P-104: Targeting the mitochondrial protease CLPP in Multiple Myeloma

Background Mitochondria are enticing potential targets against cancer, owing to their role as signaling hubs orchestrating key homeostatic functions. Of special interest is ClpP, a resident mitochondrial protease suggested to maintain OXPHOS by degrading damaged protein components and regulating the assembly of mito-ribosomes. While the exact role of ClpP in mammals remains unclear, its manipulation has been shown to induce leukemic cell death. Prompted by its distinctive expression in malignant plasma cells (PCs), we investigated the role of ClpP in maintaining mitochondrial and cellular homeostasis in multiple myeloma (MM) cells and tested it as a possible anti-myeloma target. Methods We analyzed the expression of ClpP mRNA in public and proprietary datasets of normal and malignant PCs and MM cell lines. We performed stable shRNA-mediated knockdown of ClpP (ClpPkd) in MM cell lines and analyzed its sequelae combining electron microscopy, Seahorse and ATP assays, transcriptomics, proteomics, and metabolomics. A proteolytically inactive ClpP mutant (ClpPmut) was expressed to entrap ClpP substrates for subsequent mass spectrometry identification and wet validation. Results ClpP mRNA was significantly higher in bone marrow-purified malignant vs. normal PCs, and MM cells were the highest ClpP-expressing human cancer cell lines. Attesting to a crucial role in myeloma, ClpPkd MM cell lines disappeared from culture due to rapid onset of cell cycle arrest and apoptosis. Intriguingly, toxicity in MM proved independent of the currently acknowledged ClpP-controlled mitochondrial functions, i.e., mito-ribosome assembly and OXPHOS maintenance. Indeed, Seahorse demonstrated different bioenergetics across MM lines, ranging from mixed oxidative/glycolytic to almost exclusively glycolytic. Yet, ClpPkd failed to abate ATP in glycolytic MM lines, but proved equally toxic across all lines, 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. Their integrated analysis upon ClpPkd revealed an unexpected impact of ClpPkd on protein translation in the cytosol via the processing of nuclear-encoded RNA, coupled with metabolic changes indicative of impaired fatty acid oxidation and pentose phosphate pathway. Finally, mass spectrometry of ClpPmut-entrapped partners identified myeloma-specific mitochondrial substrates, including chaperones and enzymes involved in RNA metabolism and oxidative stress. RNA-seq analyses are further characterizing the pathways impacted by ClpPkd and guiding wet validation experiments. Conclusions Overall, our data strongly suggest that ClpP is vital to MM cells due to a novel non-bioenergetic function, and that its manipulation is lethal via a broad perturbation of mitochondrial and cellular homeostasis.