Background Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by the clonal expansion of malignant progenitor cells and impaired cell differentiation. GL-V9, a nature compound derived from wogonin, has shown superior anti-tumor potential by inhibiting cancer cell growth. Azacitidine (AZA), a DNA hypomethylation agent, has demonstrated therapeutic efficacy against AML. In this study, we investigated the anti-leukemia effect and potential mechanism of AZA combined with GL-V9 in AML cells, aiming to provide evidence for future clinical treatment. Methods We employed Cell Counting Kit-8 (CCK-8) to assess cell viability, Annexin-V/PI staining followed by flow cytometry analysis to measure apoptosis. CalcuSyn analysis was used to evaluate the synergistic effect. RNA-seq was performed in U937 cells and differentially expressed genes (DEGs) were identified and subjected to KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. RT-qPCR and Western Blot were used to examine gene expression. Results Our results demonstrated significant cell proliferation arrest in U937 and MV4-11 cells treated with the combination of AZA and GL-V9, compared to the single drug ( Fig. 1A&B). CalcuSyn analysis showed a strong synergistic effect for the combination ( Fig. 1A&B). Similar results were also found in primary AML cells derived from 2 AML patients [patient 1 exhibited t(6;11) (q27;q23) chromosomal translocation, while Patient 2 presented with secondary AML, transformed from CMML (Chronic Myelomonocytic Leukemia) and featured ASXL1 and SRSF2 mutations] ( Fig. 1C&D). Moreover, AZA plus GL-V9 induced a substantial increase in cell apoptosis in U937 and MV4-11 cells compared to the single drug ( Fig. 1E&F). Consistently, the AZA+GL-V9 combination led to a notable increase in the protein level of Bax, BAD, BIM, and a decrease in BCL2 ( Fig. 1G). These data indicated the combination of AZA with GL-V9 has synergistic anti-leukemia effects in AML. To understand the underlying mechanisms of this synergy, we performed RNA-seq analysis in U937 cells, identifying 1385 and 586 DEGs (|log 2FC|≥2.0, P<0.05) upon AZA or GL-V9 treatment, respectively ( Fig. 2A). The mTOR signaling pathway exhibits prominent enrichment in the KEGG analysis of the overlapping DEGs between AZA and GL-V9 ( Fig. 2B). DDIT4, a negative regulator of mTOR emerged as one of the top DEGs in response to both AZA and GL-V9 treatment. Importantly, the AZA+GL-V9 treatment exhibited a higher expression level of DDIT4 compared to either drug alone and suppressed the phosphorylation of mTOR ( Fig. 2C). Additionally, the DDIT4 expression level was significantly decreased in AML patients, which derived from a public database (TCGA-AML, GSE13159)] ( Fig. 2D) and Zhongda Hospital (Nanjing, China) compared to the healthy controls ( Fig. 2E). To be noticed, high DDIT4 expression was associated with extended overall survival (P=0.021) ( Fig. 2F) and relapse-free survival (P=0.015) ( Fig. 2G), indicating DDIT4 may act as a tumor suppressor in AML. These findings suggested that the combination may exert the anti-leukemia effect by targeting the DDIT4/mTOR signaling pathway, as summarized in Fig. 2H. Conclusions Our study provides novel evidence of the synergistic effect on cell growth arrest and apoptosis treated with a new nature compound derived from wogonin combined with AZA in AML. Furthermore, we identified the mechanism underlying the synergy through targeting of DDIT4/mTOR signaling. These results offer preliminary support for the potential application of this combination therapy in treating AML patients.

Acute myeloid leukemia (AML) is a high-mortality malignancy with poor outcomes. Azacitidine induces cell death and demonstrates treatment effectiveness against AML. Selinexor (KPT-330) exhibited significant benefits in combination with typical induction treatment for AML patients. Here, we explore the antitumor effect of KPT-330 combined with AZA in AML through CCK-8, flow cytometry, RT-qPCR, western blot, and RNA-seq. Our results showed that KPT-330 combined with AZA synergistically reduced cell proliferation and induced apoptosis in AML primary cells and cell lines. Compared to the control, the KPT-330 plus AZA down-regulates the expression of XPO1, eIF4E, and c-MYC in AML. Moreover, the knockdown of c-MYC could sensitize the synergy of the combination on suppression of cell proliferation and promotion of apoptosis in AML. Moreover, the expression of XPO1 and eIF4E was elevated in AML patient cohorts, respectively. XPO1 and elF4E overexpression was associated with poor prognosis. In summary, KPT-330 with AZA exerted synergistic effects by suppressing XPO1/eIF4E/c-MYC signaling, which provided preclinical evidence for further clinical application of the novel combination in AML.

Background Homoharringtonine (HHT) is a plant cytotoxic alkaloid derived from the trees of the genus Cephalotaxus. HAG (HHT, low-dose cytarabine, and G-CSF), as a priming regimen, is utilized to treat acute myeloid leukemia (AML). Azacitidine (Aza), a DNA hypomethylation agent, has been a "backbone" for new combinational therapies. However, so far, it has not been well determined the efficacy and safety of adding Aza to HAG regimen. Methods This is a multi-center, single arm, phase 2 clinical trial done in 17 clinical institutions across China between Aug 2019 and Dec 2021 (ClinicalTrials.gov: NCT04248595). Induction therapy consisted of Aza (75mg/m2/d on days 1-7) was given in combination with the HAG regimen (Fig. 1A). Primary endpoints were complete remission (CR) or complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were overall survival (OS), relapse free survival (RFS), and adverse events (AEs). Mutation screening was conducted through next generation sequencing (NGS) by targeting 58 frequently mutated genes. Results A total of 112 patients, including 72 newly diagnosed (ND) (56 de novo, 16 secondary AML [sAML]), and 40 relapsed/refractory (R/R) AML were enrolled. The CR/CRi was achieved in 79.2% (57/72) in the ND and 40.0% (16/40) in R/R AML, respectively (Fig. 1B). CR/CRi were achieved in 80.4% of de novo AML (45/56) and 75.0% of sAML (12/16), respectively. In addition, the median OS and RFS of ND AML patients were 22.8m (95%CI, 12.6 to not reached) and not reached, respectively (Fig. 1C&1D), which were significantly longer than the R/R group (median OS, 10.8m [95%CI, 9.5 to 12.7], P=0.0056; median RFS 5.21m [95%CI, 4.21 to 9.64], P=0.0051) (Fig. 1C&1D). Furthermore, 94.7% (18/19) of patients with favorable-risk reached CR/CRi (Fig. 1E), with median OS and RFS were both not reached (Fig. 1F&1G). 83.9% (26/31) of patients with intermediate-risk reached CR/CRi (Fig. 1E), and the median OS and median RFS were 22.8m (95%CI, 10.5 to not reached) (Fig. 1F) and 16.9m (95%CI, 4.64 to not reached) (Fig. 1G), respectively. Whereas in those with poor-risk, 59.1% (13/22) reached CR/CRi (Fig. 1E), with the median OS 17.3m (95%CI, 3.79 to not reached) (Fig. 1F) and median RFS not reached (Fig. 1G). In this study, infection was the most common non-hematological adverse event, presenting 58.0% (65/112). Common non-hematological AEs of grade 3 or higher includes: infection (38/112, 33.9%), hemorrhage (11/112, 9.82%), fatigue (6/112, 5.36%), hypokalemia (4/112, 3.57%), cardiac arrhythmia (2/112, 1.79%), and fever (2/112, 1.79%). The median duration of neutropenia and thrombocytopenia were 11 d (IQR, 7-19d) and 16d (IQR, 11-25d) among CR/CRi patients. No patients discontinued the induction therapy due to hematological or non-hematological toxicities. The early deaths within 4 weeks of the induction treatment occurred in 1.79% (2/112). Moreover, we detected 228 mutants involving 33 genes in 103 patients at the enrollment. The most frequently mutated genes were DNMT3A (25/103), TET2 (20/103), and NPM1 (18/103) (Fig. 1H), and mostly involved in DNA methylation (DNMT3A, IDH1/2, TET2; 48/103, 46.6%), followed by signal transducers (FLT3, NRAS, KRAS, KIT, PTPN11; 36/103, 35.0%), and transcription regulation (WT1, RUNX1, CEBPA, SETBP1, GATA2; 32/103, 28.2%). Upon Aza+HAG treatment, we observed the high CR/CRi rate in ND patients with mutated BCOR (100%,7/7), KIT (100%,3/3), IDH1 (100%,7/7), NPM1 (93.3%,14/15), ASXL1 (88.9%,8/9), DNMT3A (80. 0%,16/20), RUNX1 (75.0%,6/8), FLT3 (71.4%,5/7), and TET2 (71.4%,10/14) (Fig. 1I). We further observed the clearance of mutated genes after the induction therapy and found the variant allele fraction (VAF) of mutants was dramatically reduced among the CR/CRi patients. Specifically, VAF change of FLT3 mutant from 3 of 4 available patients reduced to <0.01%, with the other one, decreased by 91.9% (Fig. 1J); 4 out of 6 patients with IDH1 mutant reduced to <0.01%, with the other two, decreased 67.6% and 35.9%, respectively (Fig. 1K); 5 out of 6 patients with ASXL1 mutant reduced to <0.01%, with the other one, decreased by 33.4% (Fig. 1L); and 10 out of 14 patients with NPM1 mutant reduced to <0.01%, respectively (Fig. 1M). Conclusion This study demonstrated that the Aza+HAG regimen is a cost-effective first-line therapy with high efficacy and well tolerance for elderly/unfit AML, especially ND AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IntroductionThis study aims to evaluate the efficacy and safety of the novel combination of Aza and HIA as the frontline induction therapy in newly diagnosed AML patients eligible for intensive chemotherapy (IC) (registered on ClinicalTrials.gov, number NCT04248595).MethodsAza (75mg/m2/d on days1-5 subcutaneous) is administered in combination with HIA [HHT 2mg/m2/d on days 4-8 intravenous over 3 hours, idarubicin 6mg/m2/d on days 4-6 intravenous, and cytarabine 100mg/m2/d on days 4-10 intravenous]. The primary endpoint was complete remission (CR) or CR with incomplete blood count recovery (CRi). Secondary endpoints were overall survival (OS), relapse-free survival (RFS), and adverse events (AEs).ResultsA total of 20 AML patients (aged 18-70 years) were enrolled between Jan 2020 and Sep 2022. 95% (19/20) of patients achieved CR/CRi, and 89.5% (17/19) had undetectable MRD, in which 94.7% (18/19) reached CR/CRi, and 88.9% (16/18) obtained MRD negative after the 1st cycle of induction therapy. Median OS and RFS were both not reached during the follow-up. The estimated 2-year OS and RFS were 87.5% (95%CI, 58.6% to 96.7%) and 87.1% (95%CI, 57.3% to 96.6%), respectively. No patient discontinued the treatment for AEs.DiscussionThis study provides preliminary evidence for this novel combination therapy as the first-line induction therapy for young or older AML patients fit for IC.

Background Previous studies have reported that a combination of Homoharringtonine (HHT), a plant cytotoxic alkaloid derived from the trees of the genus Cephalotaxus, plus intensive chemotherapy (IC) improved the clinical responses of acute myeloid leukemia (AML). Azacitidine (Aza), a DNA hypomethylation agent, has been a "backbone" for novel combinational therapies. In this trial, we assessed the efficacy and safety of adding Aza to HIA/HDA regimen in previously untreated young or eligible for IC patients with AML. Methods This is a single-center, phase 2 trial (ClinicalTrials.gov: NCT04248595). Aza (75mg/m2/d on days1-5) administered in combination with HIA/HDA [HHT 2mg/m2/d on days 4-8, idarubicin 6mg/m2/d or daunorubicin 40 mg/m2/d on days 4-6, and cytarabine 100mg/m2/d on days 4-10] regimen for patients age<60 or eligible for IC (Fig.1A) The primary endpoint was the composite CR. Secondary endpoints were overall survival (OS), relapse-free survival (RFS), and adverse events (AEs). A leukemia targeted-exome-seq panel including 58 genes and leukemia 47 fusion gene panel were used for screening. Results Between Aug, 2019 to May, 2022, a total of 20 de novo AML patients were enrolled. The CR/CRi was achieved in 95.0% (19/20) (Fig. 1B). 94.7% (18/19) reached CR/CRi after the 1st cycle of induction therapy and the median time was 25.0d (IQR, 21.0 to 27.0). The median follow-up was 628 days (range, 107 to 905) and the median OS and RFS were not reached (Fig. 1C& 1D). The estimated 2-year OS and RFS were 87.4% (95%CI, 58.1% to 96.7%) (Fig. 1C) and 82.0% (95%CI, 53.1% to 94.0%), respectively (Fig. 1D). No survival difference was observed between the favorable and intermediate versus poor-risk groups in both OS and RFS (Fig. 1E& 1F). Meanwhile, the estimated 2-year OS and RFS possibility of the favorable and intermediate-risk group was 90.9% (95%CI, 50.8% to 98.7%) and 80.0% (95%CI, 20.4% to 96.9%), respectively (Fig. 1E& 1F). While the estimated 2-year OS and RFS possibility of the poor-risk group was 85.7% (95%CI, 33.4% to 97.9%), and 75.0% (95%CI, 12.8% to 96.1%), respectively (Fig. 1E& 1F). We detected 30 mutants in 17 of 58 leukemia driver genes at the timepoint of enrollment. The median mutation number/patient was 1.50 (range, 0-4), and 17 of 20 (85.0%) patients had more than one mutation (≥1) (Fig. 1G). The most frequently mutated genes were FLT3 (ITD/TKD) (20.0%), KIT (15.0%), NRAS (15.0%), and DNMT3A (15.0%) (Fig. 1G). More than half of the patients (11/20) harbored the mutated genes involved in signal transducers (FLT3, KRAS,NRAS, KIT, PTPN11, CSF3R; 11/20, 55.0%), and all these patients achieved CR/CRi (11/11, 100%) after induction therapy (Fig 1G). It is worth noticing that, we observed a high mutant's clearance with VAF (variant allele frequency) dramatical reduction (to <0.01%) (Fig. 1H) after the induction therapy, indicating that mutations involved in signal transducers were rapidly and significantly associated with high clinical response. We also identified 2 patients with MLL-AF9 and 4 patients with CBF-β-MYH11. Both patients with MLL-AF9 reached complete molecular remission (<0.1%) after induction. 3 of 4 patients with CBF-β -MYH11 reached complete molecular remission (<0.1%), and the expression level of the CBF-β -MYH11 fusion gene in 1 patient decreased over 1000-fold at the molecular level (512.16% to 0.28%) after 1 cycle of induction. The MRD negative was 84.2% (16/19) in patients who reached CR/CRi after the 1st cycle of induction therapy (Fig. 1I, 1J). Also, MRD were negative in all 3 patients with favorable risk, 77.8% with intermediate-risk, and 87.5% with poor-risk (Fig. 1K). Infection (grade 1-4) was the most common non-hematological AE (65.0%). Other AEs of grade 3 or higher included fatigue (5.0%), decreased appetite (5.0%), and hepatic dysfunction (5.0%). The most common grade 3-4 hematological AEs were neutropenia and thrombocytopenia. The median duration of neutropenia and thrombocytopenia were 12d (IQR, 11 to 15) and 14d (IQR, 12 to 16), respectively. No patients discontinued the induction treatment and no patient died within 8 weeks of the treatment. Conclusion This trial demonstrated that the adding of Aza to HIA/HDA regimen is an effective first-line therapy for previously untreated young or fit AML patients, with high efficacy and well tolerance. This study provides preliminary results for the clinical application of this novel combination therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BackgroundAcute myeloid leukemia (AML) remains one of the most common hematological malignancies, posing a serious challenge to human health. HSPA8 is a chaperone protein that facilitates proper protein folding. It contributes to various activities of cell function and also is associated with various types of cancers. To date, the role of HSPA8 in AML is still undetermined.MethodsIn this study, public datasets available from the TCGA (Cancer Genome Atlas) and GEO (Gene Expression Omnibus) were mined to discover the association between the expression of HSPA8 and clinical phenotypes of CN-AML. A series of bioinformatics analysis methods, including functional annotation and miRNA-mRNA regulation network analysis, were employed to investigate the role of HSPA8 in CN-AML.ResultsHSPA8 was highly expressed in the AML patients compared to the healthy controls. The high HSPA8 expression had lower overall survival (OS) rate than those with low HSPA8 expression. High expression of HSPA8 was also an independent prognostic factor for overall survival (OS) of CN-AML patients by multivariate analysis. The differential expressed genes (DEGs) associated with HSPA8 high expression were identified, and they were enriched PI3k-Akt signaling, cAMP signaling, calcium signaling pathway. HSPA8 high expression was also positively associated with micro-RNAs (hsa-mir-1269a, hsa-mir-508-3p, hsa-mir-203a), the micro-RNAs targeted genes (VSTM4, RHOB, HOBX7) and key known oncogenes (KLF5, RAN, and IDH1), and negatively associated with tumor suppressors (KLF12, PRKG1, TRPS1, NOTCH1, RORA).ConclusionsOur research revealed HSPA8 as a novel potential prognostic factor to predict the survival of CN-AML patients. Our data also revealed the possible carcinogenic mechanism and the complicated microRNA-mRNA network associated with the HSPA8 high expression in AML.

The aim of the present study was to investigate the synergistic effect of LY294002 (a PI3K inhibitor) and ABT199 (a BCL2 inhibitor) on the cell cycle in acute myeloid leukemia (AML). The optimal concentration and duration of combined LY294002 and ABT199 were determined in human erythroleukemia (K562), promyelocytic leukemia (HL60) and myeloid leukemia (KG1a) cell lines. The mRNA and protein expression levels of cell cycle‑related molecules, including S‑phase kinase‑associated protein 2 (Skp2), p27, BCL2, Bax, cleaved caspase 3 (caspase‑3) and caspase 9 (caspase‑9) were detected via reverse transcription‑quantitative PCR and western blot analysis, respectively. At the molecular level, LY294002 and ABT199 combination treatment significantly downregulated Skp2, Bcl2, procaspase‑3 and procaspase‑9 expression levels, but markedly upregulated p27, Bax, cleaved caspase‑3 and caspase‑9 expression levels in K562, HL‑60 and KG1a cells. The results of the present study demonstrated that LY294002 and ABT199 combination treatment may serve as a novel therapeutic strategy for AML.

Chronic lymphocytic leukaemia (CLL) is the most prevalent leukaemia and remains incurable. Mesenchymal stem cells (MSCs) can promote tumour progression by differentiating into cancer-associated fibroblasts (CAFs). However, the mechanisms by which tumour cells induce the transition of MSCs to CAFs are still largely undefined. Exosomes can regulate recipient cellular function by mediating intracellular communication. This study aimed to investigate whether CLL cells regulate the transition of bone marrow-derived MSCs (BM-MSCs) to CAFs via exosomal miR-146a delivery. The exosomes were isolated from CLL cell line MEC-1 (CLL-Exo) and then co-cultured with BM-MSCs. The expression of α-smooth muscle actin (α-SMA) and fibroblast-activated protein (FAP) were determined by immunofluorescence, quantitative real-time polymerase chain reaction and western blot. A luciferase reporter assay was performed to verify whether ubiquitin-specific peptidase 16 (USP16) was a target of miR-146a. CLL-Exo treatment up-regulated miR-146a and down-regulated expression of CAF markers (α-SMA and FAP) and USP16. The inducing effect of CLL-Exo on CAF marker expression was compromised when miR-146a expression was inhibited in CLL-Exo. USP16 was confirmed as a direct target of miR-146a and USP16 overexpression in BM-MSCs abrogated the CLL-Exo-mediated up-regulation of CAF markers. Collectively, CLL-Exo delivered miR-146a into BM-MSCs where miR-146a mediated transition of BM-MSCs into CAFs by targeting USP16.

OBJECTIVE:To investigate the mutation rate and distribution of Homo sapiens neuroblastoma RAS viral oncogene homolog (NRAS) gene in the patients with acute myeloid leukemia.METHODS:The genomic DNA of bone marrow was screened by polymerase chain reaction (PCR) and sequencing for NRAS mutations. At the same time, the mutations of ASXL1, DNMT3A, TET2, CEBPA, FLT3, IDH2, NPM1 and c-KIT genes were also detected to analyze the relation with NRAS mutations.RESULTS:A total of 11 NRAS mutations were found in 108 patients with initial acute myeloid leukemia and the mutation rate was 10.2%, including 6 cases of G12D, 3 cases of G13D, and 2 cases of G61K. In the mutation group, the peripheral blood leukocyte count was higher (P＜0.05), more likely to occur in the M4 subtype, and the M2 subtype was mutually exclusive (P＜0.05). Moreover, the mutant group was more likely to express CD13 than the non-mutation group (P＜0.05), while no statistic difference was found in age, gender, hemoglobin level, platelet count, lactate dehydrogenase level, bone marrow blast, cytogenetics, complete remission rate and overall survival (P＞0.05).CONCLUSIONS:The mutation of NRAS gene has no effect on the prognosis of AML patients.

OBJECTIVE:To explore the rate and distribution of Runt- related transcription factor 1 (RUNX1) gene mutations in patients with acute myeloid leukemia (AML) and the correlation of these mutations with the clinical characteristics and survival outcomes of the patients. METHODS:The genomic DNA extracted from the bone marrow of 158 patients with newly diagnosed AML for PCR amplification of RUNX1 gene and sequence analysis to identify the mutations. The mutations of ASXL1, DNMT3A, TET2, FLT3, CEBPA, NPM1, IDH2, NRAS and c-KIT genes were also examined to analyze their association with RUNX1 gene mutations. RESULTS:Among the 158 AML patients, 19 (12.0%) were found to have RUNX1 mutations in A166G (9 cases), A142T (6 cases) and A162L (4 cases). RUNX1 mutations were more frequent in elderly patients (P < 0.01) and in cases of AML subtypes M4 and M5, and were associated with more frequent CD36 and CD7 expression as compared with the wild type. RUNX1 mutations were more likely to occur in patients with normal karyotype or karyotypes associated with moderate prognostic risks, but the difference was not significant (P > 0.05). The patients with RUNX1 mutations had significantly lower complete remission (CR) rate and overall survival (OS) rate than those without the mutations (P < 0.05). RUNX1 mutations were not associated with gender, white blood cell count upon diagnosis, hemoglobin level, platelet count, bone marrow blast cell ratio or lactate dehydrogenase level (P > 0.05). CONCLUSIONS:RUNX1 gene mutations are associated with an adverse prognosis of patients with AML.

Myelodysplastic syndrome (MDS) is a heterogeneous clonal disease originated from hematopoietic stem cells. Epigenetic studies had demonstrated that DNA methylation and histone acetylation were abnormal in MDS. Azacitidine is an effective drug in the treatment of demethylation.

Objective: To explore the clinical significance of real-time quantitative reverse transcription PCR (RT-qPCR) in detecting T315I BCR- ABL mutants in leukemia patients. Methods: A total of 62 leukemia patients were enrolled in this study. Peripheral blood from the patients was analyzed to determine which BCR- ABL mutants had occurred in the leukemia patients using Sanger sequencing. RT-qPCR was then introduced to detect the BCR-ABL mutants in the leukemia patients, and the results were compared with the Sanger sequencing results in terms of accuracy. Results: The T315I (C944T) BCR-ABL mutant was the most common mutation in the chronic myelogenous leukemia (CML) patients. RT-qPCR demonstrated a complete concordance with Sanger sequencing in terms of detecting the BCR-ABL mutations. An ROC curve analysis indicated that the RT-qPCR method was of value in detecting the T315I BCR-ABL mutants, and the differences were statistically significant (P<0.05). Conclusion: The RT-qPCR approach is equally accurate in detecting the T315I mutation compared with conventional Sanger sequencing. In addition, RT-qPCR is easy to carry out, is less expensive, and is more non-specific, suggesting it will have broad prospects for application in the detection and monitoring of the T315I mutation in leukemia patients in the future.

The present study investigated the function of ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) in regulating the migration and proliferation of acute myeloid leukemia (AML) cells through Wnt/β-catenin signaling, and in arsenic trioxide (ATO) treatment. The migration and proliferation of multiple ROS1-silenced leukemic cell lines was assessed, and the expression levels of proteins associated with Wnt/β-catenin signaling were determined using western blot analysis. Compared with the AML control cells, ROS1-knockdown cells exhibited increased migration and proliferation, and the significant downregulation of β-catenin expression. Additionally, ROS1 knockdown sensitized AML cells to the effects of chemotherapeutic ATO. The results of the present study demonstrated that, in leukemic cell lines, ROS1 counteracted the effects of ATO on migration and proliferation, suggesting that ROS1 may be a potential therapeutic target in patients with AML undergoing ATO treatment. The results of the present study provided novel insight into the function of ATO and ROS1 in regulating AML progression.

OBJECTIVE To analyze the promoter methylation status, mRNA expression and clinical significance of DKK-3 and WIF-1 genes in patients with acute myeloid leukemia(AML). METHODS Methylation specific polymerase chain reaction (MS-PCR) mothod was carried out to detect DKK-3 and WIF-1 gene promoter methylation status in bone marrow specimen from 56 patients with AML and 20 patients with iron deficiency anaemia(IDA) as control; then the real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of DKK-3, WIF-1 gene and β -catenin in the above-mentioned specinens, and their relationship with the clinical features and survival time was analyzed. RESULTS The promoter methylation rate of DKK-3 and WIF-1 gene in AML patients were significantly higher than that in control group(χ2=15.330,P<0.001; χ2=17.371,P<0.001). There was no relationship between DKK-3 and WIF-1 gene promoter methylation rate and AML patient's sex, age, clinical typing. The relative expression of DKK-3 and WIF-1 gene mRNA in AML group were 0.840±0.320 and 0.792±0.313, which were lower than those in control group (1.134±0.392 and 1.047±0.334) respectively, the difference was statistically significant (t=3.415,P=0.000; t=3.070, P=0.003). The relative expression of β-catenin mRNA in AML bone marrow specimens in AML group was 0.756±0.304, which was higher than that in control group(0.342±0.105), the difference was statistically significant (t=5.943, P=0.001). The expression of DKK-3 and WIF-1 gene mRNA negatively correlated with β-catenin mRNA(r=-0.543; r=-0.562). Kaplan Meier survival curve analysis showed that overall survival time in AML patients with DKK-3 gene methylation was shorter than that in the AML patients with DKK-3 gene unmethylation(χ2=3.957, P=0.042). Futhermore, the orerall survival time in AML patients with WIF-1 gene methylation was also shorter than that in AML patients with WIF-1 gene unmethylation (χ2=4.520, P=0.029). CONCLUSION Wnt/β-catenin signaling pathway is abnormally activated in AML patients, the DKK-3 and WIF-1 gene promoter methylation may be involved in Wnt pathways activation and the pathogenesis of AML.

The positive regulatory domain 1 (PRDM1) exists as two isoforms: PRDM1α and PRDM1β. The former is frequently inactivated, while the latter is overexpressed in a subset of diffuse large B-cell lymphoma (DLBCL). To investigate the possible epigenetic alteration of PRDM1α and PRDM1β expression, the methylation of these two promoter isoforms was assessed in B lymphoma cell lines and DLBCL samples. Hypomethylation of PRDM1β CpG islands was preferentially detected in lymphoma cells. However, both high and low methylation of PRDM1α CpG islands was simultaneously observed in cases of DLBCL compared with the moderate methylation of non-tumor cases. CpG 16–21-specific high methylation was correlated with low expression of PRDM1α in PRDM1β-positive DLBCL samples. Three increased and one decreased miRNAs were significantly different between cases of DLBCL and non-tumor reactive hyperplasia. Thus, our results indicate that aberrant methylation silencing of PRDM1α and hypomethylation activation of PRDM1β are frequent events in DLBCL.

OBJECTIVE To investigate the expression of DNMT1 in laryngeal squamous cell carcinoma (LSCC) and its relationship with clinicopathological factors. METHOD Ninety-six cases of LSCC samples were detected by immunohistochemical staining to analyze the expression of DNMT1 protein, while 66 cases of them were detected by real-time PCR. And the relationship between the expression of DNMT1 mRNA and clinicopathologic factors was then analyzed. RESULT The expression of DNMT1 mRNA in LSCC were up-regulated (P < 0.05). The positive rate of DNMT1 protein expression in LSCC and in peri-cancer tissue were 100% (96/96) and 36% (8/22) respectively, which showed a significant difference (P < 0.05). DNMT1 mRNA expression wasn't correlated with patients' age, gender, T stage and lympha node metastasis, but was associated with smoking habit (P < 0.05, respectively). CONCLUSION DNMT1 may initiate the oncogenesis of LSCC by increasing expression, and smoking habit may induce the expression of DNMT1 gene.