Physician scientist IRCCS Ospedale San Raffaele Milan, Lombardia, Italy
Introduction: Mitochondria orchestrate key homeostatic functions in cancer cells. Moreover, due to their bacterial origin, they can release potent immunogenic signals in the cytosol. ClpP, a resident protease of the mitochondrial matrix, has been proposed as a target in OXPHOS-dependent cancer cells, and its ablation is associated with mitochondrial DNA (mtDNA) leakage-driven cGAS-STING activation in fibroblasts. Prompted by the distinctive expression of ClpP in malignant plasma cells (PC) and by the value of activating STING to awaken immunity, we here evaluated ClpP as a possible target with both cell-intrinsic and immunostimulatory effects against multiple myeloma (MM).
Methods: We manipulated ClpP expression in MM cell lines, by both stable and inducible shRNA-mediated knockdown (KD), and assessed tumor growth in vitro and in vivo. We evaluated the effects of ClpP KD by Seahorse bioenergetic profiling, transcriptomics, proteomics, metabolomics and metabolite tracing experiments. Activation of cGAS-STING and downstream effects on immune cells were evaluated in coculture experiments of MM, dendritic and T cells.
Results: ClpP mRNA was significantly higher in bone marrow-purified malignant vs. normal PCs, and MM cell lines showed exquisite sensitivity to both acute and inducible KD of ClpP in vitro. Importantly, inducible silencing of ClpP in MM cells delayed tumor growth and prolonged survival in immunodeficient mice. Toxicity proved independent of the currently acknowledged ClpP-controlled mitochondrial functions (mainly OXPHOS surveillance and maintenance of ATP production). Indeed, an integrated proteomic and metabolomic approach revealed derangement in citric acid and urea cycles, together with depletion of the polyamines spermidine, spermine and putrescine upon ClpP KD. Concordantly, inhibition of spermidine biosynthesis or of its usage phenocopied ClpP KD toxicity MM cells. In parallel, unbiased proteomics and RNA-seq, followed by targeted biochemical validation, identified transcriptional upregulation of IFN-stimulated genes and activation of cGAS-STING upon ClpP KD. In line with a downstream secretion of type-I IFNs and proinflammatory molecules, ClpP KD MM cell supernatants significantly boosted maturation and activation of human dendritic cells and enhanced their ability to stimulate IFN- production by CD8+ T cells.
Conclusions: Overall, our data demonstrate that ClpP is essential to MM cells due to a novel non-bioenergetic function, with both intra- and extra-cellular implications. ClpP manipulation unveils an unprecedented role of mitochondrial homeostasis in regulating polyamine biosynthesis and an unexplored dependency of MM on polyamines for survival and proliferation. Furthermore, ClpP ablation activates cGAS-STING in MM cells, paving the way for the exploration of mitochondria as targets to stimulate otherwise indolent anti-tumoral immunity against myeloma.
