Abstract B112: SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response

Abstract

Abstract The goal of this study was to investigate whether SNARE proteins are necessary for immunoglobulin (Ig) free light chain (FLC) secretion, thus representing a yet unexplored therapeutic target in AL amyloidosis (AL) and Multiple myeloma (MM). Both these disorders are incurable, and in the former, pathogenesis is invariably related to deposition of Ig FLC fibrils in target organs. The molecular mechanisms underlying FLC folding/secretion have not been studied, and no therapies directly target this intrinsic vulnerability. We hypothesize that FLC secretion is at least in part mediated by SNARE proteins, and their inhibition would block FLC exocytosis, causing retention of FLC-loaded vesicles, leading to activation of a terminal unfolded protein response (UPR) and apoptosis. We therefore hypothesized that novel, effective targets for MM and AL therapy could be identified within SNAREs. As a tool to study SNARE function, we stably expressed 7 distinct, tetracycline-inducible botulinum neurotoxin (BoNT) serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1, ALMC2) and MM (U266, KMS11) cell lines. To monitor BoNT expression, a bicistronic lentiviral vector with a P2A-GFP was used. Upon doxycycline administration, we performed growth competition assay in polyclonally transduced cells and assessed kinetics of GFP expression over time and apoptosis via flow cytometry in monoclones. Western blot (WB) was used to assess cleavage of SNAREs upon expression of each BoNT. We used WB and ELISA to assess FLC secretion on a per-cell base upon BoNT expression. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and CRISPR-Cas9 gene editing to validate putative BoNT targets. By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SYNTAXIN4, and SNAP23 as the top expressed SNARE and confirmed their high expression in a panel of AL and MM cell lines. Expression of all BoNT serotypes, except BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for extent and rapidity of cytotoxicity. Concurrent cleavage of SNAP23, VAMP3, and SYNTAXIN4 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not BoNT B, blocked FLC secretion and triggered the PERK-mediated terminal UPR with induction of CHOP and GADD34 followed by massive apoptosis. We identified SNAP23 as an interacting partner of cytotoxic BoNT F, but not BoNT B. Single SNAP23 KO did not completely recapitulate cytotoxic BoNT phenotype, as it was only partially cytotoxic. Based on our preliminary data, we hypothesize SYNTAXIN4 and VAMP3 to be synthetic lethal with SNAP23. Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. Using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosis, paving the way for the design of novel therapeutics. Citation Format: Emre Karayol, Maria Moscvin, Tianzeng Chen, Peter G. Czarnecki, Annamaria Gulla, Kenneth C. Anderson, Giada Bianchi. SNAP23-dependent SNARE complex is a novel molecular target in AL Amyloidosis and Multiple Myeloma by blocking free light chain secretion and triggering a terminal unfolded protein response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B112.