Abstract B49: Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors
Authors: Singh Melissa , Donnella Hayley , Ramirez Lorimar , Chandra Joya 
Published: August 2014

Abstract
Targeting of histone modifications is clinically feasible due to the development of histone deacetylase inhibitors (HDACi), several of which are FDA-approved. In order to identify strategies to augment activity of HDACi, we tested inhibition of lysine specific demethylase-1 (KDM1A/LSD1) in combination with HDACi in malignancies where single agent activity was limited in clinical trials: acute leukemia and glioblastoma, In brain tumor cell lines, patient derived glioblastoma stem cells, and a series of acute leukemia lines, we find high expression of LSD1 protein. Using either knockdown of LSD1, or chemical inhibition via the FDA approved drug, tranylcypromine, we report significant enhancement of HDACi induced cell death. Multiple HDACi were used including vorinostat, panobinostat and entinostat, and cytoxicity was consistently heightened when combined with LSD1 inhibition. Indicative of selectivity of this therapeutic approach, augmentation of HDACi cytotoxicity occurred exclusively in tumor cells, and not in normal counterparts. Caspase activation is a feature of cell death induction by HDAC and LSD1 inhibition and pan-caspase inhibition blocks cell death. Using chemical inhibitors of caspase-8, -9 and -3 and caspase-8 deficient cells, we demonstrate a reliance upon extrinsic pathways of caspase activation by the combination therapy. However, data obtained with caspase-8 deficient cells showed a lack of protection in cells treated with LSD1 inhibitor combined with pan-HDACi (vorinostat and panobinostat), whereas there was significant protection in cells treated with entinostat (a Class I HDACi) and LSD1 inhibitor. This suggests that pan-HDACi may trigger alternate caspases, such as caspase-10, which are blocked by peptide inhibitors but are still active in caspase-8 deficient cells. To gain additional insight into the contribution of LSD1 to cytotoxicity, an apoptosis focused array and RNA-Seq were conducted. The majority of gene expression changes were seen in cells exposed to HDACi alone. However, some of these gene expression changes were further enhanced when LSD1 was also inhibited. Expression of the tumor suppressor genes, p53 and p73, was significantly reduced by HDACi alone, and by the combination of HDACi and LSD1 inhibition. Hrk, a pro-apoptotic Bcl-2 family member, known to be silenced in glioblastoma, was re-expressed by the combination therapy. Time course analyses indicated the loss of p53, p73 and increased Hrk, was an early event, occurring within 6 hours of exposure to HDACi and LSD1 inhibitors, and preceded caspase activation. Upregulation of Hrk transcript was robust and rapid. We observed a ten-fold increase within 3 h of exposure to these agents. Further studies will evaluate the contribution of these early changes to caspase activation, thereby delineating the pathway by which HDACs and LSD1 can be optimally targeted in leukemia and brain tumor model systems.
Citation Format: Melissa Singh, Hayley Donnella, Lorimar Ramirez, Joya Chandra. Mechanism of cell death induction by dual targeting of HDACs and lysine specific demethylase, LSD1, in leukemias and brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B49.