Yoo Hong Min

Protein kinase CK2α as an unfavorable prognostic marker and novel therapeutic target in acute myeloid leukemia

Introduction: Protein kinase CK2 is implicated in cellular proliferation and transformation. However, the clinical and biological significances of CK2 have not been elucidated in acute myeloid leukemia (AML). Experimental Design: We evaluated the biological significances of catalytic subunit of CK2 (CK2α) expression in leukemia cell lines and primary leukemic blasts obtained from AML patients. Results: In this study, the expression of CK2α was elevated in a substantial proportion of AML. In AML patients with normal karyotype, the disease-free survival and overall survival rates were significantly lower in the CK2α-high compared with the CK2α-low AML cases (P = 0.0252 and P = 0.0392, respectively). An induced overexpression of CK2α increased the levels of Ser473 phosphorylated (p)-Akt/protein kinase B (PKB), p-PDK1, pFKHR, p-BAD, Bcl-2, Bcl-xL, Mcl-1, and XIAP. Treatment of U937 cell line and primary AML blasts with selective CK2 inhibitor, tetrabromobenzotriazole or apigenin, reduced the levels of these molecules in a dose-dependent manner. CK2α small interfering RNA treatment also resulted in a down-regulation of p-Akt/PKB and Bcl-2 in U937 cells. Apigenin-induced cell death was preferentially observed in the CK2α-high leukemia cell lines, HL-60 and NB4, which was accompanied by cytoplasmic release of SMAC/DIABLO and proteolytic cleavage of procaspase-9, procaspase-3, procaspase-8, and poly(ADP)ribose polymerase. An induced overexpression of CK2α potentially enhanced the sensitivity of U937 cells to the apigenin-induced cell death. Apigenin-induced cell death was significantly higher in CK2α-high AML compared with CK2α-low AML (P < 0.0001) or normal bone marrow samples (P < 0.0001). Conclusion: These findings strongly suggest protein kinase CK2α as an unfavorable prognostic marker and novel therapeutic target in AML.

Treatment of high-risk acute myelogenous leukaemia by myeloablative chemoradiotherapy followed by co-infusion of T cell-depleted haematopoietic stem cells and culture-expanded marrow mesenchymal stem cells from a related donor with one fully mismatched human leucocyte antigen haplotype

Summary.  A 20‐year‐old woman with high‐risk acute myelogenous leukaemia was transplanted with granulocyte colony stimulating factor (G‐CSF)‐mobilized peripheral blood CD34+ haematopoietic stem cells and bone‐marrow‐derived mesenchymal stem cells (MSC) from her human leucocyte antigen haplotype‐mismatched father after myeloablative conditioning therapy. The patient engrafted rapidly and had no acute or chronic graft‐versus‐host disease. Since transplantation, the patient has shown an enduring trilineage haematological complete response without any evidence of leukaemia relapse at 31 months. We suggest that MSC can be used effectively for genetically haploidentical haematopoietic stem cell transplantation for acute leukaemia.

Cytoplasmic Mislocalization of p27Kip1 Protein Is Associated with Constitutive Phosphorylation of Akt or Protein Kinase B and Poor Prognosis in Acute Myelogenous Leukemia

Cyclin-dependent kinase inhibitor p27Kip1 functions at the nuclear level by binding to cyclin E/cyclin-dependent kinase-2. It was shown that Akt or protein kinase B (Akt/PKB)-dependent phosphorylation of p27Kip1 led to the cytoplasmic mislocalization of p27Kip1, suggesting the potential abrogation of its activity. Here, we evaluated the localization of p27Kip1 protein in leukemic blasts in relation to Akt/PKB phosphorylation and clinical outcomes in acute myelogenous leukemia (AML). Western blot analysis of the nuclear and cytoplasmic fractions revealed a heterogenous localization pattern of p27Kip1 in AML. Cytoplasmic mislocalization of p27Kip1 was significantly associated with the constitutive serine473 Akt/PKB phosphorylation in AML cells (P < 0.05). Transfection of U937 cells with an expression construct encoding the constitutively active form of Akt/PKB resulted in a remarkable increase in the levels of cytoplasmic p27Kip1. Whereas the transfection of U937 cells with a construct encoding dominant-negative Akt/PKB resulted in a recovery of nuclear localization of p27Kip1. Both the disease-free survival and overall survival are significantly shorter in AML cases with high cytoplasmic to nuclear ratio of p27Kip1 localization compared with the cases with low cytoplasmic to nuclear ratio (P = 0.0353, P = 0.0023, respectively). Multivariate analysis indicated that the cytoplasmic to nuclear ratio of p27Kip1 localization was an independent prognostic variable for both disease-free survival and overall survival (P = 0.043, P = 0.008, respectively). These findings additionally extend our understanding of the role of p27Kip1 in AML, and buttress the case of p27Kip1 mislocalization as a prognostic indicator and Akt/PKB/p27Kip1 pathway as a ready target for antileukemia therapy.

Phosphatase and tensin homologue phosphorylation in the C-terminal regulatory domain is frequently observed in acute myeloid leukaemia and associated with poor clinical outcome

Summary. Phosphorylation of PTEN (phosphatase and tensin homologue) affects PTEN protein stability and function. In this study, phosphorylated PTEN (pPTEN) was observed in 45 (73·8%) of 61 cases with acute myeloid leukaemia (AML). Phosphorylation of Akt and its downstream molecules [FKHR; Forkhead (Drosophila) homologue 1; and GSK‐3β; glycogen synthase kinase 3 beta] was significantly associated with pPTEN (P < 0·001). The complete remission rates were not different with respect to pPTEN, but overall survival was significantly shorter in patients with pPTEN (P < 0·05). Constitutive PTEN phosphorylation may add insight into the molecular pathogenesis of AML, and may be a new parameter for an unfavourable outcome.

Weekly rituximab followed by monthly rituximab treatment for steroid-refractory chronic graft-versus-host disease: results from a prospective, multicenter, phase II study

Background Since it was suggested that B cells play a role in the pathogenesis of chronic graft-versus-host disease, rituximab, an anti-CD20 monoclonal antibody targeting B cells, has been shown to be effective in steroid-refractory, chronic graft-versus-host disease. However, most of the data were from small numbers of patients or retrospective analyses. We, therefore, conducted a multicenter phase II study to confirm the efficacy of this treatment strategy that targets B cells. Design and Methods We diagnosed and evaluated chronic graft-versus-host disease according to the National Institute of Health criteria for clinical trials on this condition. The treatment consisted of weekly intravenous infusions of rituximab for 4 weeks followed by monthly rituximab for 4 months. We evaluated the patients’ responses and monitored their disease activity until their final visit, which was on day 365. We also assessed the patients’ subsequent quality of life and serum levels of B-cell-activating factor of the tumor necrosis factor family. Results Among 37 patients enrolled (median age, 29 years; range 8–57 years), 32 patients responded to rituximab with 8 complete and 24 partial responses. Twenty-one patients maintained their response for 1 year, so their steroid treatment was discontinued or its dose reduced (21/37, or 56.8%), and their scores representing quality of life were improved although these changes were not statistically significant. The responses were better for clinical manifestations of the skin, oral cavity and musculoskeletal system (response rate, 71.4–100%) than for other organs. However, infectious complications and primary disease relapse accounted for the majority of treatment failure. The pre-treatment serum level of B cell-activating factor of the tumor necrosis factor family was not associated with better treatment outcome (P=0.147). Conclusions Rituximab could improve clinical responses and quality of life of patients with steroid-refractory chronic graft-versus-host disease, although such patients may need active prophylaxis against infection.

Expression of Fas antigen in acute myeloid leukaemia is associated with therapeutic response to chemotherapy

Flow cytometric immunofluorescent analysis was used to assess Fas antigen (CD95) expression in blasts obtained from the bone marrow of 30 patients with acute myeloid leukaemia. The percentage of positive cells in each sample was highly variable. Fas antigen expression did not correlate with age, FAB subtype, white blood cell counts, or CD34 expression. Low expression of Fas was associated with a low complete remission rate after induction chemotherapy (62.5% in cases with <20% positive cells v 92.9% in cases with ≥ 20% positive cells, P<0.01). The main cause for not achieving remission was resistant disease. Our results suggest that the quantitation of Fas expression can be predictive of treatment outcome in acute myeloid leukaemia.

Resveratrol alters microRNA expression profiles in A549 human non-small cell lung cancer cells

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.

Comparative analysis between azacitidine and decitabine for the treatment of myelodysplastic syndromes

The present study aimed to directly compare the efficacy and safety of azacitidine and decitabine in patients with myelodysplastic syndromes (MDS). We compared the overall response rate (ORR) (complete responses, partial responses, marrow complete responses, and haematological improvements), overall survival (OS), event‐free survival (EFS), time to leukaemic transformation, and adverse outcomes between azacitidine and decitabine. To minimize the effects of treatment selection bias in this observational study, adjustments were made using the propensity‐score matching method. Among 300 patients, 203 were treated with azacitidine and 97 with decitabine. Propensity‐score matching yielded 97 patient pairs. In the propensity‐matched cohort, there were no significant differences between the azacitidine and decitabine groups regarding ORR (44% vs. 52%), OS (26 vs. 22·9 months), EFS (7·7 vs. 7·0 months), and rate of leukaemic transformation (16% vs. 22% at 1 year). In patients ≥65 years of age, survival was significantly better in the azacitidine group (P = 0·017). Patients who received decitabine experienced more frequent episodes of grade 3 or 4 cytopenia and infectious episodes. We found that azacitidine and decitabine showed comparable efficacy. Among patients ≥65 years of age, survival was significantly better in the azacitidine group (ClinicalTrials.gov Identifier: NCT01409070).

Complete Remission Status before Autologous Stem Cell Transplantation Is an Important Prognostic Factor in Patients with Multiple Myeloma Undergoing Upfront Single Autologous Transplantation

Upfront high-dose myeloablative chemotherapy followed by a single autologous stem cell transplantation (ASCT) is the standard therapy for patients under the age of 65 years with newly diagnosed multiple myeloma (MM). Because disease status after induction chemotherapy is variable, we evaluated the prognostic effect of disease status before ASCT, especially in patients who were initially chemosensitive. We retrospectively analyzed the initially chemosensitive MM patients (> or = partial remission [PR]) enrolled in the Korean Multiple Myeloma Working Party Web-based registration system (www.myeloma.or.kr). Between November 1996 and January 2007, 197 MM patients (median age 53 years) were treated with induction chemotherapy followed by a single ASCT. All patients received peripheral blood stem cell (PBSC) support after conditioning with melphalan (Mel) alone. We considered those patients with no detectable M-protein regardless of the result of immunofixation to be in complete remission (CR) in this study. The median follow-up times were 29.2 months (range, 5.4 to 103.8 months) from the day of diagnosis and 22.4 months (range, 0.4 to 96.0 months) from the day of ASCT. Before ASCT, 63 patients (32%) were in CR and 134 (68%) were in partial remission (PR). The patients in CR had significantly longer overall survival (OS) from the day of ASCT compared with those in PR (P = .0015). Among the patients who received induction chemotherapy with vincristine, adriamycin, and dexamethasone (n = 162), the same difference in OS was seen between those in CR and those in PR before ASCT (P = .0016). CR after ASCT also predicted longer OS (P = .0135); however, patients with continued CR after ASCT had significantly higher OS after ASCT compared with patient with induced CR after ASCT who were in PR before ASCT (P = .0178). Multivariate analysis indicated that remission status pre-ASCT (CR vs PR) is a significant prognostic factor for predicting OS after ASCT (P = .012, Cox proportional hazard analysis; odds ratio = 2.83; 95% confidence interval = 1.25 to 6.37). We conclude that patients with MM who are in CR before ASCT have a better OS than those in PR before ASCT. Continued CR after ASCT may be an important prognostic factor as well. Our findings suggest that the development of more effective induction regimens, including novel antimyeloma agents to improve initial response, should be pursued to enhance clinical benefits post-ASCT.

Apicidin potentiates the imatinib-induced apoptosis of Bcr–Abl-positive human leukaemia cells by enhancing the activation of mitochondria-dependent caspase cascades

Apicidin, a histone deacetylase inhibitor, is a novel cyclic tetrapeptide with potent antiproliferative activity against various cancer cells. We examined whether apicidin potentiates the imatinib‐induced apoptosis of Bcr–Abl‐positive human leukaemia cells. In K562 cells, the co‐administration of minimally toxic concentrations of imatinib and apicidin (imatinib/apicidin) for 48 h produced a marked increase in mitochondrial damage, processing of caspase cascades and apoptosis. Similar results were observed in leukaemic blasts obtained from patients with chronic myeloid leukaemia in blast crisis. Imatinib/apicidin co‐treatment for 48 h resulted in a near complete loss of the full‐length XIAP (X‐linked inhibitor of apoptosis) protein, with a corresponding increase in the 29‐kDa XIAP cleavage product. Both the degradation of XIAP and increased release of second mitochondria‐derived activator of caspase/direct IAP‐binding protein with low pI (Smac/DIABLO) into the cytosol were abrogated by pretreatment with the caspase‐3 inhibitor DEVD‐CHO. Imatinib/apicidin co‐treatment for 48 h produced a prominent decrease in Bcr–Abl protein levels in a caspase‐dependent manner. In summary, these data indicate that apicidin potentiates the imatinib‐induced apoptosis of Bcr–Abl‐positive leukaemia cells through the enhanced activation of the mitochondria‐dependent caspase cascades, accompanied by caspase‐dependent downregulation of Bcr–Abl and XIAP. These findings generate a rationale for further investigation of apicidin and imatinib as a potential therapeutic strategy in Bcr–Abl‐positive leukaemias.