGROUND: Increasing germline gene mutations have been discovered in haematological malignancies with the development of next-generation sequencing (NGS), which is critical for proper clinical management and long-term follow-up of affected individuals. Tet methylcytosine dioxygenase 2 (TET2) is one of the most common mutations in haematological neoplasms. We aimed to compare the clinical characteristics of patients with germline and somatic TET2 mutations in haematological diseases and to analyse whether germline TET2 mutations have a family aggregation and tumour predisposition. METHODS: Out of 612 patients who underwent NGS of 34 recurrently mutated genes in haematological diseases, 100 haematological patients with TET2 mutations were selected for further study. Somatic mutations were detected by NGS in bone marrow/peripheral blood genomic DNA (gDNA). Germline TET2 mutations were validated in nail/hair gDNA by Sanger sequencing. Digital data were extracted from the haematology department of the West China Hospital of Sichuan University. TET2 mutation results were analysed by referencing online public databases (COSMIC and ClinVar). RESULTS: One hundred patients were studied, including 33 patients with germline and 67 patients with somatic TET2 mutations. For germline TET2 mutations, the variant allele frequency (VAF) was more stable (50.58% [40.5-55], P < 0.0001), and mutation sites recurrently occurred in three sites, unlike somatic TET2 mutations. Patients with germline TET2 mutations were younger (median age 48, 16-82 years) (P = 0.0058) and mainly suffered from myelodysplastic syndromes (MDS) (n = 13, 39.4%), while patients with somatic TET2 mutations were mainly affected by acute myeloid leukemia (AML) (n = 26, 38.8%) (P = 0.0004). Germline TET2 mutation affected the distribution of cell counts in the peripheral blood and bone marrow (P < 0.05); it was a poor prognostic factor for MDS patients via univariate analysis (HR = 5.3, 95% CI: 0.89-32.2, P = 0.0209) but not in multivariate analysis using the Cox regression model (P = 0.062). CONCLUSIONS: Germline TET2 mutation might have a family aggregation, and TET2 may be a predisposition gene for haematological malignancy under the other gene mutations as the second hit. Germline TET2 mutation may play a role in the proportion of blood and bone marrow cells and, most importantly, may be an adverse factor for MDS patients.

The m6A RNA methyltransferase METTL3/METTL14 promotes leukemogenesis through the mdm2/p53 pathway in acute myeloid leukemia.

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA and recent studies have highlighted the importance of m6A levels in tumor development. In this study, we investigated the expression of methyltransferase-like 3 (METTL3) and 14 (METTL14), components of the RNA m6A methyltransferase complex, in samples from 89 patients with acute myeloid leukemia (AML), and followed the survival of 75 of these patients. Our results show that METTL3 and METTL14 are highly expressed in most of the patients with AML (except those with APL), and high levels of METTL3 and/or METTL14 correlated to shorter survival in the patients. In leukemia cell lines K562 and kasumi-1, both METTL3 and METTL14 promote cell proliferation and cell cycle, and the knockdown of METTL3 and METTL14 inhibits proliferation, and induces apoptosis and differentiation. Notably, the knockdown of METTL3 and METTL14 in K562 cell line leads to several changes in the expression of p53 signal pathway, including the upregulation of p53, cyclin dependent kinase inhibitor 1A (CDKN1A/p21), and downregulation of mdm2. Importantly, the m6A level of mdm2 mRNA was significant lower after knock-down of METTL3 and METTL14 examined by m6A-RIP and mdm2 qPCR assay, and the half-life of mdm2 under actinomycin-D treatment became shorter. Taken together, our study demonstrates that the lower m6A levels of mdm2 mRNA mediated by the knockdown of METTL3 and METTL14 could lead to the low stability of mdm2 mRNA transcripts and low expression of MDM2, in the end, activate p53 signal pathway. Both METTL3 and METTL14 play an oncogenic role in AML by targeting mdm2/p53 signal pathway.

High Wilms' tumor 1 associating protein expression predicts poor prognosis in acute myeloid leukemia and regulates m6A methylation of MYC mRNA.

PURPOSE: Acute myeloid leukemia (AML) is a heterogenous disease and the survival of AML patients is largely attributed to the improvement of supportive treatment. Wilms' tumor 1-associated protein (WTAP) is a nuclear protein functions in many physiological and pathological processes. Although its expression and function in many malignant diseases have been reported, its prognostic and epigenetic roles in AML are largely unknown. METHODS: Peripheral blood or bone marrow samples were collected from AML patients. The WTAP expression was detected by western blot. WTAP expression level and patients clinical features were analyzed using statistical methods. WTAP knockdown AML cells were constructed. The experiments on proliferation, tumorigenic ability, cell cycle, and apoptosis were performed. Transcriptome sequencing was performed and analyzed. M6A methylation level was measured and m6A-RIP was performed to quantify m6A methylation level of MYC mRNA. RNA stability assay was performed to measure the half-life of mRNA. RESULTS: WTAP was overexpressed in AML patients and was an independent poor-risk factor in AML (p = 0.0140). Moreover, we found that WTAP regulated proliferation, tumorigenesis, cell cycle, and differentiation of AML cells. Furthermore, WTAP made AML cells resistant to daunorubicin. In further investigations, m6A methylation level was downregulated when knocking down WTAP, and c-Myc was upregulated due to the decreased m6A methylation of MYC mRNA. CONCLUSION: High WTAP expression predicts poor prognosis in AML and WTAP plays an epigenetic role in AML.

Positive expression of PAX7 indicates poor prognosis of pediatric and adolescent AML patients.

Objective: Therapeutic advances based on risk stratification and implementation of excellent supportive care measures have significantly improved outcomes for childhood acute myeloid leukemia (AML) over the past 30 years. However, approximately half of all childhood AML cases relapse. Therefore, precise risk stratification is needed for predicting relapse potential.Methods: RNA-seq data of TARGET-AML and corresponding clinical information of pediatric and adolescent AML cases were downloaded from The Cancer Genome Atlas. Clinical information of 156 patients with gene expression data was extracted. The effects of PAX7 expression profiles on overall survival (OS) and event-free survival (EFS) were analyzed.Results: Positive expression of PAX7 indicated shorter OS and EFS, especially in patients older than 14 years. Furthermore, positive PAX7 expression also predicted shorter OS and EFS in intermediate- and low-risk group patients, compared to patients with negative PAX7 expression. In addition, patients who have received allogenic hematopoietic stem cell transplantation (allo-HSCT) in the first complete remission had better outcome than those who did not receive HSCT.Conclusions: Positive PAX7 expression in pediatric and adolescent AML patients indicates a poor outcome. Hence, the detection of PAX7 expression profiles is helpful for further stratification of intermediate- and low-risk groups.

[Relationship of Cystic Fibrosis Transmembrane Conductance Regulator Expression with Clinical Features and Prognosis in Patients with Acute Leukemia].

OBJECTIVE: To investigate the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in patients with acute leukemia and its relationship to clinical features and prognosis of acute leukemia. METHODS: A total of115 patients with acute leukemia were enrolled in the experimental group and 20 healthy individuals were used as control. Peripheral blood or bone marrow samples were collected, and mononuclear cells were isolated. The expression of CFTR protein was detected by Western blot. The relationships of CFTR protein expression to clinical features and prognosis was analyzed. RESULTS: The expression of CFTR protein was not detected in peripheral blood mononuclear cells of normal control, while it was positive in more than half of acute leukemias including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), but negative in the patients with acute promyelocytic leukemia (M3). In the patients with AML, there was no difference in peripheral white blood cells (WBC), peripheral blast cells, platelet and hemoglobin (HGB) between CFTR-positive and CFTR-negative patients. There was no relationship between the expression of CFTR protein and gene mutations such as NPM1, CEBPA, FLT3-ITD, and C-Kit. Complete remission (CR) rate after two course in CFTR-negative patients was slightly higher than that in positive patients. The survival time of CFTR-negative patients was little longer than that of positive patients, but the difference was not statistically significant. CONCLUSIONS: The expression of CFTR protein seems not associated with clinical features, treatment response and prognosis in the patients with acute leukemia.

Prognostic value of SRSF2 mutations in patients with de novo myelodysplastic syndromes: A meta-analysis.

BACKGROUND: The recent application of gene-sequencing technology has identified many new somatic mutations in patients with myelodysplastic syndromes (MDS). Among them, serine and arginine rich splicing factor 2 (SRSF2) mutations belonging to the RNA splicing pathway were of interest. Many studies have already reported the potential prognostic value of SRSF2 mutations in MDS patients, with controversial results. Therefore, a meta-analysis was performed to investigate their prognostic impact on MDS. METHODS: Databases, including PubMed, Embase and the Cochrane Library, were searched for relevant studies published up to 14 October 2016. Overall survival (OS) was selected as the primary endpoint, and acute myeloid leukemia (AML) transformation was the secondary endpoint. We extracted the corresponding hazard ratios (HRs) and their 95% confidence intervals (CIs) for OS and AML transformation from multivariate Cox proportional hazards models. The combined HRs with their 95% CIs were calculated using fixed or random effect models. RESULTS: A total of 10 cohort studies, covering 1864 patients with de novo MDS and 294 patients with SRSF2 mutations, were included in the final meta-analysis. Our results indicated that SRSF2 mutations had an adverse prognostic impact on OS (p<0.0001) and AML transformation (p = 0.0005) in the total population. Among the MDS patients with low or intermediate-1 risk defined according to the International Prognostic Scoring System (IPSS), SRSF2 mutations predicted a shorter OS (p = 0.009) and were more likely to transform to AML (p = 0.007). CONCLUSIONS: This meta-analysis indicates an independent, adverse prognostic impact of SRSF2 mutations on OS and AML transformation in patients with de novo MDS. This also applies to the subgroup of low- or intermediate-1-IPSS risk MDS. The identification of mutations in SRSF2 can improve current risk stratification and help make treatment decisions.

[The Roles of Glut5 in Imatinib Resistance in the Ph+ Acute Lymphoblastic Leukemia Cell].

OBJECTIVES: To explore the possible roles of glucose transport 5 (Glut5) in imatinib resistance in the Ph+ acute lymphoblastic leukemia cell (Ph+ ALL). METHODS: The gene chip technique was used to detect different gene expression between Ph+ ALL cell line SUP-B15/R (imatinib resistant cell line) and SUP-B15/S (imatinib sensitive cell line), the gene of solute carrier family 2 member 5 (SLC2A5) and its coded protein Glut5 were screened out and were reconfirmed by qPCR and Western blot assay. The imatinib half maximal inhibitory concentration (IC50) to SUP-B15/S cells with or without fructose treatment was further detected by MTT assay, simultaneously signal pathway gene was detected by qPCR assay. RESULTS: Metabolism related gene SLC2A5 was screened out with gene chip technique and the Western blot assay and qPCR confirmed the high expression of SLC2A5 gene and its coded protein Glut5 in SUP-B15/R cells. IC50 values of imatinib to SUP-B15/S cells after treatment with 25 µmol/L fructose were increased from (44.50±2.38) µmol/L to (64.71±1.69) µmol/L, in the meanwhile, PI3K and AKT mRNA level also increased in fructose treated SUP-B15/S cells compared to the control. CONCLUSIONS: High expression of SLC2A5 and Glut5 protein in SUP-B15/R cells leads to increased fructose absorption, and further activates PI3K/AKT pathway which cause the SUP-B15 cell resistance to imatinib.

High CFTR expression in Philadelphia chromosome-positive acute leukemia protects and maintains continuous activation of BCR-ABL and related signaling pathways in combination with PP2A.

Cystic fibrosis transmembrane conductance regulator (CFTR) is classified as an anion channel transporter of Cl- and HCO3-. Through interactions with its PDZ domain, CFTR is capable of regulating other proteins, such as protein phosphatase 2A (PP2A). The aberrant expression and mutation of CFTR have been observed in several tumor, but not in philadelphia chromosome-positive(Ph+) acute leukemia, including Ph+ B cell acute lymphoblastic leukemia(Ph+ B-ALL) and chronic myelogenous leukemia blast crisis phases (CML-BC). In this study, we demonstrated the mean expression level of CFTR in Ph+ acute leukemia cells was markedly higher than that in Ph- B-ALL and CML-chronic phase cells. CFTRinh-172, a classic CFTR inhibitor, down-regulated the expression of CFTR, p-BCR-ABL and classical Wnt/ß-catenin signaling in Ph+ acute leukemia cells, while imatinib had no effect on CFTR. Importantly, reduced efficacy of CFTRinh-172 was closely associated with elevated PP2A phosphatase activity. Furthermore, we confirmed an interaction between CFTR and the PP2AA subunit in K562 cells. In addition, we demonstrated CFTR and PP2AA interact in the cytosol, resulting in PP2A complex inactivation and increased degradation of PP2A substrates via the lysosomal/proteasome pathway. In conclusion, our results showed CFTR was highly expressed in Ph+ acute leukemia, which protected and maintained the continuous activation of BCR-ABL and the canonical Wnt/ß-catenin signaling pathway by decreasing PP2A phosphatase activity. According to this working model of the CFTR-PP2A-BCR-ABL axis, targeting the CFTR protein will activate PP2A and may offer a new treatment strategy for Ph+ acute leukemia, especially for patients exhibiting high levels of CFTR expression.

High-expressing cystic fibrosis transmembrane conductance regulator interacts with histone deacetylase 2 to promote the development of Ph+ leukemia through the HDAC2-mediated PTEN pathway.

The aberrant expression or mutation of CFTR has been shown to be involved in several tumors, but how mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains unclear. Here, we report the interaction between CFTR and HDAC2, and its involvement in the development of Ph+ leukemia. We first detected the physical interaction and co-localization of CFTR and HDAC2 in Ph+ leukemia cell lines. And treatment with CFTRinh-172, a CFTR inhibitor, reduced the expression of HDAC2 protein in K562 and SUP-B15 cells, which could be partially recovered by MG132, a proteasome inhibitor. Additionally, high expression levels of HDAC2 protein were observed in CFTR cDNA transfected HEK-293 and Ba/F3 cells. Next, we found that HDAC2 bound in the promoter region of the PTEN gene, and treatment with HDAC2 inhibitor or CFTRinh-172 resulted in an increase in PTEN mRNA and protein levels and a decrease in PDK1 and mTOR (downstream signaling of PTEN) activity. Finally, the MTT assay revealed that CFTRinh-172 could strengthen the anti-proliferation effect of HDAC2 inhibitor on Ph+ leukemia cells. Altogether, this study provides strong evidence that high-expression CFTR plays an important role in the development of Ph+ leukemia through the HDAC2-mediated PTEN pathway.

Niemann-Pick disease type C1(NPC1) is involved in resistance against imatinib in the imatinib-resistant Ph+ acute lymphoblastic leukemia cell line SUP-B15/RI.

Niemann-Pick disease type C1 (NPC1) is involved in cholesterol trafficking and may normally function as a transmembrane efflux pump. Previous studies showed that its dysfunction can lead to cholesterol and daunorubicin accumulation in the cytoplasmic endosomal/lysosomal system, lead to Niemann-Pick disease and resistance to anticancer drugs. In the present study, NPC1 was shown by microarray analysis to be more highly expressed in the Ph+ acute lymphoblastic leukemia cell line SUP-B15/RI, an imatinib-resistant variant of SUP-B15/S cells without bcr-abl gene mutation established in our lab. Further investigation revealed a defect in the functional capacity of the NPC1 protein demonstrated by filipin staining accompanied by a lower intracellular imatinib mesylate(IM) concentration by high-performance liquid chromatography in SUP-B15/RI compared with SUP-B15/S cells. Furthermore, U18666A, an inhibitor of NPC1 function, was used to block cholesterol trafficking to imitate the NPC1 defect in SUP-B15/S cells, leading to higher NPC1 expression, stronger filipin fluorescence, lower intracellular IM concentrations and greater resistance against IM. Samples from non-mutated relapsed Ph+ ALL patients also showed higher NPC1 expression compared with IM-sensitive patients. Our experiment may reveal a new mechanism of IM resistance in Ph+ ALL.

Ribavirin Inhibits the Activity of mTOR/eIF4E, ERK/Mnk1/eIF4E Signaling Pathway and Synergizes with Tyrosine Kinase Inhibitor Imatinib to Impair Bcr-Abl Mediated Proliferation and Apoptosis in Ph+ Leukemia.

The eukaryotic translation initiation factor 4E (eIF4E), which is the main composition factor of eIF4F translation initiation complex, influences the growth of tumor through modulating cap-dependent protein translation. Previous studies reported that ribavirin could suppress eIF4E-controlled translation and reduce the synthesis of onco-proteins. Here, we investigated the anti-leukemic effects of ribavirin alone or in combination with tyrosine kinase inhibitor imatinib in Philadelphia chromosome positive (Ph+) leukemia cell lines SUP-B15 (Ph+ acute lymphoblastic leukemia cell line, Ph+ ALL) and K562 (chronic myelogenous leukemia cell line, CML). Our results showed that ribavirin had anti-proliferation effect; it down-regulated the phosphorylation levels of Akt, mTOR, 4EBP1, and eIF4E proteins in the mTOR/eIF4E signaling pathway, and MEK, ERK, Mnk1 and eIF4E proteins in ERK/Mnk1/eIF4E signaling pathway; reduced the expression of Mcl-1 (a translation substrates of eIF4F translation initiation complex) at protein synthesis level not mRNA transcriptional level; and induced cell apoptosis in both SUP-B15 and K562. 7-Methyl-guanosine cap affinity assay further demonstrated that ribavirin remarkably increased the eIF4E binding to 4EBP1 and decreased the combination of eIF4E with eIF4G, consequently resulting in a major inhibition of eIF4F complex assembly. The combination of ribavirin with imatinib enhanced antileukemic effects mentioned above, indicating that two drugs have synergistic anti-leukemic effect. Consistent with the cell lines, similar results were observed in Ph+ acute lymphoblastic primary leukemic blasts; however, the anti-proliferative role of ribavirin in other types of acute primary leukemic blasts was not obvious, which indicated that the anti-leukemic effect of ribavirin was different in cell lineages.

The antileukemia effect of metformin in the Philadelphia chromosome-positive leukemia cell line and patient primary leukemia cell.

In recent years, there have been considerable research advances on the antileukemic mechanisms of the antidiabetic drug metformin. Our current studies have shown that metformin suppresses cell viability, induces apoptosis, and downregulates the mTORC1 signaling pathway both in the Ph+ALL cell line and primary blasts from Ph+ ALL patients, as well as the CML cell lines K562 (imatinib-sensitive) and K562R (imatinib-resistance). We have also shown that metformin activates the ERK pathway in Ph+ALL cells, SUP-B15, a side effect that can be overcome by U0126 (MEK1/2 inhibitor) or imatinib. Moreover, this activation of ERK signaling in SUP-B15 induces autophagy. Inhibition of the autophagic process by 3-MA, promoting the death of these cells, suggests that autophagy may be a cytoprotective factor in cell survival after metformin treatment. Finally, metformin is shown to potentiate the anticancer efficacy of imatinib in Ph+ALL and CML cells, resensitizing the CML imatinib-resistance cells to imatinib. Overall, our data suggest that metformin represents a promising and attractive agent for Ph+ALL or CML therapy.