Natural product discovery: investigations of immunogenic cell injury and cell selectivity
Balsamo, Joseph Anthony
Natural products isolated from bacteria impact many current anti-cancer medical interventions. Some natural products induce injury processes in malignant tumors that recruit and activate host immune cells to produce an adaptive anti-cancer immune response by a process known as immunogenic cell death. However, challenges remain to delineate forms of cell death and injury that best promote durable anti-tumor immunity. To evaluate functional consequences of injury to immunogenicity a new ten-color, fluorescent cell barcoding compatible module was developed to measure six immunogenic cell injury signals: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). These injury signatures were correlated to three markers of immunogenic cell death, ATP, HMGB1, and calreticulin, to catalog secondary metabolite induced injury profiles. Natural products with known mechanisms of action induced specific injury in leukemia models and afforded a proof-of-concept discovery paradigm that identified compound specific injury within chemically complex metabolomic extracts. Species within the Actinobacteria phyla were chemically or biologically stimulated to engage biosynthesis of and discover potentially immunogenic compounds. This discovery scheme first used multiplexed activity profiling to leverage fluorescent cell barcoding and fluorescent antibodies to compare stimulus specific compound production and dose dependent responses of cell injury markers. Active extracts were subsequently assessed for injury specific activity with multiplexed activity metabolomics, a platform that identifies bioactive secondary metabolites within mass spectrometric chromatographically-fractionated metabolomes. This workflow uncovered an unexpected injury profile induced by the macrolide narbomycin, discovered DNA damage induced by the pyridine-pyrrolidine alkaloid siderochelin, observed non-specific cytotoxicity exhibited by the filipin family of polyene macrolides, and identified a unique injury response induced by the anthracycline isoquinocycline b. Follow up single cell chemical biological analysis of isoquinocycline b demonstrated differential cell type and subtype targeting among anthracyclines in healthy peripheral blood mononuclear cells and leukemia patient samples. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulated injury response pathways that can contribute to cancer immunogenicity and be extended to investigate the therapeutic potential for both new and known compounds in human leukemias.