Efficacy of LSD1 Directed Agents is Enhanced with Kinase Signaling Inhibition in Glioblastoma Stem Cells

Abstract

<b>Abstract ID 17899</b> <b>Poster Board 120</b> Lysine-specific demethylase 1 (LSD1) is an epigenetic eraser that removes methyl marks from lysine residues on histones (H3K4 and H3K9) and is implicated in the regulation of tumor initiating cells. In glioblastoma (GBM), LSD1 is overexpressed in the tumor initiating cells, glioblastoma stem cells (GSCs), and LSD1 directed therapy via small molecule inhibition can affect tumorgenicity. However, there is no clinically viable strategy to treat GBM with LSD1 inhibition yet. To design an effective LSD1 treatment approach for clinical use, treatment strategies should proactively evade resistance and prevent tumor regrowth, such as the concurrent inhibition of LSD1 with inhibition of compensatory effects. In liquid cancer models, mitogen-activated protein kinase (MAPK) signaling activation functions as a resistance mechanism to LSD1 inhibition. We find this pathway relevant in GBM as it is frequently hyperactivated, in part via mutations and/or amplification of receptor tyrosine kinases (RTKs). Therefore, the present study sought to understand the relationship between LSD1 and RTK/MAPK signaling and to evaluate the combination efficacy of LSD1 inhibition and kinase signaling inhibition in GBM. Upon LSD1 knockdown in human GBM cells, transcriptional changes were examined using RNA-seq and GSEA. The GSEA identified several kinase signaling processes that were enriched for LSD1 expression, including those related to transmembrane RTK activity and RTK binding. Western blot analysis was used to determine effect of LSD1 inhibition on components of the MAPK signaling pathway in GSCs and a pediatric high-grade glioma cell model. Pharmacological inhibition of LSD1 was assessed as a single agent with five candidates, including GSK-LSD1, ORY-1001, ORY-2001, IMG-7289 and tranylcypromine. Next, we evaluated the effect of LSD1 inhibition combined with osimertinib, a kinase inhibitor directed against the epidermal growth factor receptor (EGFR). Our results demonstrate that MAPK activity can be modulated via inhibition of LSD1, and perhaps support the emergence of a resistant subpopulation. We observed an increase in phosphorylated ERK1/2 (pERK) following pharmacological LSD1 inhibition in GSC17 and SU-DIPG-XIII cells. The LSD1 inhibitor, ORY-2001, caused increased pERK relative to an untreated control in both the GSC17 and SU-DIPG-XIIIs (mean = 11.9 and 2.17, respectively) To further understand the relationship between LSD1 and kinase signaling, we found several protein tyrosine phosphatases non-receptors (PTPNs) downregulated upon LSD1 knockdown, including PTPN1/6/7 (fold change = -0.39, -0.89, and -1.64, respectively). PTPNs act as negative regulators of kinase signaling cascades and can regulate the activity of RTKs and their downstream signaling components. Lastly, the concurrent inhibition of LSD1 and EGFR showed synergy <i>in&nbsp;vitro</i> compared to single agent inhibition. The combination of ORY-1001 and osimertinib had the highest synergy score of 38.7 in the GSC17s. In summary, the results from this study emphasize the importance of preemptive therapeutic strategies to improve efficacy and avoid therapeutic resistance that is common in GBM. Future studies are aimed at defining the mechanism by which LSD1 regulates kinase signaling at the EGFR and to evaluate the combination treatment <i>in&nbsp;vivo</i> using orthotopic xenograft models of the GSCs. This work was supported by NIH awards R21 NS093387, P50 CA127001, R21 NS111058, and R33 NS111058 and by CPRIT award DP160014.