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Small-molecule activating SIRT6 elicits therapeutic effects and synergistically promotes anti-tumor activity of vitamin D3 in colorectal cancer.

ABSTRACT: Colorectal cancer (CRC) is the leading cause of cancer death; however, targets with broad anti-CRC effects are limited. Sirtuin6 (SIRT6) is a conserved nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that is widely pathologically downregulated in CRC, but its pharmacological effect in CRC remains undefined due to the lack of small-molecule SIRT6 activators. We searched for a compound activating SIRT6 and investigated its anti-CRC effect in various models. Methods: We identified an allosteric SIRT6 activator, MDL-811. Its ability to enhance SIRT6 deacetylation at protein and cellular levels was evaluated by Fluor de Lys (FDL) and western blots. We assessed the proliferation of 26 CRC cell lines and patient-derived organoids (PDOs) treated with MDL-811. In vivo efficacy of MDL-811 was evaluated in HCT116 cell line- and patient-derived xenografts as well as a spontaneous CRC model. RNA sequencing and real-time quantitative PCR assays were performed to analyze gene expression changes in MDL-811-treated HCT116 cells. Along with controls in SIRT6-overexpressing HCT116 cells, the SIRT6-mediated histone H3 deacetylation at the Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene locus was assessed by chromatin immunoprecipitation (ChIP) in MDL-811-treated HCT116 cells. A combination therapy against CRC based on the downstream gene of SIRT6 activation was evaluated in cells and mouse models. Results: MDL-811 significantly activated SIRT6 histone H3 deacetylation (H3K9Ac, H3K18Ac, and H3K56Ac) in vitro and had broad antiproliferative effects on diverse CRC cell lines and PDOs. More importantly, the in vivo anti-tumor efficacy of MDL-811 was demonstrated across cell line- and patient-derived xenografts and in the APCmin/+ spontaneous CRC model. Mechanically, we identified a new downstream target gene of SIRT6 in CRC, CYP24A1. Based on these findings, a combination drug strategy with MDL-811 to synergistically enhance the anti-CRC effect of vitamin D3 was validated in vitro and in vivo. Conclusions: Our data provide proof of concept that targeting SIRT6 using a small-molecule activator is an attractive therapeutic strategy for CRC and that MDL-811 could be a promising lead compound for further preclinical and clinical studies of treatments for CRC.

SUBMITTER: Shang J

PROVIDER: S-EPMC7255010 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Small-molecule activating SIRT6 elicits therapeutic effects and synergistically promotes anti-tumor activity of vitamin D3 in colorectal cancer.

Shang Jialin J Zhu Zhehui Z Chen Yingyi Y Song Jinglue J Huang Yuji Y Song Kun K Zhong Jie J Xu Xinyuan X Wei Jiacheng J Wang Chengxiang C Cui Long L Liu Chen-Ying CY Zhang Jian J

Theranostics 20200427 13

Colorectal cancer (CRC) is the leading cause of cancer death; however, targets with broad anti-CRC effects are limited. Sirtuin6 (SIRT6) is a conserved nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that is widely pathologically downregulated in CRC, but its pharmacological effect in CRC remains undefined due to the lack of small-molecule SIRT6 activators. We searched for a compound activating SIRT6 and investigated its anti-CRC effect in various models. Methods ...[more]

PMID: 32483423

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Project description:Cladribine (2CdA), a synthetic purine analog interfering with DNA synthesis, is a medication used to treat hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia. Entinostat, a selective class I histone deacetylase (HDAC) inhibitor, shows antitumor activity in various human cancers, including hematological malignancies. The therapeutic potential of cladribine and entinostat against multiple myeloma (MM) remains unclear. Here we investigate the combinatorial effects of cladribine and entinostat within the range of their clinical achievable concentrations on MM cells. While either agent alone inhibited MM cell proliferation in a dose-dependent manner, their combinations synergistically induced anti-proliferative/anti-survival effects on all MM cell lines (RPMI8226, U266, and MM1.R) tested. Further studies showed that the combinations of cladribine and entinostat as compared to either agent alone more potently induced mitotic catastrophe in the MM cells, and resulted in a marked increase of the cells at G1 phase associated with decrease of Cyclin D1 and E2F-1 expression and upregulation of p21waf-1. Apoptotic ELISA and western blot analyses revealed that the combinations of cladribine and entinostat exerted a much more profound activity to induce apoptosis and DNA damage response, evidenced by enhanced phosphorylation of histone H2A.X and the DNA repair enzymes Chk1 and Chk2. Collectively, our data demonstrate that the combinations of cladribine and entinostat exhibit potent activity to induce anti-proliferative/anti-survival effects on MM cells via induction of cell cycle G1 arrest, apoptosis, and DNA damage response. Regimens consisting of cladribine and/or entinostat may offer a new treatment option for patients with MM.AbbreviationsMM, multiple myeloma; HCL, hairy cell leukemia; HDAC, histone deacetylase; Ab, antibody; mAb, monoclonal Ab; FBS, fetal bovine serum; CI, combination index; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PARP, poly(ADP-ribose) polymerase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt.

Project description:BACKGROUND:Typically minor ABO incompatible platelet products are transfused without any incident, yet serious hemolytic transfusion reactions occur. To mitigate these events, ABO 'low titer' products are used for minor ABO incompatible transfusions. We sought to understand the role of IgM/IgG and complement activation by anti-A on extravascular hemolysis. METHODS:Samples evaluated included (i) Group O plasma from a blood donor whose apheresis platelet product resulted in an extravascular transfusion reaction, (ii) Group O plasma from 12 healthy donors with matching titers that activated complement (N?=?6) or not (N?=?6), and (iii) Group O sera from 10 patients with anti-A hemolysin activity. A flow cytometric monocyte erythrophagocytosis assay was developed using monocytes isolated by immunomagnetic CD14-positive selection from ACD whole blood of healthy donors. Monocytes were frozen at -?80 °C in 10% dimethyl sulfoxide/FBS and then thawed/reconstituted on the day of use. Monocytes were co-incubated with anti-A-sensitized fluorescently-labeled Group A1?+?RBCs with and without fresh Group A serum as a source of complement C3, and erythrophagocytosis was analyzed by flow cytometry. The dependency of IgM/IgG anti-A and complement C3 activation for RBC erythrophagocytosis was studied. Anti-A IgG subclass specificities were examined for specific samples. RESULTS:The plasma and sera had variable direct agglutinating (IgM) and indirect (IgG) titers. None of 12 selected samples showed monocyte-dependent erythrophagocytosis with or without complement activation. The donor sample causing a hemolytic transfusion reaction and 2 of the 10 patient sera with hemolysin activity showed significant erythrophagocytosis (>?10%) only when complement C3 was activated. The single donor plasma and two sera demonstrating significant erythrophagocytosis had high IgM (??128) and IgG titers (>?1024). The donor plasma anti-A was IgG1, while the patient sera were an IgG3 and an IgG1 plus IgG2. CONCLUSION:High anti-A IgM/IgG titers act synergistically to cause significant monocyte erythrophagocytosis by activating complement C3, thus engaging both Fc?- and CR1-receptors.

Dataset Information

Small-molecule activating SIRT6 elicits therapeutic effects and synergistically promotes anti-tumor activity of vitamin D3 in colorectal cancer.

Publications

Small-molecule activating SIRT6 elicits therapeutic effects and synergistically promotes anti-tumor activity of vitamin D<sub>3</sub> in colorectal cancer.

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