Abstract 1892: Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung cancer

Abstract

Abstract Heme is a central biosynthetic molecule with important implications in diverse molecular and cellular processes, including oxygen utilization and metabolism. Heme carries out a vital role in mitochondrial energy generation through oxidative phosphorylation (OXPHOS) by acting as a cofactor or prosthetic group for complexes II, II, and IV in the electron transport chain. Lung cancer is the leading cause of cancer related death in the US. Our lab has previously shown that non-small cell lung cancer (NSCLC) cells exhibit an elevated heme flux characterized by elevated heme synthesis and import. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. To target this elevated heme flux, we have engineered recombinant bacterial HasA hemophores as heme-sequestering proteins (HeSPs). HeSPs bind to and sequester heme with an extremely high (pM) affinity, thereby disrupting this elevated heme flux. HeSPs display potent anti-tumor activity in mouse models of NSCLC and triple-negative breast cancer (TNBC). Here, we utilized the Pichia pastoris expression platform to achieve high-yield expression. P. pastoris is a methylotrophic yeast that has minimal endogenous protein secretion and potentially high-yields for heterologous protein expression, due to a tightly regulated alcohol oxidase gene (AOX1). The P. pastoris expression platform is generally easier to handle and requires simpler fermentation conditions than mammalian cell cultures, making it an ideal candidate for expression of recombinant bacterial proteins without endotoxin contamination.We utilized a secretion signal to direct secretion of HeSP2 into the culture medium to allow for easier protein purification. Purification was performed through ion exchange chromatography. We utilized native PAGE and heme-binding spectrum to verify that these yeast-expressed HeSPs adopt a similar conformation to those expressed in bacteria and maintain their heme-binding activity. HeSPs expressed through this P. pastoris expression system demonstrated remarkable anti-cancer efficacy in proliferation assays on NSCLC cells and SCLC (small cell lung cancer) cells. Experiments are underway to test their anti-tumor efficacy in mouse models. These experiments may suggest a novel approach to generate effective anti-cancer agents. Citation Format: Narges Salamat, Adnin Ashrafi, Tianyuan Wang, Zakia Akter, Hanling Gong, Li Zhang. Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1892.