A SNAP23-Dependent SNARE Complex Mediates Free Light Chain Secretion in AL Amyloidosis and Multiple Myeloma and Its Blockade Triggers a Terminal Unfolded Protein Response and Massive Apoptosis

Background

AL amyloidosis (AL) is an incurable plasma cell (PC) disorder with only one FDA-approved therapy. Deposition of immunoglobulin free light chain (FLC) fibrils in target organs is the sole pathogenic mechanism in AL. However, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, the specific targets of botulinum neurotoxin (BoNT), mediate the docking and fusion of secretory vesicles. We hypothesized that targeting specific sets of SNAREs via BoNT will block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR) and apoptosis in AL cells. We postulate that SNAREs are potential novel targets in AL and other plasma cell disorders.

Materials and Methods

We stably expressed 7 distinct, inducible BoNT serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1 and ALMC2) and multiple myeloma (MM, KMS11) cell lines via tetracycline-inducible (Tet-On) bicistronic lentiviral vectors with a P2A-GFP which allowed us to use GFP as a biomarker of BoNT expression. We established monoclones of successfully transduced cells for each BoNT. We performed flow cytometry to assess the kinetics of GFP induction and assess for apoptosis via AnnexinV/DAPI staining. Western blot (WB) was used to assess the cleavage of SNAREs upon expression of each BoNT serotype. We used WB and ELISA to assess FLC secretion per cell upon expression of each BoNT serotype. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and used co-IP for confirmation. We used RNP-based CRISPR-Cas9 gene editing to knock out (KO) putative BoNT targets.

Results

By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SNAP23, and SYNTAXIN4 as the top expressed SNAREs. Their high expression was retained in a large panel of AL and MM cell lines. Expression of all BoNT serotypes, except for BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for the extent and rapidity of cytotoxicity. Dual cleavage of SNAP23 and VAMP3 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not non-toxic BoNT B, led to decreased FLC secretion and increased FLC retention and triggered the PERK pathway of the UPR with increased expression of CHOP and GADD34, preceding the onset of apoptosis. We identified SNAP23 as an interacting partner of highly cytotoxic BoNT F, but not non-toxic BoNT B. KO experiment showed single SNAP23 KO to be partially toxic for AL and MM cell lines but did not recapitulate the effects of cytotoxic BoNTs entirely. Based on our preliminary data, we hypothesize VAMP3 and SYNTAXIN4 to be synthetic lethal with SNAP23.

Conclusions

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. By using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosisin AL cell lines, thus representing a novel, promising molecular target in AL amyloidosis and multiple myeloma.