Identification of Endoplasmic Reticulum Stress Inducing Agents by Antagonizing Autophagy: A New Potential Strategy for Identification of Anti-Cancer Therapeutics in B-Cell Malignancies.
Authors: Mahoney Emilia , Cole Sara L , Maddocks Kami , Flynn Joseph M. , Jones Jeffrey A. , Zhang Xiaoli , Byrd John C. , Johnson Amy J. 
Published: October 2019

Abstract
Abstract 2473
Chronic lymphocytic leukemia, a B-cell malignancy, is the most common adult leukemia and it lacks curative therapy. There are numerous ongoing investigations aimed at finding new therapeutics for this disease.
Endoplasmic reticulum (ER) is an organelle which plays a vital function in the cellular processes, such as calcium homeostasis, calcium mediated signaling pathways, protein synthesis, folding and modification, synthesis of lipids necessary for cellular structure and signaling. There has been growing interest in developing therapeutic agents that are able to target the endoplasmic reticulum in cancer cells, inducing a stress response that ultimately leads to cell death. Several such agents (imatinib in CML, bortezomib in AML, resveratrol in MM) have been described for use in hematologic malignancies.
ER stress also induces autophagy as a strategy of survival of the cancer cells when exposed to the ER stress inducing agents. Autophagy consists in formation of double membrane vesicles called autophagosomes, which engulf damaged organelles and proteins within the cytoplasm and subsequently fuse with lysosomes, their cargo being then digested by the lysosomal enzymes.
It is of interest to inhibit autophagy to increase the efficacy of the ER stress inducing agents. Nelfinavir, an FDA-approved, HIV protease inhibitor which has been recently described to have anti-cancer properties, has the ability to induce ER stress. No previous literature has described nelfinavir activity in chronic lymphocytic leukemia (CLL) cells.
We demonstrate here that nelfinavir induces significant morphological changes in endoplasmic reticulum by using confocal fluorescence microscopy for calnexin (protein marker for ER) in a time series experiment, where primary CLL cells were treated in vitro with vehicle (DMSO), nelfinavir, thapsigargin (ER stress inducer) or F-ara-A (fludarabine) for 2, 4, 6, 8 or 24 hours. These morphological changes were also observed using live cell imaging of primary CLL cells stained with ER Tracker Red and treated with vehicle (DMSO), nelfinavir, thapsigargin and F-ara-A for 12 hours. Nelfinavir-induced ER stress response in CLL was confirmed by the increased ER stress marker gene expression IRE1, XBP1, GRP78 and CHOP, detected by RT-PCR analysis, as well as an immunoblot for Grp78. This stress response is followed however by minimal cytotoxicity, as shown by viability assay performed on CLL cells stained with Annexin V and PI. We also observed an induction of autophagy in CLL cells in the presence of nelfinavir, detected by confocal fluorescence microscopy for LC3 and immunoblot for both LC3 and p62/SQSTM1, two markers of autophagy flux. Interestingly, the autophagy inhibitor chloroquine, an anti-malarial drug, increases significantly nelfinavir cytotoxicity.
Endoplasmic reticulum (ER) stress is one mechanism of tumor cytotoxicity in CLL and other cancers for which new agents are being sought. We identify a potential strategy to enhance identification of agents which induce ER stress by combination with the anti-malarial drug chloroquine that is applicable to both repurposed drugs and also new molecules.

Disclosures:
No relevant conflicts of interest to declare.