Targeting the Secretory Pathway of the Proteostasis Network as a Novel Therapeutic Approach in AL amyloidosis

Amyloid light chain (AL) amyloidosis is a plasma cell disorder, whose pathogenesis is related to the overproduction and extracellular deposition of misfolded immunoglobulin (Ig) free light chain (FLC) fibrils in target organs, leading to progressive organ failure. Although treatable, AL amyloidosis is incurable, with only one FDA approved therapy, highlighting an urgent, unmet clinical need. Targeting the proteostasis network (PN) has long since been a therapeutic interest in plasma cell disorders, as plasma cells suffer from baseline proteotoxicity. Exploiting this intrinsic vulnerability is key to the development of therapies with curative potential. This intercalated MSc thesis endeavoured to target the PN as a therapeutic approach to AL amyloidosis. In particular, the molecular mechanisms underlying FLC secretion, for which therapeutic strategies directly targeting do not exist. It is postulated that AL plasma cells are highly dependent on the PN to sustain FLC secretion. Thus, identification of novel therapeutics within the PN secretory pathway would be selectively toxic to AL plasma cells by blocking FLC exocytosis and irreversibly compromising proteostasis. Identification of molecular machinery involved in FLC secretion involved a combination of broad searches in genomic databases (UniProt and GeneOntology), extrapolation of existing literature and collaboration with bioinformatics labs. Criteria for involvement included an “entity-based” and “domain-based” approach. This culminated in an annotated list of 1208 genes involved in the secretory pathway of plasma cells. A high throughput electroporation system for the purpose of CRISPR screening was optimised for the amyloidosis cell line (ALMC1). Variables optimised for maximal transfection efficacy and viability included voltage, pulse length, pulse number, cell concentration and type of electroporation buffer. Following optimization, genes with known toxicity in other plasma cell dyscrasias were tested as positive control candidates. Downstream analysis involved viability readouts, such as spectrophotometer fluorescence and flow cytometry, as well as knockout confirmation via western blot analyses. To fortify the rationale and provide clinical relevance for targeting FLC secretion as a novel therapeutic in AL amyloidosis, a clinical study was conducted on the prognostic value of FLC burden in AL patients. It was postulated that patients with higher FLC burden would respond more rapidly and deeply to Bortezomib-containing (PN disrupting) regimens, as FLC-derived proteotoxicity is the foundation of Bortezomib effectiveness in plasma cell disorders. Analysis of patient FLC serum levels over time suggested that, in the current treatment landscape, patients with high baseline FLC levels responded equally, if not better than, patients with low baseline FLC levels. These findings carry significant implications in the development of future staging systems, and warrant further investigation of therapies targeting the PN.