Abstract 6283: Evaluating the potency of a first-in-class covalent antagonist of the H3K9me3 reader protein MPP8 in bladder cancer
Authors: Tineo Stephany Gonzalez , Kemper Ryan M. , Tripathi Surya K. , Buttery Peter H. , Kim William Y. , James Lindsey I. , Crona Daniel J. 
Published: June 2023

Abstract
Background: Bladder cancer (BC) is a common and deadly disease, and despite recent treatment advances, metastatic BC (mBC) remains incurable. Mutations in genes that encode epigenetic/chromatin modifier proteins are common in mBC, with &gt;90% of tumors harboring at least one inactivating mutation. The epigenetic reader protein MPP8 recognizes the histone-3-lysine-9-trimethyl (H3K9me3) region of target gene promoters, and recruits transcription factors associated with cell proliferation and metastasis. UNC7713 was developed as a covalent antagonist to disrupt MPP8 binding to H3K9me3. Here, we explored whether UNC7713 potently inhibits cell proliferation, migration, and viability in preclinical models of BC.
Methods: 5637 cells were treated with ascending concentrations of UNC7713 (0.1 nM-100 µM), negative control compound UNC7716, or 0.1% DMSO negative control. Cell viability was measured using CellTiter-Glo™ after 48 h and 96h incubations. IC50 values were calculated using a four-parameter non-linear regression model in GraphPad. Cells were stained with Annexin V and propidium iodide (PI) to evaluate apoptosis versus necrosis signaling by flow cytometry after 48 h incubation of UNC7713. Flow cytometry experiments were performed on a Thermo Attune NxT, and data were analyzed using FlowJo. 5637 cells were grown in a monolayer, treated with UNC7713 (25 nM-100 nM), and then subjected to a wound healing assay to evaluate cell migration after 48 h. To evaluate effects on cell proliferation, 5637 cells were treated with four doses of UNC7713 (25 nM-100 nM), and colony formation was evaluated after 10 days using crystal violet and analyzed by Fiji. For wound healing, images were captured by an Olympus IX83 inverted microscope and analyzed by Fiji.
Results: UNC7713 achieved submicromolar potency for reduction of cell viability in 5637 cells and was nearly 200x more potent than UNC7716 at 48 h (IC50: 0.28 μM vs. 56.03 μM). Potency was also maintained at 96 h (0.18 µM). Concentrations of UNC7713 as low as 75 nM caused over 2.5x greater apoptotic signaling than 20 µM UNC7716 after 48 h (18.5% vs. 7.3% Annexin V and PI positive cells). Next, cells treated with UNC7713 did not migrate to initiate wound closure, but instead caused cell death, increasing the size of the initial wound. Cells treated with UNC7713 at concentrations as low as 50 nM caused dramatic wound expansion compared to 0.1% DMSO and 20 µM UNC7716 (70% wound expansion for UNC7713 vs. 100% wound closure for both controls). Last, after 10 days &gt;90% fewer colonies were detected in cells treated with UNC7713 (25 nM-100 nM) when compared to 20 µM UNC7716 and 0.1% DMSO controls.
Conclusions: These preliminary data support further inquiry into the role of MPP8 in BC. Future studies will focus on identifying molecular mechanisms that underlie UNC7713’s ability to inhibit cell proliferation, migration, and viability in preclinical models of BC.
Citation Format: Stephany Gonzalez Tineo, Ryan M. Kemper, Surya K. Tripathi, Peter H. Buttery, William Y. Kim, Lindsey I. James, Daniel J. Crona. Evaluating the potency of a first-in-class covalent antagonist of the H3K9me3 reader protein MPP8 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6283.