Inhibition of the Sec61 Translocon Promotes Immunoglobulin Light Chain Dependent Cell Stress and Apoptosis: Therapeutic Implications for AL Amyloidosis

Immunoglobulin light chain (AL) amyloidosis is an incurable hematologic disorder characterized by the production and secretion of misfolding immunoglobulin light chains (LC) by a small and slowly proliferating clone of abnormal plasma cells. These LCs misfold and form β-pleated amyloid fibrils in healthy tissues, causing major organ dysfunction and death. Treatment for AL amyloidosis is largely focused on the elimination of diseased plasma cells using proteosome inhibitors, immunomodulatory agents and monoclonal antibodies. However, these treatments have limited efficacy in AL amyloidosis, making the development of novel therapeutic strategies a significant clinical challenge, especially for the subset of patients with cardiac involvement at time of diagnosis. We explored whether inhibition of LC secretion with small molecule inhibitors of the Sec61 translocon could be an effective strategy in this disease. In addition to directly lowering the circulating levels of the toxic LC in patients, we hypothesize that inhibition of LC translocation would be a potent stressor to AL cells. This was based on the fact that plasma cells are among the most highly secretory cells in the human body. The increased secretory capacity of plasma cells is accompanied by an increased reliance on the ubiquitin-proteasome system to degrade misfolded proteins, including misfolded LCs, and concomitant sensitivity to perturbations that cause proteotoxic stress. Work from others previously demonstrated that a natural compound Sec61 inhibitor (mycolactone) could drive apoptosis in myeloma cells (Domenger et al, EMBO Molecular Medicine, 2018). Here we set out to confirm that Sec61 inhibition induced apoptosis also occurred in the context of AL amyloidosis, and to test the hypothesis that inhibition of LC translocation through the Sec61 channel contributed to apoptosis induction. We began by confirming that two Sec61 inhibitor tool compounds (MG-001 and MG-006) effectively block LC secretion from the AL amyloidosis cell line, ALMC-1, and validated that the inhibition of LC by these compounds was Sec61 dependent. MG-001 and MG-006 reduced ALMC-1 cell viability, and induced apoptosis and cell stress gene signatures in ALMC-1 cells, both as single agents and synergistically with the standard of care proteasome inhibitor, bortezomib. In contrast, we did not observe apoptosis in response to MG-001 or MG-006 in peripheral blood mononuclear cells. We next asked whether inhibition of LC translocation (rather than other Sec61 clients) contributed to MG-001 and MG-006 induced cytotoxicity. To do this we employed two approaches-in the first approach we knocked down the expression of LC with siRNA (previously shown to be a stressor in itself, Zhou et al, Blood, 2014) and treated with MG-001. Knocking down LC partially reversed the effect of MG-001 on cell viability, indicating that inhibition of LC translocation was involved MG-001 induced cytotoxicity. As a second approach, we screened our compound library for molecules that selectively inhibited the secretion of certain LC variable domains but not others. One tool compound (MG-566) was able to selectively inhibit the secretion of an IGLV1-44 LC over the majority of other known Sec61 clients. Treatment of an IGLV1-44-LC secreting cell line (KHM1B) with the selective compound MG-566 induced a similar stress response signature to MG-001 and MG-006 (which are not selective for particular Sec61 clients). MG-566 did not induce a stress response in cell lines secreting LC with other IGLV domains. This suggests that selective LC inhibition is sufficient to induce a stress response, and future work will be aimed at determining the mechanism by which this stress is induced. In summary, our work demonstrates that inhibition of co-translational translocation of LC via inhibition of Sec61 blocks its secretion and promotes stress and apoptosis in AL cells, thus providing a strong rationale for pursuing LC secretion inhibitors as a therapeutic strategy for AL Amyloidosis.

Antonia:NGM Biopharmaceuticals: Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Yu:Harpoon Therapeutics (Merk): Ended employment in the past 24 months. Henderson:Pfizer: Ended employment in the past 24 months. Zancanella:Global Blood Therapeutics (Pfizer): Ended employment in the past 24 months.