Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

Background Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. Methods Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. Results In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. Conclusions Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

Expression and Clinical Significance of Hypoxia-related Factors HIF-1a, Gli-1 and MMP9 in Breast Cancer

Objective: To investigate the expression and clinical significance of hypoxia inducible factors HIF-1a, Gli-1 and MMP9 in breast cancer. Methods: Eighty patients with invasive ductal carcinoma of the breast and 40 normal tissues adjacent to cancer were selected. Immunohistochemical methods were used to detect the expression of HIF-1a, Gli-1 and MMP9 in breast cancer and normal tissues adjacent to cancer, and their relationship with clinicopathological features of breast cancer and prognosis was explored. Results: The positive rates of HIF-1a, Gli-1 and MMP9 in breast cancer tissues were significantly higher than those in normal breast tissues. HIF-1a, Gli-1 and MMP9 expressions are positively correlated in breast cancer. Conclusion: HIF-1a, Gli-1 and MMP9 proteins are involved in the pathogenesis of breast cancer.

Formulation Comprising Arsenic Trioxide and Dimercaprol Enhances Radiosensitivity of Pancreatic Cancer Xenografts

Objective: To investigate the efficacy of a formula comprising arsenic trioxide and dimercaprol (BAL-ATO) as a radiosensitizing agent in model mice with pancreatic cancer xenografts. Methods: Female BALB/c nude mice bearing SW1990 human pancreatic cancer xenografts were divided into four treatment arms, including control, radiotherapy (RT), BAL-ATO, and RT + BAL-ATO groups. Survival and tumor volume were analyzed. We also assessed apoptosis in tumor samples by live imaging and detected hypoxia by confocal laser microscope observation. We further investigated the mechanisms of BAL-ATO action in RT by detecting affected proteins by western blot and immunohistochemistry assays. Results: Median survival was significantly longer in the RT + BAL-ATO group (64.5 days) compared with the control (49.5 days), RT (39 days), and BAL-ATO (48 days) groups ( P < 0.001). RT + BAL-ATO inhibited the growth of tumors in mice by 73% compared with the control group, which was significantly higher than the rate of inhibition following RT alone (59%) ( P < 0.01). Further analysis showed an improved microenvironment in terms of hypoxia in tumors treated with BAL-ATO alone or RT + BAL-ATO. Expression of signaling molecules associated with pancreatic cancer stem cells, including CD24, CD44, ALDH1A1, Gli-1, and Nestin, was detected in tumors treated with BAL-ATO alone or in combination with RT. Conclusion: These data suggest that BAL-ATO function as a radiosensitizer in mice with pancreatic cancer xenografts, via mechanisms involving hypoxia reduction and inhibition of signaling pathways associated with pancreatic cancer stem cells. BAL-ATO may thus be a promising radiosensitizing agent in patients with pancreatic cancer.

MBRS-18. TUMOR SUPPRESSOR p53 DEFINES THE THERAPEUTIC RESPONSES IN TREATMENT OF MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common primary pediatric malignant brain tumor. Current molecular analysis classifies MB into 4 groups, classic (WNT), sonic hedgehog (Shh), group 3, and group 4. Furthermore, atypical p53 signaling is associated with disease progression and confers poor prognosis. This study investigated the correlation of mutational status of p53 and iSO17q with disease progression and metastatic potential. In addition, we used small molecule inhibitors of PI3K (Buparlisib; BKM120) and HDAC (LBH-589) on a p53-mutant MB cell line to find novel therapeutic targets. Efficacy of these drugs were assessed using functional assays (cell proliferation, migration, cell cycle and drug resistance). MB tumors (n=53) were evaluated for GLI-1, GAB-1, NPR, KV1, YAP expression and mutant p53 via immunohistochemistry and correlated to patient outcomes. Results demonstrated that: 1) high expression of GAB-1 and YAP were found in the Shh group, while KV1 expression was present in all subtypes; 2) mutant p53 expression was present in various subsets of MB with no apparent correlation with metastasis or disease progression; 3) patients displaying iSO17q (determined by fluorescence in situ hybridization (FISH) technique) exhibited metastatic disease; 4) LBH-589 and BKM120 caused both time and dose-dependent inhibition of MB cell proliferation and migration; 5) combined treatment of BKM120 and LBH-589 had a synergistic effect; 6) MB cells demonstrated drug-resistance to BKM120. In conclusion, these findings underscore use of Buparlisib and LBH-589 in treatment of MB. Further, the role of mutant p53 in disease progression remains elusive, whereas presence of iSO17q defines metastatic potential.

Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

Background: Dysregulation of epigenetics plays important roles in leukemogenesis and progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate acetylation and deacetylation of nuclear histone, aberrant activation of HDAC results in uncontrolled proliferation and differentiated blockage, HDAC inhibitors have been investigated as therapeutic drugs for treatment of AML. Methods: Cell growth was assessed by CCK-8 assay, apoptosis was determined by flow cytometry in AML cell lines, CD45+ and CD34+CD38- cells from patient’s samples after stained with Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI). EZH2 expression was silenced by short hairpin RNA (shRNA). The pathway changes were detected by western blot. The effect of chidamide or EZH2 shRNA in combination with adriamycin was studies in vivo in nude mice model bearing leukemia.Results: In this study, we investigated the antileukemia activities of HDAC inhibitor chidamide and its combinatorial effect with cytotoxic agent in AML. We demonstrated in vitro and in vivo that chidamide suppressed expression of EZH2, exerted potential antileukemia activity and increased the sensitivity AML cells and AML stem/progenitor cells to chemotherapeutic drug through Smo/Gli-1 pathway. In addition to decrease the expression of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologicall by chidamide or genetically by shEZH2 decreased the activity of Smo/Gli-1 pathway, and increased chemotherapeutic sensitivity in AML cells. Conclusions: Inhibition of EZH2 by chidamide has antileukemia activity and increases chemosensitivity, it provides a potential strategy to improve chemotherapeutic effect in AML.