Epigenetic activation of the elongator complex sensitizes gallbladder cancer to gemcitabine therapy

Background Gallbladder cancer (GBC) is known for its high malignancy and multidrug resistance. Previously, we uncovered that impaired integrity and stability of the elongator complex leads to GBC chemotherapy resistance, but whether its restoration can be an efficient therapeutic strategy for GBC remains unknown. Methods RT-qPCR, MS-qPCR and ChIP-qPCR were used to evaluate the direct association between ELP5 transcription and DNA methylation in tumour and non-tumour tissues of GBC. EMSA, chromatin accessibility assays, and luciferase assays were utilized to analysis the DNA methylation in interfering PAX5-DNA interactions. The functional experiments in vitro and in vivo were performed to investigate the effects of DNA demethylating agent decitabine (DAC) on the transcription activation of elongator complex and the enhanced sensitivity of gemcitabine in GBC cells. Tissue microarray contains GBC tumour tissues was used to evaluate the association between the expression of ELP5, DNMT3A and PAX5. Results We demonstrated that transcriptional repression of ELP5 in GBC was highly correlated with hypermethylation of the promoter. Mechanistically, epigenetic analysis revealed that DNA methyltransferase DNMT3A-catalysed hypermethylation blocked transcription factor PAX5 activation of ELP5 by disrupting PAX5-DNA interaction, resulting in repressed ELP5 transcription. Pharmacologically, the DNA demethylating agent DAC eliminated the hypermethylated CpG dinucleotides in the ELP5 promoter and then facilitated PAX5 binding and reactivated ELP5 transcription, leading to the enhanced function of the elongator complex. To target this mechanism, we employed a sequential combination therapy of DAC and gemcitabine to sensitize GBC cells to gemcitabine-therapy through epigenetic activation of the elongator complex. Conclusions Our findings suggest that ELP5 expression in GBC is controlled by DNA methylation-sensitive induction of PAX5. The sequential combination therapy of DAC and gemcitabine could be an efficient therapeutic strategy to overcome chemotherapy resistance in GBC.

Direct reprogramming of epithelial cell rests of malassez into mesenchymal-like cells by epigenetic agents

The DNA demethylating agent, 5-Azacytidine (5Aza), and histone deacetylase inhibitor, valproic acid (Vpa), can improve the reprogramming efficiencies of pluripotent cells. This study aimed to examine the roles of 5Aza and Vpa in the dedifferentiation of epithelial cell rests of Malassez (ERM) into stem-like cells. Additionally, the ability of stem-like cells to differentiate into mesenchymal cells was evaluated. ERM was cultured in embryonic stem cell medium (ESCM) with 1 µM of 5Aza, or 2 mM of Vpa, or a combination of 5Aza and Vpa. The cells stimulated with both 5Aza and Vpa were named as progenitor-dedifferentiated into stem-like cells (Pro-DSLCs). The Pro-DSLCs cultured in ESCM alone for another week were named as DSLCs. The stem cell markers were significantly higher in the DSLCs than the controls (no additions). The mRNA and protein levels of the endothelial, mesenchymal stem, and osteogenic cell markers were significantly higher in the Pro-DSLCs and DSLCs than the controls. The combination of a demethylating agent and a deacetylated inhibitor induced the dedifferentiation of ERM into DSLCs. The Pro-DSLCs derived from ERM can be directly reprogrammed into mesenchymal-like cells without dedifferentiation into stem-like cells. Isolated ERM treated with epigenetic agents may be used for periodontal regeneration.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.