Multiple myeloma (MM) remains an incurable malignancy despite major therapeutic advances. Immunotherapies are limited by the immunosuppressive tumor microenvironment The cGAS–STING pathway, a cytosolic DNA sensor inducing type I interferon (IFN-I), is a promising strategy to convert “cold” tumors into immunogenic ones. We showed that silencing the mitochondrial protease ClpP in MM cells induces intrinsic cytotoxicity and cGAS–STING–dependent IFN-I activation with immunostimulatory effects. We hypothesize that perturbation of mitochondrial homeostasis may unveil novel immunogenic vulnerabilities in MM.

To dissect the immunogenic mechanism of ClpP silencing, we will assess the role of cytosolic mtDNA and mtRNA leakage. Moreover, ad-hoc engineered fluorescent IFN-I reporter cell lines and a custom sgRNA library targeting the mitochondrial proteome will be used in a CRISPR/Cas9 screen to identify genes whose silencing induces immunogenic effects

First, we tested if mtDNA release via VDAC (Voltage-Dependent Anion Selective Channel) is the cause of the cGAS-STING response observed upon ClpP manipulation. To this aim, we silenced ClpP in presence of an inhibitor of VDAC, a component of the mitochondrial permeability transition pore (mPTP) essential for mtDNA leakage. We found that VDAC inhibition was not sufficient in preventing the IFN-I response activation, suggesting mtDNA leakage is linked to other mPTP components. Interestingly, we observed that treatment with the STING agonist ADU-S100 induces a significantly weaker IFN-I response in MM cells compared to THP1, a monocytic leukemia cell line. On the opposite, treatment with poly(I:C), a synthetic dsRNA analog, successfully triggered a potent IFN-I response in both cell lines. These findings, together with our published evidence of sizeable but modest cGAS-STING activation upon ClpP inhibition, suggest the exsistence of an unprecedented epigenetic regulation or signaling defect of the cGAS-STING axis in MM cells, limiting the expression of interferon-stimulated genes (ISGs) in response to cytosolic DNA. Based on these observations and considering our preliminary data on mitochondrial vulnerabilities in MM cell, we designed a sgRNAs library targeting the entire nuclear-encoded mitochondrial proteome to perform a CRISPR/Cas9 screening aimed at identifying novel targets whose silencing induces a potent activation of IFN responses in MM cells.

This project will investigate novel mitochondrial vulnerabilities in MM cells to offer new actionable targets and broaden the therapeutic arsenal against this tumor. Targeting mitochondrial homeostasis may represent a novel approach to couple a direct cytotoxic effect together with immunogenic stimulus.

This work was supported by grants to T.P. from Fondazione Cariplo (Giovani Ricercatori 2023- 1298), and to S.C. from Fondazione AIRC (Investigator Grant 2024-30332).