Quantitative Proteomic Profiling Of Novel Anti-Cancer Small Molecule Inhibitors Of Sec61: Mechanistic Investigation And Biomarker Discovery.

Abstract Secreted and transmembrane (TM) proteins play key roles in malignant transformation and tumor growth. During translation, the majority of these proteins require translocation through the Sec61 protein complex into the endoplasmic reticulum (ER) for further processing. Thus, Sec61 represents a novel therapeutic target for cancer treatment through selective blockade of protein secretion. The structural analogs KZR-261 and KZR-834 were identified through a medicinal chemistry campaign and found to have broad in vitro anti-tumor activity and efficacy in mouse xenograft models. KZR-261 was nominated for clinical development and we describe here the proteomic assessment and in vitro pharmacodynamics of these novel anti-cancer agents. We utilized quantitative proteomic methods to study inhibition of protein secretion and global modulation of protein homeostasis across multiple tumor cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors. Cells were treated with KZR-834 or KZR-261 followed by subcellular fractionation of cytosolic and membrane/ER proteomes. The membrane/ER fraction was expected to be enriched for Sec61 client proteins. Subsequent proteomic profiling was performed with Tandem Mass Tag 6-plex (TMTsixplex™). Across tumor cell lines, a total of 2222 Sec61 client proteins were detected in the membrane/ER proteome, representing >30% of the annotated Sec61 clientome. Of these proteins, 5-17% were inhibited by >50% upon treatment, indicating that only a small portion of the total flux of secreted and TM proteins in cells was affected by our compounds. KZR-261 and KZR-834 preferentially targeted secreted and type 1 TM proteins relative to multi-pass TM proteins, which may be related to the smaller size of Sec61 targeting sequences in these proteins. Additionally, Ingenuity®Pathway Analysis (Qiagen) of the cytosolic proteome from tumor cell lines indicated that activation of the ER stress response was induced by KZR-834, which were further verified by gene expression and western blot analyses. In PBMCs treated with KZR-261, multiple type 1 TM proteins (IL-7 receptor, L-selectin, ICAM-1, HLA-DR) and the secreted protein beta-2-microglobulin were identified as sensitive cell surface targets. Flow cytometric analysis of PBMCs also showed a significant reduction in surface expression of those proteins in multiple cell populations. Our results highlight quantitative proteomic profiling as a valuable tool towards elucidation of the mechanism of pleiotropically-acting molecules. By assessing global proteomic changes in non-transformed cells, pharmacodynamic markers were identified that will be employed in the Phase 1 trial of KZR-261 in solid tumors. Citation Format: Yu Qian, Jennifer Whang, Janet Anderl, Henry W. Johnson, Christopher J. Kirk, Eric Lowe, Dustin McMinn, Beatriz Millare, Tony Muchamuel, Jinhai Wang. Quantitative proteomic profiling of novel anti-cancer small molecule inhibitors of Sec61: Mechanistic investigation and biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 323.