Fig. 1. Plasma cells from patients with AL amyloidosis are primed for apoptosis and dependent on pro-survival proteins based on BH3 profiling.
a Analysis overview: mononuclear cells were isolated from bone marrow aspirates from patients diagnosed with AL amyloidosis. One portion was stained with anti-CD38 and anti-CD138 antibodies then permeabilized with digitonin and exposed to BH3 peptides. After 60 minutes of exposure, cells were fixed and stained with anti-cytochrome c, anti-kappa and anti-lambda antibodies and analyzed by flow cytometry to determine the percentage of cells that had released cytochrome c (indicating MOMP) in response to each BH3 peptide. A second portion of cells were treated in vitro with drugs for either 24 or 48 h and then stained with anti-CD38, anti-CD138, anti-kappa, anti-lambda antibodies, as well as Annexin V. Viability of cells was then assessed by flow cytometry. Created with BioRender.com. b Mononuclear cells were isolated from patient-derived bone marrow aspirates across 40 patients that were not being treated at time of sample collection and BH3 profiled. Cells were exposed to pro-apoptotic BH3-only sensitizer peptides BIM, BID, and PUMA for 1 h, then cytochrome c loss was measured by flow cytometry in clonal plasma cells (n = 40). c Patient samples were stratified into treatment naïve or relapsed subgroups according to their treatment status at the time of collection (n = 9, n = 31). d Plasma cells were BH3 profiled and exposed to peptides that inhibit pro-survival proteins to determine anti-apoptotic protein dependence. e Patient samples were stratified based on their treatment status at the time of collection. f Comparison of BCL-2/BCL-XL dependence versus MCL-1 dependence across 40 patients that were not being treated at time of sample collection. P-values were calculated using one-way ANOVA with Holm-Sidak’s adjustment for b, d and two-way ANOVA for c.