Yan-Hua Wang

Lack of nucleophosmin mutation in patients with myelodysplastic syndrome and acute myeloid leukemia with chromosome 5 abnormalities

Nucleophosmin (NPM1) gene exon 12 mutations are frequently present in patients with acute myeloid leukemia (AML) with normal karyotype. The NPM1 gene is located on chromosome 5q35, which is often affected in myeloid malignancies including myelodysplastic syndrome (MDS). This suggests that the NPM1 gene is a one of the target genes affected by chromosome 5 abnormalities and play a role in the development of MDS. It has not been clarified whether MPM1 mutations are present in patients with MDS and AML with chromosome 5 abnormalities. Therefore, we carried out a mutational analysis on the NPM1 gene exon 12. NPM1 mutations were not detected in the 28 patients with MDS and AML with chromosome 5 abnormalities.

Activation of STAT5 confers imatinib resistance on leukemic cells through the transcription of TERT and MDR1.

We used two imatinib resistant cell lines, K562-ADM cells, which over-express P-glycoprotein (a product of the ABCB1 gene, more commonly known as MDR1), and K562-hTERT cells, which over-express the telomerase reverse transcriptase (TERT), as models to show that the acquisition of multidrug resistance in CML is associated with the enhanced phosphorylation of signal transducer and activator of transcription 5 (STAT5). The induction of P-glycoprotein expression that occurred in response to adriamycin treatment was accompanied by increased phosphorylation of BCR-ABL and STAT5, as well as increased telomerase protein expression. Intriguingly, a ChIP assay using an anti-STAT5 antibody revealed direct binding of STAT5 to the promoter regions of both the human TERT gene and the MDR1 gene in K562-ADM cells. Conversely, silencing of endogenous STAT5 expression by siRNA significantly reduced both the expression of P-glycoprotein and telomerase activity and resulted in the recovery of the imatinib sensitivity of K562-ADM cells. These findings indicate a critical role for STAT5 in the induction of P-glycoprotein and in the modulation of telomerase activity in drug-resistant CML cells. Furthermore, primary leukemic cells obtained from patients in blast crisis showed increased levels of phospho-STAT5, P-glycoprotein and telomerase. In contrast, none of these proteins were detectable in the cells obtained from patients in the chronic phase. Together, these findings indicate a novel mechanism that contributes toward multidrug resistance involving STAT5 as a sensor for cytotoxic drugs in CML patients.

Inhibition of PRAME expression causes cell cycle arrest and apoptosis in leukemic cells

The preferentially expressed antigen of melanoma (PRAME) is known as a tumor-associated antigen, but its function in leukemia remains unclear. We investigated the function with small interfering RNA (siRNA)-induced knockdown of PRAME in a K562 cell line. After PRAME siRNA transfection, proliferation was suppressed and cell cycle analysis showed G(0)/G(1) arrest, followed by apoptosis. PRAME siRNA-treated cells also showed changes in the genes affecting erythroid differentiation. We examined the PRAME expression levels and the S phase population of 32 acute leukemia patients at the time of diagnosis and relapse. An increase of the S phase population was accompanied by an increase of PRAME expression at relapse. Our results suggest that PRAME plays an important role in disease progression in acute leukemia.

Aberrant methylation of the RIZ1 gene in myelodysplastic syndrome and acute myeloid leukemia.

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p=0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p=0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.

Significance of lung resistance-related protein in the clinical outcome of acute leukaemic patients with reference to P-glycoprotein

Lung resistance‐related protein (LRP) overexpression in leukaemic blast cells from acute leukaemia patients and the effect of LRP or P‐glycoprotein (P‐gp) on the clinical outcome of acute leukaemia were investigated individually by dividing patients into four groups. The complete remission rate of group I (LRP and P‐gp both negative) was 81·7%, group II (only LRP positive) 87·5%, group III (only P‐gp positive) 87·1% and group IV (LRP and P‐gp both positive) 40·0%. There were no statistical differences between group I and groups II or III, but a significant difference was observed between groups I, II or III and group IV. Median overall survival in group IV was significantly shorter (4·6 months) than in groups I, II or III, although no significant differences were observed between group I and groups II or III (18·9, 20·5 and 31·8 months). There was a tendency for disease‐free survival in group III to be longer than that in groups I, II or IV. The reasons for these findings are discussed. Our present results indicate that the co‐existence of LRP and P‐gp strongly influenced the effectiveness of induction chemotherapy and long‐term prognosis, whereas the isolated presence of LRP or P‐gp did not.

Level of DNA topoisomerase IIα mRNA predicts the treatment response of relapsed acute leukemic patients

The DNA topoisomerase IIalpha (Topo IIalpha) is known as a target enzyme for many chemotherapeutic agents. We investigated the Topo IIalpha mRNA expression by real-time RT-PCR in 37 paired samples at diagnosis and at relapse of acute leukemic patients in relation to drug sensitivity and clinical outcome. The Topo IIalpha levels in leukemic blasts at relapse were significantly higher than that at diagnosis, especially in ALL. The increase in the Topo IIalpha level at relapse was significant in cases which could not achieve a second remission, but not significant in cases which achieved a second remission. These results suggest that the change of Topo IIalpha expression in leukemic blasts at relapse may predict therapeutic responsiveness.

Aberrant methylation and decreased expression of the RIZ1 gene are frequent in adult acute lymphoblastic leukemia of T-cell phenotype

Abstract Retinoblastoma protein-interacting zinc finger, RIZ1, is a tumor suppressor gene that is inactivated in various solid tumors. However, the role of the RIZ1 gene has not been well examined in adult acute lymphoblastic leukemia (ALL). We analyzed the expression and promoter methylation status of the RIZ1 gene in patients with newly diagnosed ALL by quantitative real-time reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR, respectively. RIZ1 expression in 67 cases of ALL (mean 1.043) was decreased compared with that in normal bone marrow (mean 1.471) (p = 0.030). Methylation was detected in 11 of 71 patients (15.5%) but not in healthy controls. Methylation was associated with decreased RIZ1 expression in many ALL cases examined, but this was not statistically significant. In T-ALL, RIZ1 methylation was more frequent (63.6%) than in B-ALL (6.7%) (p < 0.0001) and the decrease of RIZ1 expression was more significant than in B-ALL (p = 0.045). 5-Aza-2′-deoxycytidine treatment of MOLT-4 cells with RIZ1 methylation induced demethylation of RIZ1 and restoration of expression. Forced RIZ1 expression in T-ALL cell lines suppressed cell growth accompanied by G2/M arrest and apoptosis. No mutations were found by PCR-single strand conformation polymorphism analysis in hotspots of the gene. These results suggest that RIZ1 is inactivated in adult ALL, and this inactivation is associated with methylation in T-ALL.

Histone deacetylase inhibitor panobinostat induces calcineurin degradation in multiple myeloma

Multiple myeloma (MM) is a relapsed and refractory disease, one that highlights the need for developing new molecular therapies for overcoming of drug resistance. Addition of panobinostat, a histone deacetylase (HDAC) inhibitor, to bortezomib and dexamethasone improved progression-free survival (PFS) in relapsed and refractory MM patients. Here, we demonstrate how calcineurin, when inhibited by immunosuppressive drugs like FK506, is involved in myeloma cell growth and targeted by panobinostat. mRNA expression of PPP3CA, a catalytic subunit of calcineurin, was high in advanced patients. Panobinostat degraded PPP3CA, a degradation that should have been induced by inhibition of the chaperone function of heat shock protein 90 (HSP90). Cotreatment with HDAC inhibitors and FK506 led to an enhanced antimyeloma effect with a greater PPP3CA reduction compared with HDAC inhibitors alone both in vitro and in vivo. In addition, this combination treatment efficiently blocked osteoclast formation, which results in osteolytic lesions. The poor response and short PFS duration observed in the bortezomib-containing therapies of patients with high PPP3CA suggested its relevance to bortezomib resistance. Moreover, bortezomib and HDAC inhibitors synergistically suppressed MM cell viability through PPP3CA inhibition. Our findings underscore the usefulness of calcineurin-targeted therapy in MM patients, including patients who are resistant to bortezomib.

Overexpression of lung resistance-related protein and P-glycoprotein and response to induction chemotherapy in acute myelogenous leukemia

Lung resistance-related protein (LRP) and P-glycoprotein (P-gp) are associated with multidrug resistance. P-gp overexpression reduces intracellular anticancer drug concentrations and is correlated with low remission rates. However, whether the presence of LRP influences the response to induction chemotherapy remains controversial. Therefore, we investigated the relationship of LRP and P-gp overexpression with the response to induction chemotherapy. Univariate analysis revealed that there was a significant difference between complete remission rates for acute myelogenous leukemia patients depending on their blast cell expressions, between LRP positive versus negative, P-gp positive versus negative, and LRP/P-gp double positive versus other groups. Crude odds ratios (ORs) for complete remission were 0.390, 0.360, and 0.307 for LRP positive, for P-gp positive, and LRP/P-gp double positive patients, respectively. After controlling the confounding variables by stepwise multivariate logistical regression analysis, the presence of LRP/P-gp double positivity and P-gp positivity were found to be independent prognostic factors; adjusted ORs were 0.233 and 0.393, respectively. Furthermore, the monoclonal antibody against LRP significantly increased daunorubicin acumulation (P=0.004) in the nuclei of leukemic blast cells with LRP positivity in more than 10% of the cells. An LRP reversing agent, PAK-104P, was found to increase the daunorubicin content with marginal significance (P=0.060). The present results suggest that not only the presence of P-gp, but also LRP in leukemic blast cells is a risk factor for resistance to induction chemotherapy. Inhibiting LRP function, similar to the inhibition of P-gp function, will be necessary to improve the effectiveness of induction chemotherapy.

Knockdown of the Wnt receptor Frizzled-1 (FZD1) reduces MDR1/P-glycoprotein expression in multidrug resistant leukemic cells and inhibits leukemic cell proliferation

Multidrug resistance (MDR) is a major obstacle to leukemia treatment. The Frizzled-1 (FZD1) Wnt receptor is involved in MDR in some solid cancers, but has rarely been reported to act in acute myeloid leukemia (AML). We investigated whether the knockdown of FZD1 affects MDR1 expression and P-glycoprotein (P-gp) function in multidrug resistant leukemic cell lines, as well as FZD1 and MDR1/P-gp expression in leukemic cells taken from patients with AML (n = 112). FZD1 knockdown significantly reduced MDR1 expression through the Wnt/β-catenin pathway, disrupted the P-gp efflux function, induced the recovery of sensitivity to chemotherapeutic agents, and hindered cell proliferation in cell lines. FZD1 expression in leukemic cells was significantly higher in patients experiencing relapse (n = 34) than in those with no relapse (n = 44, P = .003). Leukemic cells unable to achieve complete response (CR) showed an increased expression of MDR1 and P-gp, compared to patients who achieved CR. Obtaining CR in patients with higher FZD1 expression at diagnosis is difficult. Moreover, they tend to present instances of relapse, suggesting that AML cells with increased FZD1 expression are resistant to chemotherapy. We conclude that the activated FZD1 observed in leukemic cells likely confers acquired drug resistance, whereas FZD1 silencing may be more effective in reversing MDR.