Targeting the Mitochondrial Protease Clpp Unveils Mitochondrial Vulnerabilities in Multiple Myeloma

Mitochondria are enticing potential targets against cancer, owing to their role as signaling hubs orchestrating key homeostatic functions. Of special interest is ClpP, a major protease of the mitochondrial matrix, recently found essential for leukemic cell viability via maintenance of oxidative phosphorylation (OXPHOS) activity. Prompted by its distinctive expression in malignant plasma cells (PC), we investigated the role of ClpP in mitochondrial and cellular homeostasis in multiple myeloma (MM) cells and tested it as a possible anti-myeloma target.

MM cells were the highest ClpP-expressing human cancer cell lines. Moreover, we found ClpP mRNA significantly higher in bone marrow-purified PCs from newly diagnosed MM patients as compared to normal PCs from healthy donors. Attesting to a crucial role in myeloma, both acute shRNA-mediated knockdown (kd) and CRISPR/Cas9 knockout led to apoptosis and reduced proliferation. Intriguingly, toxicity in MM proved independent of the currently acknowledged ClpP-controlled mitochondrial functions, i.e., mito-ribosome assembly and OXPHOS maintenance. Indeed, not only did Seahorse analyses demonstrate no effect of ClpP kd on OXPHOS activity, but the toxicity of ClpP ablation extended to MM lines displaying a predominantly glycolytic metabolism, thus unveiling an energy-independent vulnerability. To unbiasedly define the role of ClpP in MM, we undertook a threefold orthogonal approach employing RNA-seq, proteomics, and metabolomics upon ClpP kd.

Metabolomic profiling showed significantly lower abundance of the active polyamines spermidine, spermine and putrescine in ClpP kd MM cells. Pathway enrichment analyses and arginine tracing experiments confirmed deranged polyamine biosynthesis as the main metabolic consequence of ClpP kd. Accordingly, RNA-seq and proteomics revealed concerted downregulation of polyamine biosynthetic enzymes, unveiling an unprecedented link between mitochondrial homeostasis and polyamine biosynthesis in the cytosol. Causally implicating polyamine depletion in MM cell toxicity, competitive inhibition of their biosynthesis with difluoromethylornithine led to growth arrest in a wide panel of MM cell lines. Spermidine is the precursor of the unusual amino acid hypusine, required for the post-translational activation of translation initiation factor eIF5A. Of note, specific inhibition of spermidine-dependent eIF5A hypusination with N-guanyl-diaminoheptane caused rapid apoptosis of MM cells.

In parallel, unbiased Gene Set Enrichment Analysis of RNA-seq upon ClpP kd identified highly significant up-regulation of GO terms describing a broad immune signature, including high type-I interferon (IFN) responses. Notably, all representative IFN-stimulated gene (ISG)-encoded proteins detected in parallel proteomic analysis were also significantly higher, along with MHC-I molecules. We also found that ClpP kd in MM cells causes cGAS-STING activation, as demonstrated by increased TBK1 phosphorylation and upregulation of a classical type-I IFN response, including heightened expression of IFNB1 (IFN-β) and a broad ISG panel. These data are consistent with the recent discovery of cGAS-STING activation associated to nucleoid disruption and mtDNA stress in Clpp^-/- fibroblasts and establish ClpP as a privileged entry point to study the immune effects of mitochondria-induced cGAS-STING activation in MM.

Overall, our data strongly suggest that ClpP is essential to MM cells due to a novel non-bioenergetic function, with both intra- and extra-cellular implications. Its manipulation unveils an unprecedented role of mitochondrial homeostasis in regulating polyamine biosynthesis and suggests an unexplored dependency of MM on polyamines for survival and proliferation. Furthermore, ClpP ablation generates intracellular activation of inflammatory pathways, paving the way for the exploration of mitochondria as targets to achieve cGAS-STING activation and increased anti-tumoral immunity against MM.

Munshi:Pfizer: Consultancy; GSK: Consultancy; Takeda Oncology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Janssen: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Legend: Consultancy.