Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

C1Q labels a highly aggressive macrophage-like leukemia population indicating extramedullary infiltration and relapse.

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor ß1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells.

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.

Transcriptome-wide subtyping of pediatric and adult T cell acute lymphoblastic leukemia in an international study of 707 cases.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of T cell progenitors, known to be a heterogeneous disease in pediatric and adult patients. Here we attempted to better understand the disease at the molecular level based on the transcriptomic landscape of 707 T-ALL patients (510 pediatric, 190 adult patients, and 7 with unknown age; 599 from published cohorts and 108 newly investigated). Leveraging the information of gene expression enabled us to identify 10 subtypes (G1­G10), including the previously undescribed one characterized by GATA3 mutations, with GATA3R276Q capable of affecting lymphocyte development in zebrafish. Through associating with T cell differentiation stages, we found that high expression of LYL1/LMO2/SPI1/HOXA (G1­G6) might represent the early T cell progenitor, pro/precortical/cortical stage with a relatively high age of disease onset, and lymphoblasts with TLX3/TLX1 high expression (G7­G8) could be blocked at the cortical/postcortical stage, while those with high expression of NKX2-1/TAL1/LMO1 (G9­G10) might correspond to cortical/postcortical/mature stages of T cell development. Notably, adult patients harbored more cooperative mutations among epigenetic regulators, and genes involved in JAK-STAT and RAS signaling pathways, with 44% of patients aged 40 y or above in G1 bearing DNMT3A/IDH2 mutations usually seen in acute myeloid leukemia, suggesting the nature of mixed phenotype acute leukemia.

Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL2012 trial).

As all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are widely accepted in treating acute promyelocytic leukemia (APL), deescalating toxicity becomes a research hotspot. Here, we evaluated whether chemotherapy could be replaced or reduced by ATO in APL patients at different risks. After achieving complete remission with ATRA-ATO-based induction therapy, patients were randomized (1:1) into ATO and non-ATO groups for consolidation: ATRA-ATO versus ATRA-anthracycline for low-/intermediate-risk patients, or ATRA-ATO-anthracycline versus ATRA-anthracycline-cytarabine for high-risk patients. The primary end point was to assess disease-free survival (DFS) at 3 y by a noninferiority margin of -5%; 855 patients were enrolled with a median follow-up of 54.9 mo, and 658 of 755 patients could be evaluated at 3 y. In the ATO group, 96.1% (319/332) achieved 3-y DFS, compared to 92.6% (302/326) in the non-ATO group. The difference was 3.45% (95% CI -0.07 to 6.97), confirming noninferiority (P < 0.001). Using the Kaplan-Meier method, the estimated 7-y DFS was 95.7% (95% CI 93.6 to 97.9) in ATO and 92.6% (95% CI 89.8 to 95.4) in non-ATO groups (P = 0.066). Concerning secondary end points, the 7-y cumulative incidence of relapse (CIR) was significantly lower in ATO (2.2% [95% CI 1.1 to 4.2]) than in non-ATO group (6.1% [95% CI 3.9 to 9.5], P = 0.011). In addition, grade 3 to 4 hematological toxicities were significantly reduced in the ATO group during consolidation. Hence, ATRA-ATO in both chemotherapy-replacing and -reducing settings in consolidation is not inferior to ATRA-chemotherapy (https://www.clinicaltrials.gov/, NCT01987297).

Effect of needling technique on diabetic gastroparesis and transmembrane protein 16A / 中国针灸

OBJECTIVE@#To observe the therapeutic effect of needling technique (acupuncture for regulating spleen and stomach) on diabetic gastroparesis (DGP), and to explore its possible mechanism.@*METHODS@#A total of 128 patients with DGP were randomized into an observation group (64 cases, 4 cases dropped off) and a control group (64 cases, 4 cases dropped off). On the basis of intervention on controlling blood glucose by western medication, needling technique was adopted at Zhongwan (CV 12), Zusanli (ST 36), Yinlingquan (SP 9), Xuehai (SP 10), Sanyinjiao (SP 6), Diji (SP 8), etc. in the observation group, once a day. Mosapride citrate dispersible tablet 5 mg was given orally 3 times a day in the control group. The treatment was given 6 times a week in the both groups, and totally 4-week treatment was required. Before and after treatment, the DGP symptom score, serum content of transmembrane protein 16A (ANO1) were observed, and the clinical therapeutic effect and the safety were evaluated in the both groups.@*RESULTS@#After treatment, the each subitem score (belching, abdominal distension, inappetence, nausea and vomiting, epigastric pain, abnormal defecation) and the total score of DGP symptom were decreased in both groups (<0.05), the subitem scores of belching, abdominal distension, inappetence, nausea and vomiting and the total score in the observation group were lower than those in the control group (<0.05). After treatment, the serum contents of transmembrane protein 16A were reduced in both groups (<0.05), and that in the observation group was lower than the control group (<0.05). The total effective rate was 86.7% (52/60) in the observation group, which was superior to 70.0% (42/60) in the control group (<0.05). Subcutaneous hematoma occurred in 5 cases in the observation group, which was improved after cold compress without other particular intervention.@*CONCLUSION@#The therapeutic effect of needling technique on improving symptoms in patients with diabetic gastroparesis is superior to mosapride citrate dispersible tablet, its mechanism may be related to alleviating the damage of interstitial cells of Cajal (ICC).

Anthracycline dose optimisation in patients with diffuse large B-cell lymphoma: a multicentre, phase 3, randomised, controlled trial.

BACKGROUND: Anthracycline dose optimisation in the treatment of diffuse large B-cell lymphoma has rarely been tested. We aimed to find out whether R-CEOP70 was non-inferior to R-CHOP50 with less cardiotoxicity, and whether R-CEOP90 had a superior efficacy to R-CHOP50 or R-CEOP70 with acceptable toxic effects. METHODS: In this multicentre, phase 3, randomised, controlled study (NHL-001), patients with newly diagnosed diffuse large B-cell lymphoma or follicular lymphoma grade 3B were enrolled from 20 centres of the Multicenter Hematology-Oncology Programs Evaluation System in China. Young patients (16-60 years) were randomly assigned 1:1:1 (block size of six) to six courses of R-CHOP50, R-CEOP70, or R-CEOP90, and older patients (61-80 years) were assigned 1:1 (block size of four) to R-CHOP50 or R-CEOP70. Patients were randomly assigned using computer-assisted permuted-block randomisation. Investigators and patients were not masked to treatment assignment. In the R-CHOP50 group, patients were given rituximab 375 mg/m2 intravenously on day 0, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1·4 mg/m2 (maximum dose 2 mg) intravenously on day 1, and prednisone 60 mg/m2 (maximum dose 100 mg) orally from day 1-5; in the R-CEOP70 group, epirubicin 70 mg/m2 replaced doxorubicin; and in the R-CEOP90 group, high dose epirubicin 90 mg/m2 replaced doxorubicin. All patients received two additional courses of rituximab 375 mg/m2 intravenously every 21 days. Consolidation radiotherapy was given to patients with bulky disease at diagnosis or residual disease at the end of treatment. The primary endpoint was 2-year progression-free survival. The non-inferiority margin for R-CEOP70 versus R-CHOP50 was defined by hazard ratio [HR] as the upper limit of its 95% CI being no greater than 1·50. Analysis of efficacy and safety were of the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01852435. FINDINGS: From May 15, 2013, to March 16, 2016, a total of 648 patients were enrolled, including 404 (62%) young patients (R-CHOP50 [n=135], R-CEOP70 [n=134], or R-CEOP90 [n=135]), and 244 (38%) older patients (R-CHOP50 [n=122] or R-CEOP70 [n=122]). Four patients were excluded from the study for consent withdrawal and one patient for misdiagnosis before treatment. The 2-year progression-free survival in the R-CHOP50 group was 72·5% (95% CI 66·6-77·6) and in the R-CEOP70 group was 72·4% ([66·5-77·5]; HR 1·00 [0·73-1·38]; p=0·99). The non-inferiority was met and adverse events were similar between the two groups. Fewer patients in the R-CEOP70 group (14 [13%] of 110) presented with over 10% decrease in left ventricular ejection fraction (LVEF) than those in the R-CHOP50 group (31 [29%] of 108) at 3 years after remission. For young patients, the 2-year progression-free survival in the R-CEOP90 group was 88·8% (82·1-93·1) and was significantly improved compared with the R-CHOP50 group (75·9% [67·7-82·3]; 0·44 [0·25-0·76]; p=0·0047) and the R-CEOP70 group (77·4% [69·4-83·7%]; 0·49 [0·27-0·86]; p=0·017). Grade 3-4 neutropenia occurred more frequently in the R-CEOP90 group (97 [72%] of 134) than in the R-CHOP50 group (87 [65%] of 133) and R-CEOP70 group (84 [63%] of 133) in young patients but without further increase of clinically significant infections. Fewer patients in the R-CEOP70 group (7 [11%] of 66) and in the R-CEOP90 group (10 [13%] of 79) presented with more than 10% decrease in LVEF than those in the R-CHOP50 group (17 [26%] of 66) at 3 years after remission. INTERPRETATION: R-CEOP70 could serve as an alternative regimen to R-CHOP50 with mild long-term cardiotoxicity. Young patients with diffuse large B-cell lymphoma might benefit from high-dose epirubicin. Epirubicin is an alternative drug to doxorubicin in regular R-CHOP with mild long-term cardiotoxicity. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program, Shanghai Commission of Science and Technology, Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support, Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine, Clinical Research Plan of Shanghai Hospital Development Center, and Chang Jiang Scholars Program.

2-Bromopalmitate targets retinoic acid receptor alpha and overcomes all-trans retinoic acid resistance of acute promyelocytic leukemia.

Fatty acid oxidation dependency of leukemia cells has been documented in recent studies. Pharmacologic inhibition of fatty acid oxidation, thereby, displays significant effects in suppressing leukemia. 2-Bromopalmitate, a palmitate analogue, was initially identified as an inhibitor of fatty acid oxidation, and recently recognized as an inhibitor of protein palmitoylation. However, the effects of 2-Bromopalmitate on leukemia and its cellular targets remain obscure. Herein, we discover in cultured cell lines, a transplantable mouse model, and primary blasts that 2-Bromopalmitate presents synergistic differentiation induction with all-trans retinoic acid in acute promyelocytic leukemia. Moreover, 2-Bromopalmitate overcomes all-trans retinoic acid resistance in all-trans retinoic acid-resistant cells and leukemic mice. Mechanistically, 2-Bromopalmitate covalently binds at cysteine 105 and cysteine 174 of retinoic acid receptor alpha (RARα) and stabilizes RARα protein in the presence of all-trans retinoic acid which is known to induce RARα degradation, leading to enhanced transcription of RARα-target genes. Mutation of both cysteines largely abrogates the synergistic effect of 2-Bromopalmitate on all-trans retinoic acid-induced differentiation, demonstrating that 2-Bromopalmitate promotes all-trans retinoic acid-induced differentiation through binding RARα. All-trans retinoic acid-based regimens including arsenic trioxide or chemotherapy, as preferred therapy for acute promyelocytic leukemia, induce adverse events and irreversible resistance. We expect that combining all-trans retinoic acid with 2-Bromopalmitate would be a promising therapeutic strategy for acute promyelocytic leukemia, especially for overcoming all-trans retinoic acid resistance of relapsed acute promyelocytic leukemia patients.

[Down-regulation of Annexinâ ¡Expression in NB4 Cells Induced by all-trans Retinoic Acid and Its Mechanisms].

OBJECTIVE: To explore effect of all-trans retinoic acid(ATRA) on annexin Ⅱ expression in NB4 cells and to analyze the luciferase activity of annexinⅡ promoter in condition of ATRA-induced treatment. METHODS: NB4 cells were cultured in vitro, the transcriptional or translational expression levels of Annexin Ⅱ in NB4 cells treated with 1 µmol/L ATRA at different time points were detected by RT-PCR or Western blot respectively. Annexin Ⅱ-promoter was constructed, the recombinant plasmids pGL4.15 -Annexin Ⅱ -promoter were transfected into NB4 cells with electroporation, and after being treated with 1 µmol/L ATRA for 24 hours the luciferase acttivity of Annexin Ⅱ promoter was determined by luciferase activity assay. RESULTS: The transcriptional expression of Annexin Ⅱ was down-regulated after 48 h. The translation expression of Annexin Ⅱ was slowly weakened after 24 h, and it was seriously reduced after 48 h. Further, Luciferase activity of AnnexinⅡ promoter in NB4 cells treated with 1 µmol/L ATRA was down-regulated, and showed a decreased tendency at indicated time points. CONCLUSION: All-trans retinoic acid can induce the down-regulation of AnnexinⅡ expression on the membrane of NB4 cells, and the activity of Annexin Ⅱpromoter is down-regulated too. This study provide a basis for further study of molecular mechanism.

Genomic Profiling of Adult and Pediatric B-cell Acute Lymphoblastic Leukemia.

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.

The Philadelphia chromosome in leukemogenesis.

The truncated chromosome 22 that results from the reciprocal translocation t(9;22)(q34;q11) is known as the Philadelphia chromosome (Ph) and is a hallmark of chronic myeloid leukemia (CML). In leukemia cells, Ph not only impairs the physiological signaling pathways but also disrupts genomic stability. This aberrant fusion gene encodes the breakpoint cluster region-proto-oncogene tyrosine-protein kinase (BCR-ABL1) oncogenic protein with persistently enhanced tyrosine kinase activity. The kinase activity is responsible for maintaining proliferation, inhibiting differentiation, and conferring resistance to cell death. During the progression of CML from the chronic phase to the accelerated phase and then to the blast phase, the expression patterns of different BCR-ABL1 transcripts vary. Each BCR-ABL1 transcript is present in a distinct leukemia phenotype, which predicts both response to therapy and clinical outcome. Besides CML, the Ph is found in acute lymphoblastic leukemia, acute myeloid leukemia, and mixed-phenotype acute leukemia. Here, we provide an overview of the clinical presentation and cellular biology of different phenotypes of Ph-positive leukemia and highlight key findings regarding leukemogenesis.

Exome sequencing identifies somatic mutations of DDX3X in natural killer/T-cell lymphoma.

Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL.

ATO/ATRA/anthracycline-chemotherapy sequential consolidation achieves long-term efficacy in primary acute promyelocytic leukemia.

The combination of all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3, ATO) has been effective in obtaining high clinical complete remission (CR) rates in acute promyelocytic leukemia (APL), but the long-term efficacy and safety among newly diagnosed APL patients are unclear. In this retrospective study, total 45 newly diagnosed APL patients received ATRA/chemotherapy combination regimen to induce remission. Among them, 43 patients (95.6%) achieved complete remission (CR) after induction therapy, followed by ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment with a median follow-up of 55 months. In these patients, the estimated overall survival (OS) and the relapse-free survival (RFS) were 94.4% ± 3.9% and 94.6 ± 3.7%, respectively. The toxicity profile was mild and reversible. No secondary carcinoma was observed. These results demonstrated the high efficacy and minimal toxicity of ATO/ATRA/anthracycline-based chemotherapy sequential consolidation treatment for newly diagnosed APL in long-term follow-up, suggesting a potential frontline therapy for APL.

A distinct glucose metabolism signature of acute myeloid leukemia with prognostic value.

Acute myeloid leukemia (AML) is a group of hematological malignancies with high heterogeneity. There is an increasing need to improve the risk stratification of AML patients, including those with normal cytogenetics, using molecular biomarkers. Here, we report a metabolomics study that identified a distinct glucose metabolism signature with 400 AML patients and 446 healthy controls. The glucose metabolism signature comprises a panel of 6 serum metabolite markers, which demonstrated prognostic value in cytogenetically normal AML patients. We generated a prognosis risk score (PRS) with 6 metabolite markers for each patient using principal component analysis. A low PRS was able to predict patients with poor survival independently of well-established markers. We further compared the gene expression patterns of AML blast cells between low and high PRS groups, which correlated well to the metabolic pathways involving the 6 metabolite markers, with enhanced glycolysis and tricarboxylic [corrected] acid cycle at gene expression level in low PRS group. In vitro results demonstrated enhanced glycolysis contributed to decreased sensitivity to antileukemic agent arabinofuranosyl cytidine (Ara-C), whereas inhibition of glycolysis suppressed AML cell proliferation and potentiated cytotoxicity of Ara-C. Our study provides strong evidence for the use of serum metabolites and metabolic pathways as novel prognostic markers and potential therapeutic targets for AML.

Prognostic significance of 2-hydroxyglutarate levels in acute myeloid leukemia in China.

The 2-hydroxyglutarate (2-HG) has been reported to result from mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes and to function as an "oncometabolite." To evaluate the clinical significance of serum 2-HG levels in hematologic malignancies, acute myeloid leukemia (AML) in particular, we analyzed this metabolite in distinct types of human leukemia and lymphoma and established the range of serum 2-HG in appropriate normal control individuals by using gas chromatograph-time-of-flight mass spectrometry. Aberrant serum 2-HG pattern was detected in the multicenter group of AML, with 62 of 367 (17%) patients having 2-HG levels above the cutoff value (2.01, log2-transformed from 4.03 µg/mL). IDH1/2 mutations occurred in 27 of 31 (87%) AML cases with very high 2-HG, but were observed only in 9 of 31 (29%) patients with moderately high 2-HG, suggesting other genetic or biochemical events may exist in causing 2-HG elevation. Indeed, glutamine-related metabolites exhibited a pattern in favor of 2-HG synthesis in the high 2-HG group. In AML patients with cytogenetically normal AML (n = 234), high 2-HG represented a negative prognostic factor in both overall survival and event-free survival. Univariate and multivariate analyses confirmed high serum 2-HG as a strong prognostic predictor independent of other clinical and molecular features. We also demonstrated distinct gene-expression/DNA methylation profiles in AML blasts with high 2-HG compared with those with normal ones, supporting a role that 2-HG plays in leukemogenesis.

[Down-regulation of transcription factor PU.1 via abnormal epigenetic modification in chronic myeloid leukemia].

OBJECTIVE: To investigate the underlying mechanism and clinical significance of PU.1 down-expression in chronic myeloid leukemia (CML) patients. METHODS: Different methylation status of PU.1 promoter region containing 20 CpG islands in normal individuals, CML chronic phase and blast crisis patients, complete cytogenetic remission patients after imatinib treatment, and blast crisis bone marrow K562 CML cells was detected by bisulfite sequencing. Semi-quantitative PCR was used to detect the PU.1 mRNA expression in normal controls, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells. Indirect immune fluorescence and Western blot were used to analyze the exprtession of PU.1 protein in normal individuals, CML chronic phase and blast crisis patients, and blast crisis bone marrow K562 CML cells. RESULTS: Aberrant methylation in the promoter region of transcription factor PU.1 was found in both CML chronic phase and blast crisis phase bone marrow cells, as well as in CML blast K562 cells. Down-expression of PU.1 mRNA and protein levels was found in above cells. No methylation in the promoter region of PU.1 was observed in normal individuals, and the PU.1 mRNA and protein expressions were not reduced at all. Furthermore, high methylation status of bone marrow cells was even observed in the CML patients who acquired complete cytogenetic remission. CONCLUSIONS: The results of our study indicate that the epigenetic modification of PU.1 in CML patients and K562 cell line might be responsible for the down-expression of PU.1. The data suggest that aberrant methylation of PU.1 plays a role in CML pathogenesis, therefore, it might serve as a useful biomarker and potential target in therapy for chronic myeloid leukemia.

[Establishment of stable subline of K562 cells overexpressing high mobility group B1 protein].

This study was aimed to establish a stable subline of K562 cells (K562-HMGB1) overexpressing HMGB1 protein and K562-HMGB1 sublines served as control, so as to provide a basis for exploring the role of hmgb1 gene in occurrence and development of leukemia and their mechanism. Protein-coding gene of hmgb1 was amplified by PCR with cDNA as template, which was synthesized by reverse transcription from total RNA extracted from U937 cells. The PCR-amplified hmgb1 gene was ligated into PMD18-T vector (PMD18-T-HMGB1 vector), and then transformed into E. coli strain DH5α. DH5α containing PMD18-T-HMGB1 vector were grown on LB agar plate supplemented with 100 µg/ml ampicillin overnight. The single ampicillin-selected DH5α clone was picked for culturing overnight and then harvested for plasmid extraction. The extracted plasmid was characterized to contain hmgb1 gene digested with the desired restriction enzymes of KpnI/XhoI. The correctness of hmgb1 sequence was confirmed with DNA sequencing. The insert of hmgb1 gene contained in PMD18-T-HMGB1 vector was cut out with restriction enzymes of KpnI/XhoI and then ligated into eukaryotic expression vector pcDNA3.1 to form pcDNA3.1-HMGB1 vector. 10µg of pcDNA3.1-HMGB1 or pcDNA3.1 plasmid was separately electroporated into K562 cells. At 48 hours after electroporation the cells were cultured with G418 at a final concentration of 800 µg/ml for over 2 weeks. Finally stably transfected sublines of K562 cells containing hmgb1 gene (K562-HMGB1), and of K562 containing pcDNA3.1 vector (K562-pcDNA3.1) served as a control, were obtained. The transcriptional or translational expression of hmgb1 gene was detected with RT-PCR or Western blot, respectively, to testify transfected efficiency and validity of stable subline of K562-HMGB1. The results indicated that the eukaryotic expression vector pcDNA3.1-HMGB1 plasmid was successfully constructed and was electroporated into K562 cells. The transcriptional or translational expression of hmgb1 gene in the stable subline of K562 cells containing hmgb1 gene was overexpressed. It indicated that stable subline of K562-HMGB1 cells was successfully established. It is concluded that the stable sublines of K562-HMGB1 cells or K562-pcDNA3.1 cells are successfully established, which provides a basis for exploring the roles and mechanisms of hmgb1 gene in leukemogenesis and development of leukemia.

[Screening drugs for regulating tissue factor gene expression].

This study was purposed to screen the drugs for regulating tissue factor (TF) gene expression through establishing stable cell line with luciferase gene having TF promoter transcription activity, so as to provide the basis for further studying the molecular mechanism of screened drugs. A series of luciferase reporter gene plasmids under control of 5'-truncated TF promoter (including -2174 bp - +128 bp, -684 bp - +128 bp, -247 bp - +128 bp and -201 bp - +128 bp) were constructed. The above plasmids were separately electroporated into U937 cells to establish stably transfected sublines. The function of stable cell line was testified by treatment with ATRA, the luciferase gene activity was analyzed by treating established cell line with bortezomib (BTZ) and CDA-II, and drugs for regulating TF gene expression were screened. The results indicated that the BTZ of 5 nmol/L could activate TF gene transcription activity, up-regulate the expression level of TF transcripts; CDA-II of 1 mg/ml could suppress TF gene transcription activity, down-regulate the expression level of TF transcripts. The functional analysis of TF promoter transcription revealed that the region of regulating TF promoter transcription activity by BTZ and CDA-II was between -201 to 0 bp. It is concluded that stable cell line U937 expressing luciferase activity of TF promoters is established, the novel drugs regulating TF gene expression are screened out by means of this established cell line. This study provides basis for screening the new drugs and further studying their molecular mechanisms.

Establishing SCID mouse models of B-cell non-Hodgkin's lymphoma.

BACKGROUND AND OBJECTIVE: Recently, the incidence of non-Hodgkin's lymphoma (NHL) is increasing, in which most are aggressive. It is limited for promoting the efficacy of conventional chemotherapy on NHL. In this study, mouse models of B-cell NHL were established for determining the efficacy and mechanisms of novel therapies. METHODS: Diffuse large B-cell lymphoma SU-DHL-4 cells and Burkitt's lymphoma Daudi cells were injected into SCID (severe combined immunodeficiency) mice through the tail veins to observe the presentations and requirements for establishing mouse models. The Daudi-cell lymphoma mice were divided into control group and rituximab group, and the latter received treatment of rituximab. The tumor onset and survival time of mice were investigated. RESULTS: The median onset time of SU-DHL-4-cell lymphoma in SCID mice was 39.5 days, which presented cachexia, weight loss, erect hair, tardiness and enlarged tumors in the abdomen, rump or pelvic limb, but without tumor cell infiltration in the liver, spleen or bone marrow. The median onset time of Daudi-cell lymphoma in SCID mice was 30.5 days, which were characterized by paralyzed lower limbs and died about 9.5 days after paralysation. Most organs such as the liver, kidney, spleen and bone marrow were infiltrated by a number of Daudi cells. After treatment of rituximab, Daudi cells presented typical characteristics of apoptosis. The median paralysis time and survival time of mice with Daudi-cell lymphoma were significantly longer in rituximab group than in control group (52.5 days vs. 30.5 days, 76.5 days vs. 40 days, p < 0.05). CONCLUSION: SCID mouse models of B-cell lymphoma can be successfully established with either SU-DHL-4 cells or Daudi cells.