HDAC inhibitors in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy characterized by uncontrolled proliferation, differentiation arrest, and accumulation of immature myeloid progenitors. Although clinical advances in AML have been made, especially in young patients, long-term disease-free survival remains poor, making this disease an unmet therapeutic challenge. Epigenetic alterations and mutations in epigenetic regulators contribute to the pathogenesis of AML, supporting the rationale for the use of epigenetic drugs in patients with AML. While hypomethylating agents have already been approved in AML, the use of other epigenetic inhibitors, such as histone deacetylases (HDAC) inhibitors (HDACi), is under clinical development. HDACi such as Panobinostat, Vorinostat, and Tricostatin A have been shown to promote cell death, autophagy, apoptosis, or growth arrest in preclinical AML models, yet these inhibitors do not seem to be effective as monotherapies, but rather in combination with other drugs. In this review, we discuss the rationale for the use of different HDACi in patients with AML, the results of preclinical studies, and the results obtained in clinical trials. Although so far the results with HDACi in clinical trials in AML have been modest, there are some encouraging data from treatment with the HDACi Pracinostat in combination with DNA demethylating agents

Frequent and simultaneous epigenetic inactivation of TP53 pathway genes in acute lymphoblastic leukemia

Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (n = 48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-29-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (p = 0.001) and mortality (p,0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML

Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium

The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms. Methods and results: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56+), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56−). Gene expression revealed up-regulation of pro-angiogenic (PGF), antiapoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (pb0.05) and a decrease in apoptosis of cardiomyocytes (pb0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-β, and IGF-1 1 month after transplant. Conclusions: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium. © 2008 European Society of Cardiology. Published by Elsevie

Methylation status of Wnt signaling pathway genes affects the clinical outcome of Philadelphia-positive acute lymphoblastic leukemia

The clinical significance of aberrant promoter methylation of the canonical Wnt pathway antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5, Wif1, Dkk3, and Hdpr1) and also putative tumor-suppressor gene Wnt5a, belonging to the non-canonical Wnt signaling pathway, was investigated in a large series of 75 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia by methylationspecific polymerase chain reaction. At least one methylated gene was observed in cells from 66% (49/75) of patients (methylated group). Disease-free survival and overall survival at 9 years were 51 and 40%, respectively, for the unmethylated group and 3 and 2%, respectively, for the methylated group (both P < 0.0001). Multivariate analysis demonstrated that the Wnt methylation profile was an independent prognostic factor predicting disease-free survival (P = 0.007) and overall survival (P = 0.039). Abnormal DNA methylation of promoter-associated CpG islands in the Wnt signaling pathway is very common in Philadelphia chromosome-positive acute lymphoblastic leukemia and potentially defines subgroups with distinct clinical characteristics

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia

Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies

BCR-ABL1-induced expression of HSPA8 promotes cell survival in chronic myeloid leukaemia

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML

Down-Regulation of hsa-miR-10a in Chronic Myeloid Leukemia CD34+ Cells Increases USF2-Mediated Cell Growth

MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34+ cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML

Preclinical activity of LBH589 alone or in combination with chemotherapy in a xenogeneic mouse model of human acute lymphoblastic leukemia.

Histone deacetylases (HDACs) have been identified as therapeutic targets due to their regulatory function in chromatin structure and organization. Here, we analyzed the therapeutic effect of LBH589, a class I-II HDAC inhibitor, in acute lymphoblastic leukemia (ALL). In vitro, LBH589 induced dose-dependent antiproliferative and apoptotic effects, which were associated with increased H3 and H4 histone acetylation. Intravenous administration of LBH589 in immunodeficient BALB/c-RAG2(-/-)γc(-/-) mice in which human-derived T and B-ALL cell lines were injected induced a significant reduction in tumor growth. Using primary ALL cells, a xenograft model of human leukemia in BALB/c-RAG2(-/-)γc(-/-) mice was established, allowing continuous passages of transplanted cells to several mouse generations. Treatment of mice engrafted with T or B-ALL cells with LBH589 induced an in vivo increase in the acetylation of H3 and H4, which was accompanied with prolonged survival of LBH589-treated mice in comparison with those receiving vincristine and dexamethasone. Notably, the therapeutic efficacy of LBH589 was significantly enhanced in combination with vincristine and dexamethasone. Our results show the therapeutic activity of LBH589 in combination with standard chemotherapy in pre-clinical models of ALL and suggest that this combination may be of clinical value in the treatment of patients with ALL