Yun Woong Ko

Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable.

Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable

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.

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.

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.

Constitutive phosphorylation of FKHR transcription factor as a prognostic variable in acute myeloid leukemia

The transcription factor FKHR, which is controlled by Akt-PKB signaling, is involved in regulating cell cycle progression and cell death. In this study, the phosphorylation of FKHR was observed in 45 (73.8%) of 61 patients with acute myeloid leukemia (AML). The phosphorylation of Akt-PKB was found to be significantly associated with phospho-FKHR (P<0.001). Patients with phospho-FKHR had a significantly shorter overall survival than those without (P<0.05). In conclusion, the constitutive phosphorylation of FKHR was observed in the majority of AML, and the detection of phospho-FKHR might provide a new tool for identifying AML patients with an unfavorable outcome.

Elevated S-phase kinase-associated protein 2 protein expression in acute myelogenous leukemia: Its association with constitutive phosphorylation of phosphatase and tensin homologue protein and poor prognosis

Purpose: The F-box protein S-phase kinase-associated protein 2 (Skp2) positively regulates the G1-S phase transition by controlling the stability of several G1 regulators, such as p27Kip1. However, the clinical significance of Skp2 in patients with acute myelogenous leukemia (AML) remains unknown. Experimental Design: We examined the clinical and biological significance of Skp2 expression in AML and evaluated the relationship between Skp2 and p27Kip1 expression and phosphatase and tensin homologue (PTEN) phosphorylation. Results: Western blot analysis showed that high Skp2 expression was observed in 57 (57.6%) cases and significantly correlated with unfavorable cytogenetics (P = 0.035) but not with age, white blood cell count, serum lactic dehydrogenase level, and the French-American-British subtype. An inverse correlation was not observed between Skp2 and p27Kip1 expression. However, p27Kip1 protein was preferentially localized to cytoplasm in the high-Skp2-expression group. The cytoplasmic to nuclear ratio of p27Kip1 expression was significantly correlated with the levels of Skp2 expression (P < 0.001). The frequency of PTEN phosphorylation was significantly higher in the high-Skp2-expression group compared with the low- Skp2-expression group (P = 0.035). The Skp2 overexpression was significantly associated with shorter disease-free survival and overall survival (P = 0.0386 and P = 0.0369, respectively). Multivariate analysis showed that Skp2 expression was an independent prognostic factor both in the disease-free survival and overall survival. Conclusion: These findings suggest that Skp2 expression is an independent marker for a poor prognosis in AML. The presence of a positive correlation between Skp2 and phosphorylated PTEN suggests that an aberration in the PTEN/Skp2 signaling pathway might be operating in AML.

Difference in the expression of Fas/Fas-ligand and the lymphocyte subset reconstitution according to the occurrence of acute GVHD

Acute graft-versus-host disease (aGVHD) remains a major barrier to a wider application of allogeneic bone marrow transplantation (BMT). Although this complication is mainly dependent on donor-derived T lymphocytes, very little information is available concerning the mechanism of lethality. In this study, we investigated both the expression of Fas/Fas-ligand (FasL) and lymphocyte subset reconstitution in patients who underwent HLA-matched related allogeneic BMT (n = 16) and normal donors (n = 10), and several distinct features were observed. First, the reconstitutions of CD3+ and CD56+ cells were different between the aGVHD+ and aGVHD− group. In particular, the percentage of CD3−CD56+ cells was significantly decreased in patients with aGVHD (P < 0.01). second, the expansion of cd8+ (P = 0.01) and CD8+ CD28− T cells (P = 0.03) was a characteristic finding in patients with aGVHD. Finally, we found that the percentages of Fas+CD8+, Fas+HLA-DR+ and FasL+ CD8+ cells were significantly increased. Fas antigen was highly coexpressed on most of the lymphocyte subsets, especially on CD8+ cells (P < 0.01), and also, significantly higher coexpression of fasl on cd8+ cells was found in patients with aGVHD (P < 0.01). in summary, an increase in the percentage of cd8+ cells which express Fas and its ligand in patients with aGVHD after BMT points to a possible role for the Fas/FasL pathway in the effector phase of aGVHD.

AC133 antigen as a prognostic factor in acute leukemia

AC133 is a novel 5-transmembrane antigen present on a CD34((bright)) subset of human hematopoietic stem cells (HSCs) and it is also expressed on the subset of CD34 positive (CD34(+)) leukemias. But the clinical significance of AC133 expression on leukemic blasts is not yet known. We investigated the expression of AC133 antigen on blast cells of acute leukemia. Forty-one cases of acute leukemia were examined for expression of AC133, CD34, and other antigens using multicolor flow-cytometry. Samples were considered positive if at least 20% of the cells specifically stained with monoclonal antibodies (MoAbs) revealed a higher fluorescence intensity compared to cells of corresponding negative control samples (=20% cut-off level). 14/36 (38.9%) acute myelogenous leukemia (AML) samples and 6/20 (30%) acute lymphoblastic leukemia (ALL) samples were positive for AC133, the difference was not significant. All AC133 positive (AC133(+)) leukemias expressed CD34, whereas 13 of 33 CD34(+) leukemias were negative for AC133, and AC133(+)/CD34(-) leukemia was not found. Expression rates of CD31, CD62L, CD62E, CD105 and CD144 were significantly higher in AC133(+) leukemia compared to those of AC133(-) leukemia (P=0.045, P<0.001, P<0.001, P<0.001, P=0.003, respectively), but bcl-2, CXCR-1, CXCR4, VLA-4, CD106 expression rates were not significantly different between AC133(+) and AC133(-) leukemias. None of the clinical prognostic markers such as age, hemogram, lactate dehydrogenase, and chromosomal aberration were significantly different between AC133(+) and AC133(-) leukemias. CR rates of AC133(+) AML and AC133(-) AML were not significantly different, although there was a trend toward higher CR rates in AC133(-) AML (18/22[81.8%] AC133(-) AML versus 9/14[64.3%] AC133(+) AML), but the 1-year relapse rate of AC133(+) AML was significantly higher than that of AC133(-) AML (8/9 (88.9%) versus 7/19 (36.8%), P=0.016). Median disease-free survival (DFS) times of AC133(+) and AC133(-) AML were significantly different (11 and 18 months, respectively, P=0.006), although overall survival (OS) times were not significantly different (AC133(+) 15 months versus AC133(-) 20 months, respectively, P=0.06). Similar results regarding clinical outcomes were found when AC133(+)/CD34(+) and AC133(-)/CD34(+) were analyzed separately, but the difference did not attain statistical significance. In ALL, 9/11 (81.8%) AC133(-) and 2/4 (50%) AC133(+) cases achieved CR, but the difference was not significant. Four of 11 AC133(-) ALL (36.4%) and 2 of 3 AC133(+) ALL (66.7%) relapsed within 1 year. In survival analysis, median DFS time and OS time of the AC133(+) group were 7 and 18 months, respectively, and these were not significantly different from those of the AC133(-) group (median DFS 15, OS 22 months, respectively). Our results demonstrate that AC133 expression in AML blasts is associated with poor clinical outcomes in terms of higher early relapse and shorter disease-free survival, suggesting that the AC133 antigen might provide the prognostic stratification of acute leukemia. However, to verify the effect of AC133 expression on the therapeutic outcomes of adult acute leukemia, further study including more cases is needed.