Acute lymphoblastic leukemia (ALL) samples exhibit an activated PI3K/Akt pathway, which suggests a general role of Akt in the development of leukemia. We have previously used western blot analysis to show that the catalytic topoisomerase (topo) inhibitor, 3EZ, 20Ac-ingenol, induced DNA damage response (DDR), which activated ATR, downregulated p-Akt through upregulation of PTEN level, and led to cell cycle arrest or apoptosis. In this study, we used ATR or PTEN siRNA and observed that the specific cell arrest and apoptosis of BALL-1 cells in DDR caused by 3EZ, 20Ac-ingenol was dependant on activation of ATR and downregulation of nuclear p-Akt through upregulation of PTEN. Moreover, some B cell lymphomas among ALLs overexpress cyclin D1. The DDR induced during the S-phase with 3EZ, 20Ac-ingenol treatment was increased by the intra S-phase checkpoint response that was triggered by the loss of nuclear cyclin D1 regulation in BALL-1 cells overexpressing cyclin D1. Although topo 1 catalytic inhibitors induce a decatenation checkpoint and subsequent G2/M phase arrest, the decatenation checkpoint caused by 3EZ, 20Ac-ingenol induced apoptosis only in the BALL-1 cells that accumulated cyclin D1.
Drug resistance to tyrosine kinase inhibitors (TKIs) is currently a clinical problem of chronic myelogenous leukemia (CML). Bcr-Abl protein depletion is considered as a way to overcome drug resistance to TKIs. In our study, Chk1 inhibitors, AZD7762 and MK-8776, had strong antitumor effects on CML cell line KBM5 and imatinib-resistant form KBM5T315I. Moreover, Chk1 inhibitors showed a strong cytotoxic effect on leukemia cells from primary CML and imatinib-resistance CML patients, but low cytotoxic effect on normal human mononuclear cells. Then, we found that Chk1 inhibitors induced apoptosis and increased DNA damage in CML cell lines with the degradation of the Bcr-Abl protein. Using the proteasome inhibitor and an immunoprecipitation assay, we found that Chk1 inhibitors triggered the degradation of Bcr-Abl through ubiquitination, which is depending on E3 ubiquitin ligase CHIP. At last, MK-8776 showed a significant tumor-suppressive effect of KBM5T315I cell in xenograft tumor models. Taking together, these findings suggest that targeting Chk1 may overcome TKIs resistance for the treatment of CML.
The present study explored the effects of Forkhead box Q1 (FOXQ1) on cell proliferation, cell cycle and apoptosis via the Sonic hedgehog (Shh) pathway in Natural killer/T-cell lymphoma (NKTCL). Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect FOXQ1 expression in 117 NKTCL patients and 120 healthy controls. Additionally, FOXQ1 expression in NKTCL cell lines (HANK-1, NK-92, SNK-6, SNT-8 and YT) was determined by western blotting and qRT-PCR. SNK-6 cells were transfected with FOXQ1-shRNA or Shh pathway inhibitor Cyclopamine/recombinant protein Shh. Cell counting kit-8 (CCK-8) and 5-bromo-2-deoxy-uridine (BrdU) incorporation assays were conducted to detect cell proliferation, flow cytometry was used to determine the cell cycle and cell apoptosis, and western blotting was used to detect protein expression. FOXQ1 expression was higher in NKTCL patients than in healthy controls, which was related to Ann Arbor stage, bone marrow involvement and the 5 year survival rate in NKTCL patients. Moreover, FOXQ1 expression, pathological type, Ann Arbor stage, B symptom and bone marrow involvement were independent risk factors in NKTCL. Shh pathway-related proteins were down-regulated after transfection of SNK-6 cells with FOXQ1-shRNA. Additionally, SNK-6 cell proliferation was greatly reduced, the cell cycle was blocked at the G0/G1 phase, and the expression of CyclinD1 and CyclinE was markedly decreased, while an increase in cell apoptosis with elevated Bcl-2-associated X protein (Bax) and Caspase-3 and reduced B-cell lymphoma/leukemia-2 (Bcl-2) were also observed. However, no significant alterations were observed between the FOXQ1-shRNA + Shh and Blank groups. The inhibition of FOXQ1 restricted NKTCL cell proliferation and growth but induced apoptosis via blocking the Shh signaling pathway.
Organic anion-transporting polypeptide 1A2 (OATP1A2) is involved in the cellular uptake of methotrexate (MTX). Genetic variation in solute carrier organic anion transporter family member 1A2 (SLCO1A2, the coding gene of OATP1A2) has important implications for the elimination of MTX. We investigated the association between a microRNA (miRNA) binding site polymorphism (rs4149009 G>A) in the 3'-untranslated region (3'-UTR) of SLCO1A2 with the serum MTX concentrations in Chinese children with acute lymphoblastic leukemia (ALL). Genotyping for SLCO1A2 rs4149009 G>A in 141 children with ALL was performed using the Sequenom MassARRAY system. Serum MTX concentrations were determined by fluorescence polarization immunoassay. The percentages of MTX level >= 1 mu mol/L at 42 h were compared among the AA, GA, and GG genotypes. The minor allele frequency observed in this study (33.0%) was significantly lower than that in the African samples reported in the 1000 Genomes Project (57.4%, P=0.00). The incidence rate of delayed MTX elimination was significantly higher in patients with the GG genotype (23.1%) compared with the AA genotype (0.0%, P=0.03). Bioinformatics tools predicted that the rs4149009 A allele would disrupt the putative binding sites of hsa-miR324-3p and hsa-miR-1913. These results indicate that the rs4149009 G>A polymorphism might affect MTX pharmacokinetics by interfering with the function of miRNAs.
Porcine anti-human lymphocyte immunoglobulin (pALG) and rabbit anti-human thymocyte immunoglobulin (rATG) are the only two ATGs for severe aplastic anemia (SAA) treatment in China. 148 treatment-naive SAA patients who received ATG combined with cyclosporine A (CsA) therapy were analysed retrospectively. The patients were divided into a pALG group (n=114) and a rATG group (n=34). After three months, the pALG and rATG groups had an overall response (OR) of 65.8% and 44.1%, respectively (P=0.023); after six months, the OR reached 74.6% and 64.7%, respectively (P=0.361). The pALG group had markedly better time-related efficacy than the rATG group (P=0.03). The overall survival (OS) and event-free survival (EFS) between groups had no significant difference (P>0.1). The pALG and rATG groups did not significantly differ in terms of recurrence (8.8% vs. 5.9%, P=0.734) or PNH clonal transformation (5.3% vs. 2.9%, P=1.000), whereas a significant difference was found in the incidence of MDS/AML transformation (2.6% vs. 11.8%, P=0.049). We found that pALG achieved a better time-related efficacy than rATG for the treatment of SAA; nonetheless, no significant difference in the OS or EFS of pALG compared with rATG.
Bone marrow (BM) failure is often seen in leukemia patients, indicating an abnormal hematopoietic process. However, hematopoiesis in leukemic milieus is largely unknown. In the present study, we utilized one of the most frequent leukemogenic translocations MLL-AF9 to induce leukemia and investigated the hematopoiesis and the activity of hematopoietic stem and progenitor cells (HSPCs) in a leukemic milieu. We found that the phenotypes of the non-leukemic population in leukemic BM were drastically different than normal BM, including blockage of differentiation and a drastically reduced Lin-/Sca+/c-kit+ (LSK) population that contains all HSPCs in leukemic BM. Further, transplantation assays demonstrated that stem cell function of HSPCs from leukemic BM was significantly compromised. Intriguingly, BM from a patient-derived xenograft leukemia model and from immunocompromised mice transplanted with murine MLL-AF9 cells, showed comparable percentage of hematopoietic stem cells (HSCs) to normal controls, indicating that an immunocompetent microenvironment is critical for leukemia-induced loss of HSPCs. Mechanistically, we found that the non-leukemic cells from leukemic BM possessed a more inflammatory profile than either leukemic cells or normal BM counterparts. Co-culturing or co-transplantation with non-leukemic cells from leukemic BM impaired the stem cell function of normal HSPCs in vitro and in vivo respectively, suggesting that the highly inflammatory non-leukemic population in leukemic BM not only is functionally abnormal but displayed a 'leukemia-like' characteristic. Finally, we tested the effect of the anti-inflammation drug diclofenac on leukemia mice. However, no phenotypic changes of HSPCs were observed upon diclofenac treatment due to only mild repression of inflammatory genes by diclofenac, further indicating that inflammation is a powerful negative regulator of HSPCs. Together, our results suggest that leukemia impairs normal hematopoiesis and inflammation as well as immune cells play a critical role in leukemia-induced BM failure.
3q26.2/EVI1 rearrangements resulting in EVI1 overexpression play an important role in leukemogenesis and are associated with treatment resistance and a poorer prognosis in patients with acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia and BCR-ABL negative myeloproliferative neoplasms. In this study, we aim to explore the clinicopathological features of myelodysplastic/myeloproliferative (MDS/MPN) neoplasms with 3q26.2/EVI1 rearrangements and determine the potential impact of these cytogenetic abnormalities on treatment response and survival. The study group included 12 cases of MDS/MPN with 3q26.2 rearrangements detected by conventional karyotyping. There were 7 men and 5 women with a median age of 67 years (range, 51-79 years) at time of initial MDS/MPN diagnosis. Ten cases were classified as chronic myelomonocytic leukemia (CMML) and 2 were MDS/MPN, unclassifiable. Among CMML cases, 5 (50%) were proliferative type and 5 (50%) were dysplastic type. Based on blast counts, these 10 CMML were: CMML-0 (n=2), CMML-1 (n=3), and CMML-2 (n=5). Eleven (92%) patients had 3q26 rearrangements at the initial diagnosis. Inv(3)(q21q26.2) was most common, identified in 7(58%) patients, followed by t(3; 21)(q26.2; q22) in 2 patients and 1 patient each with t(3; 3)(q21; q26.2), t(2; 3)(p21; q26-27), and t(3; 6)(q26.2; q26). Six (50%) patients had 3q26.2 rearrangements as a sole cytogenetic abnormality and 6 (50%) patients had additional cytogenetic abnormalities. Molecular studies revealed DNMT3A mutations in all 3 patients assessed and RAS mutations in 2 of 8 (25%) patients. No mutations in ASXL1 (n=3), TET2 (n=3), FLT3 ITD/D835 (n=10), and CEBPA (n=7) were detected. Most patients received hypomethylating agent based chemotherapy. The median follow-up was 11.5 months (range, 1.5-24 months) and at time of last follow-up, 11 (92%) died with a median survival of 13.4 months (range, 1.5-24 months). The only patient alive had a relatively short follow-up of 2.4 months and showed disease progression at the last visit. In conclusion, 3q26.2/EVI1 rearrangements are a rare event and usually present at time of initial diagnosis in MDS/MPN. The presence of 3q26.2/EVI1 rearrangements in MDS/MPN is associated with rapid disease progression, poor response to treatment, and a poor prognosis.
Genomic alterations underlying chemotherapy resistance remains poorly characterized in pediatric acute myeloid leukemia (AML). In this study, we used whole exome sequencing to identify gene mutations associated with chemo-resistance in 44 pediatric AML patients. We identified previously unreported mutations involving epigenetic regulators such as KDM5C, SRIT6, CHD4, and PRPF6 in pediatric AML patients. Despite low prevalence in general pediatric AML, mutations involving epigenetic regulators including splicing factors, were collectively enriched as a group in primary chemo-resistance AML patients. In addition, clonal evolution analysis of secondary chemo-resistance AML patients reveals dominant clone at diagnosis could survive several course of intensified chemotherapy. And gain of new mutations in genes such as MVP, TCF3, SS18, and BCL10, may contribute to chemo-resistance at relapse. These results provide novel insights into the genetic basis of treatment failure in pediatric AML.
The aim of our study was to evaluate the impact of oral arsenic (the realgar-indigo naturalis formula, RIF) and all-trans retinoic acid (ATRA) on coagulopathy in acute promyelocytic leukemia (APL) compared with intravenous arsenic trioxide (ATO) and ATRA during induction. Mitoxantrone was added to all the patients at a dose of 1.4 mg/m(2) per day for 5-7 days. D-dimer levels, prothrombin time (PT), fibrinogen (Fbg) levels and the platelet count were comparably analyzed among 83 newly diagnosed APL patients treated with RIF (n = 45) or with ATO (n = 38). Since induction therapy with RIF and ATRA, the median levels of Fbg, PT and platelets were recovered to the normal range within 4 days, 10 days and 28 days, respectively. The last day of platelet and plasma transfusion was day 12 (range: 0-24 days) and day 3 (range: 0-27 days), respectively. Among the 42 patients with a disseminated intravascular coagulation (DIC) score = 4, the consumption of transfused platelets was less in the RIF group than that in the ATO group (P = 0.037). In the 17 patients with a DIC score< 4, prompt recovery of Fbg levels (P = 0.028) was observed in the RIF group compared with that in the ATO group (P = 0.401). RIF and ATO showed similar effects on the recovery of coagulopathy in APL patients. RIF had a potential beneficial effect in accelerating the recovery of thrombocytopenia and hypofibrinogenemia for subclinical DIC patients.
A morphometric analysis was performed on aspirated clots of bone marrow to identify the presence of micromegakaryocytes after immune staining with a monoclonal antibody raised against CD41. Quantitative and qualitative abnormalities of micromegakaryocytes were assessed based on both standard staining and CD41 immune staining. Micromegakaryocytes were largely detected in bone marrow from patients with myelodysplastic syndrome (MDS), while almost no micromegakaryocytes were present in aplastic anemia. CD41 immune staining clearly improved the efficiency of micromegakaryocyte detection under any conditions, showing strong potential as a tool for the auxiliary diagnosis of MDS and differentiation of MDS from pancytopenia, particularly aplastic anemia(AA).
Background: Decitabine is widely used in the treatment of acute myeloid leukemia (AML) in elderly patients. Low-dose Vitamin C has also been indicated to induce DNA demethylation M the cellular level. However, little is known whether low-dose Vitamin C has a synergistic effect with decitabine in clinic. Methods: The effect of combined low-dose Vitamin C and decitabine on cell proliferation, the cell cycle, apoptosis and the expression level and activity of TET2 was investigated in HL60 and NB4 human leukemic cells. Additionally, we analyzed the clinical outcomes of 73 elderly AML patients who received A-DCAG (intravenous Vitamin C [IVC] plus DCAG [n = 39]) or DCAG (n = 34) treatment. Results: We found that low-dose Vitamin C and decitabine has a synergistic efficacy on proliferation, apoptosis, TET2 expression and activity, compared to drug-alone treatment in HL60 and NB4 cell lines in vitro. In clinic, feasibility and safety evaluations revealed that patients who received A-DCAG regimen have a higher complete remission (CR) rate than those who received the DCAG regimen (79.92% vs. 44.11%; P = 0.004) after one cycle of chemotherapy. The median overall survival (OS) was better in the A-DCAG group compared with the DCAG group (15.3 months vs. 9.3 months, P = 0.039). Patients with adverse cytogenetics did benefit from CR. There was no clinically significant additional toxicity observed with the addition of IVC. Conclusion: On the basis of these results, the addition of IVC M low doses to DCAG appeared to improve CR and prolong OS, compared with DCAG, in elderly patients with AML.
Regulatory T cells (Tregs) characterized by the transcription factor forkhead box P3 (FoxP3) are crucial for maintaining immune tolerance and preventing autoimmunity. However, FoxP3 does not function alone and Helios is considered a potential candidate for defining Treg subsets. In this study, we investigated the expression and function of Helios for identifying Tregs in childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL). Our results demonstrated that patients with pre-B ALL had a higher percentage of Helios(+) FoxP3(+) CD4(+) Tregs. And there was a positive correlation between the expression of Helios and the suppressive function of Tregs, the risk gradation of ALL. Helios in combination with CD4 and FoxP3 may be an effective way to detect functional Tregs in pre-B ALL by promoting the secretion of transforming growth factor (TGF)-beta 1. Furthermore, Helios(+) Tregs could regulate angiogenesis in the BM niche of pre-B ALL via the VEGFA/VEGFR2 pathway. We also found Helios(+) Tregs decreased apoptosis rate of nalm-6 cells by up-regulating the expression of antiapoptosis protein Bcl-2. In summary, these data strongly imply the physiological importance of Helios expression in Tregs, and suggest that the manipulation of Helios may serve as a novel strategy for cancer immunotherapy.
Objective: To investigate the frequencies and biological characteristics of CD25 positive hematopoietic stem cells (HSC) in myelodysplastic syndromes. Methods: The expression of CD25 on HSC in bone marrow derived from patients with untreated MDS patients, untreated AML patients and normal controls were accessed by flow cytometry (FCM). The correlation analysis was done between CD25(+) HSC and clinical parameters in MDS patients. Results: The expression of CD25 on HSC (CD34(+) CD38-cells) in MDS patients (28.81%) was significantly higher than that in normal controls (9.41%, P=0.020), which similar to that in AML patients (32.54%, P=0.410). The CD25 expression on HSC was positively correlated with the CD123 expression on HSC (r=0.602, P=0.008). The expression of CD25 on HSC in high-risk MDS group (53.27%) based on IPSS score was significantly higher than that in low-risk MDS group (18.66%, P=0.003). In MDS patients, CD25(+) HSC were negatively correlated with the counts of neutrophils (r=-0.684, P=0.002) and platelets (r=-0.561, P=0.015), while positively correlated with the percentage of blasts in bone marrow (r=0.596, P=0.009). The CD25 expression on erythroblasts had a significant positive correlation with red blood cell counts in MDS patients (r=0.536, P=0.012). Conclusions: CD25 was over-expressed on HSC in MDS patients, especially in high-risk MDS patients. Increased CD25(+) HSC was correlated with progression of MDS. Low-expression of CD25 on erythroblasts might correlate with anemia in MDS patients. CD25 could be a specific marker of LSC in MDS, and could involve in the mechanisms of development and progression of MDS.
Tyrosine kinase inhibitors have achieved unprecedented efficacy in the treatment of chronic myeloid leukemia (CML); however, imatinib resistance has emerged as a major problem in the clinic. Because the overexpression of BCR-ABL1 critically contributes to CML pathogenesis and drug resistance, targeting the regulation of BCR-ABL1 gene expression may be an alternative therapeutic strategy. In this study, we found that the transcriptional repressor MXD1 showed low expression in CML patients and was negatively correlated with BCR-ABL1. Overexpression of MXD1 markedly inhibited the proliferation of K562 cells and sensitized the imatinib-resistant K562/G01 cell line to imatinib, with decreased BCR-ABL1 mRNA and protein expression. Further investigation using reporter gene analysis showed that MXD1 significantly inhibited the transcriptional activity of the BCR-ABL1 gene promoter. Taken together, these data show that MXD1 functions as a negative regulator of BCR-ABL1 expression and subsequently inhibits proliferation and sensitizes CML cells to imatinib treatment.