Regulation of AURKC expression by CpG island methylation in human cancer cells.

AURKC, a member of the Aurora kinase gene family, is highly expressed in testis but is either moderately expressed or repressed in most somatic cells. Varying expression of AURKC has been observed in human cancers, but the underlying mechanisms of differential expression have been investigated only to a limited extent. We investigated the role of promoter CpG methylation in the regulation of AURKC gene expression in human cancer cells, in relation to a recently reported AURKC transcription repressor PLZF/ZBTB16, implicated in transformation and tumorigenesis. AURKC and PLZF/ZBTB16 expression profiles were investigated in reference to CpG methylation status on the AURKC promoter experimentally, and also in The Cancer Genome Atlas (TCGA) dataset involving multiple cancer types. AURKC promoter showed dense to moderate hypermethylation correlating with low to moderate expression of the gene in normal somatic cells and cancer cell lines, while testis with high expression revealed marked hypo-methylation. Treatment with the demethylating agent, 5-aza-dC, but not the histone deacetylase (HDAC) inhibitor, TSA, led to elevated expression in cancer cell lines, indicating that promoter DNA methylation negatively regulates AURKC expression. High expression of PLZF in PLZF-transfected cells treated with 5-aza-dC only partially repressed expression of AURKC despite 5-aza-dC also inducing elevated PLZF expression. Analyses of the TCGA data showed differential expression of AURKC in multiple cancer types and stronger correlation of AURKC expression with CpG methylation compared to PLZF levels. These findings demonstrate that differential promoter CpG methylation is an important mechanism regulating AURKC expression in cancer cells.

Residual DNA methylation at remission is prognostic in adult Philadelphia chromosome-negative acute lymphocytic leukemia.

Pretreatment aberrant DNA methylation patterns are stable at time of relapse in acute lymphocytic leukemia (ALL). We hypothesized that the detection of residual methylation alterations at the time of morphologic remission may predict for worse prognosis. We developed a real-time bisulfite polymerase chain reaction assay and analyzed the methylation levels of p73, p15, and p57(KIP2) at the time of initial remission in 199 patients with Philadelphia chromosome-negative and MLL(-) ALL. Residual p73 methylation was detected in 18 (9.5%) patients, p15 in 33 (17.4%), and p57(KIP2) in 7 (3.7%); 140 (74%) patients had methylation of 0 genes and 48 (25%) of more than or equal to 1 gene. In 123 (65%) patients, matched pretreatment samples were also studied and compared with remission ones: in 82 of those with initial aberrant methylation of at least one gene, 59 (72%) had no detectable methylation at remission and 23 (28%) had detectable residual methylation. By multivariate analysis, the presence of residual p73 methylation was associated with a significant shorter duration of first complete remission (hazard ratio=2.68, P= .003) and overall survival (hazard ratio=2.69, P= .002). In conclusion, detection of epigenetic alterations allows the identification of patients with ALL with standard risk but with poor prognosis.

Antileukemia activity of the combination of 5-aza-2'-deoxycytidine with valproic acid.

Aberrant DNA methylation of promoter associated CpG islands is a common phenomenon in human leukemias and cooperates with histone code changes in the control of gene expression. 5-Aza-2'-deoxycytidine (DAC) is a hypomethylating agent with significant antileukemia activity in humans. Recently, valproic acid (VPA) has been shown to be a histone deacetylase inhibitor and to have potential antineoplastic activity. In this report, we study the in vitro effects of the combination of DAC and VPA on the leukemic cell lines HL-60 and MOLT4. DAC alone induced growth inhibition and apoptosis at doses of 1 microM, an effect observed with VPA at doses of 1 mM. Each drug alone had the capacity to induce the expression of p57KIP2 and p21CIP1. DAC mediated hypomethylation of p57KIP2 was not required to induce p57KIP2 gene expression, and treatment with DAC resulted in the induction of p21CIP1. VPA induced global histone acetylation, an effect enhanced by the addition of DAC. The combination of DAC and VPA had a synergistic effect in terms of growth inhibition, induction of apoptosis and reactivation of p57KIP2 and p21CIP1. These results suggest that the combination of DAC and VPA could have significant antileukemia activity in vivo.

p15 mRNA expression detected by real-time quantitative reverse transcriptase-polymerase chain reaction correlates with the methylation density of the gene in adult acute leukemia.

Cyclin-dependent kinase inhibitor p15 is frequently inactivated by either methylation or deletion in patients with acute leukemia. To examine pathologic and clinical significance of the p15 gene inactivation, we established a quantitative assay of p15 mRNA expression in the bone marrow cells by real-time quantitative reverse transcriptase-polymerase chain reaction. p15 mRNA expression in 14 patients with precursor B-cell acute lymphoblastic leukemia (PBC-ALL) well correlated with status of deletion and methylation in the p15 gene analyzed by Southern blotting. Furthermore, two patients with PBC-ALL and 11 acute myeloblastic leukemia (AML) were quantitatively examined for p15 gene methylation using bisulfite genomic sequencing. The data showed that p15 mRNA expression significantly correlated with the CpG island methylation density. Among 108 AML patients, p15 mRNA expression was significantly lower in the myeloid lineage (M1, M2, M3) than the monocytic lineage (M4, M5) (P = 0.0019). Above all, the majority of M3 patients showed low p15 expression compared with M1 and M2 patients (P = 0.029). These observations suggest that quantitative analysis of p15 mRNA will be useful to evaluate transcriptional repression of the p15 gene caused by various degrees of methylation.

Aberrant DNA methylation of a cell cycle regulatory pathway composed of P73, P15 and P57KIP2 is a rare event in children with acute lymphocytic leukemia.

Aberrant DNA methylation of multiple promoter associated CpG islands is a frequent phenomenon in acute lymphocytic leukemia (ALL). Recently, methylation of a cell cycle control pathway composed of P73, P15 and P57KIP2 has been shown to confer poor prognosis to adult patients with ALL. Using bisulfite PCR methods, we have explored the prevalence of methylation of this pathway in a cohort of children with ALL (N=20), and compared these results with those observed in a group of adult patients (N=53). P73 was methylated in 4 (20%) pediatric patients, P15 in 3 (15%), and P57KIP2 in 2 (10%). These compared to 14 (26%), p=0.5, 16 (30%), p=0.04 and 20 (37%), p=0.04, respectively in adult patients. Methylation of two or more genes was not observed in any pediatric patient, but in 15 (28%) adult patients (p=0.003). Poor survival of adult patients was associated with methylation of > or =2 genes (p=0.003). These results indicate that differences in DNA methylation of specific molecular pathways may contribute to the prognostic differences known to occur between pediatric and adult patients with ALL.

Successful treatment of Aspergillus liver abscesses in a patient with acute monoblastic leukemia using combination antifungal therapy including micafungin as a key drug.

While anti-cancer chemotherapy has improved the survival of patients with hematologic malignancies, it has also exposed such patients to the risk of life-threatening infection due to neutropenia. In intensive chemotherapy for leukemia, invasive aspergillosis resulting in death is infrequently observed. In such cases, aggressive diagnostic and therapeutic intervention is required. Herein, we report a case of Aspergillus liver abscesses in a patient with acute monoblastic leukemia. The patient presented with febrile neutropenia and concomitantly with an elevated serum beta-D: -glucan level during chemotherapy. The abscesses were finally diagnosed by liver biopsy. Although antifungal monotherapy of voriconazole or liposomal amphotericin B, both of which are recommended for invasive aspergillosis, showed a poor response, when combined with micafungin, an echinocandin, both had a highly favorable effect against the infection. Therefore, our clinical experience suggests that the serum test is useful for the rapid diagnosis of invasive aspergillosis, especially in deep tissues, and that combination antifungal therapy with micafungin should be considered when initial monotherapy for fungal infection shows an insufficient effect.

Downregulation of JUNB mRNA expression in advanced phase chronic myelogenous leukemia.

JUNB inactivation in transgenic mice results in a myeloproliferative disorder that closely resembles human chronic myelogenous leukemia (CML). It has been reported that downregulation of JUNB expression is a universal phenomenon in patients with CML due aberrant DNA methylation of its promoter. Based on this, we studied methylation and gene expression levels of JUNB in CML. We analyzed the methylation status of the JUNB gene in 6 cell lines and in 102 patients with CML using several bisulfite PCR assays. JUNB expression was analyzed using real-time PCR and gene expression profiling. JUNB methylation was not observed in any of the cell lines studied, and only in 3% of patients with CML. Despite the lack of JUNB methylation, JUNB was expressed at low levels both in CML cell lines (median dCT -6.86; range -5.87 to -9.61), and in patients with CML in blastic phase (BP) (median dCT -3.95; range -1.48 to -6.29) (p = 0.002). Finally, we evaluated JUNB expression in 82 additional patients with CML by gene expression arrays. We found that JUNB was significantly downregulated in advanced phase CML in contrast to chronic phase CML (median log ratio difference in expression = 0.53). Overall, our results indicate that JUNB expression is downregulated in advanced phase CML through a mechanism independent from DNA methylation.

Antileukemia activity of the combination of an anthracycline with a histone deacetylase inhibitor.

We studied the cellular and molecular effects of the combination of an anthracycline with 2 different histone deacetylase inhibitors (HDACIs): vorinostat (suberoylanilide hydroxamic acid) and valproic acid (VPA). The 10% inhibitory concentration (IC(10)) of idarubicin was 0.5 nM in MOLT4 and 1.5 nM in HL60 cells. Concentrations above 0.675 microM of vorinostat resulted in at least 80% loss of cell viability in both cell lines. Concentrations of 1.5 to 3 mM of VPA induced 50% to 60% loss in viability in HL60 and 80% in MOLT4 cells. The combination of idarubicin with vorinostat at 0.075 microM or VPA at 0.25 mM resulted in at least an additive loss of cell viability in both lines. Vorinostat (0.35 microM) and VPA (0.25 mM) in combination with idarubicin (0.5 nM) resulted in a significant increase in apoptotic cells in MOLT4 cells. The combination resulted in an increase in histone H3 and H4 acetylation at 24 hours, phosphorylated H2AX, as well as in the induction of p21(CIP1) mRNA. No effect on cell cycle transition was observed. Of importance, the cellular and molecular effects observed were independent of the sequence used. In summary, the combination of an anthracycline with an HDACI should have significant clinical activity in patients with leukemia.

The absence of the p15INK4B gene alterations in adult patients with precursor B-cell acute lymphoblastic leukaemia is a favourable prognostic factor.

We examined deletion and methylation of the p15INK4B (p15) and p16INK4A (p16) genes, using Southern blotting and methylation-specific polymerase chain reaction (PCR), in 70 untreated adult patients with precursor B-cell acute lymphoblastic leukaemia (PBC-ALL) and analysed the relationship between their genetic changes and clinical outcome. Methylation and homozygous deletion of the p15 gene were detected in 30 (43%) and 18 (26%) patients, while those of the p16 gene were found in 16 (23%) and 11 (16%) patients respectively. Thirteen out of 17 patients with wild-type p15 gene showed expression of p15 mRNA, whereas 31 out of 39 patients with alteration (deletion and methylation) of the p15 gene showed no p15 mRNA expression by reverse transcription-PCR, suggesting that alterations of the p15 gene are highly associated with loss of p15 mRNA expression. Disease-free survival (DFS) at 4 years in patients with wild-type p15 gene is 33%, compared with 4% of those with p15 gene alterations (P = 0.049). Multivariate analysis showed that the absence of p15 gene alterations was an independent significant favourable prognostic factor for longer DFS (P = 0.0001). These results suggest that alterations in the p15 but not p16 gene can be used as a genetic prognostic indicator in PBC-ALL.