Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: Immunostimulatory activity and regulation of extracellular 2’3’-cGAMP in human multiple myeloma

Multiple myeloma (MM) is a multi-step hematological malignancy arising from the clonal proliferation of antibody-secreting cancerous plasma cells in the bone marrow. The progression of the disease, from the benign condition known as monoclonal gammopathy of undetermined significance (MGUS) to frank MM, is in part due to the gradual loss of tumor immunosurveillance caused by pathological alterations of the bone marrow microenvironment, which affect most cell populations, including mesenchymal stromal cells (BMSCs) and Natural Killer (NK) cells. A promising frontier in cancer immunotherapy is represented by the cytosolic DNA-sensing cGAS-STING pathway, a crucial defense mechanism of the innate immunity. The second messenger 2’3’-cGAMP (or simply cGAMP), the endogenous activator of the STING protein, is released by damaged tumor cells and uptaken by immune or stromal cells, which respond by secreting type I Interferons (IFNs) and other immunostimulating cytokines that can orchestrate a potent antitumor response. However, cGAMP is actively degraded by extracellular hydrolases like ENPP1, overexpressed in certain types of cancer and associated with metastasis development, which can therefore protect tumor cells from STING-mediated tumor clearance. During my Ph.D. I demonstrated the ability of MM cell lines to secrete cGAMP in the extracellular environment upon stimulation with the genotoxic drug doxorubicin (Dox). In addition, I investigated how Natural Killer cells and bone marrow mesenchymal stromal cells (BMSCs) respond to the treatment with exogenous cGAMP, showing that NK cells from healthy donors increase their cytotoxicity upon overnight treatment, whereas BMSCs from MM patients increase the expression of IFN-β and other cytokines. I further demonstrated that the MM cell line SKO-007(J3) undergoes apoptosis when challenged with high doses of cGAMP, while a lower-dose stimulation results in the increase of β-galactosidase activity, a typical marker of cellular senescence. Secondly, I collected evidence for a role of the ectoenzyme CD38, an important marker and therapeutic target of MM, in the enzymatic degradation of cGAMP. Inhibition of CD38 enzymatic activity on Dox-treated MM cell lines causes the accumulation of cGAMP in the conditioned medium (CM), as measured through competitive ELISA. Moreover, stimulation of patients-derived BMSCs with CM from CD38-inhibited MM cells increases the expression of IFN-β and other STING-activated genes; pre-treatment of the CM with snake venom phosphodiesterase (SVPDE), which can hydrolyze cGAMP, abrogates this effect. Ultimately, in collaboration with Dr. Annamaria Sandomenico (Institute of Biostructure and Bioimaging, National Research Council, Napoli, Italy), I accumulated biochemical data that further corroborate the hypothesis of cGAMP being substrate of CD38. In particular, incubation of human recombinant CD38 in the presence of increasing concentrations of cGAMP induces quenching at 345 nm, which corresponds to the maximum of fluorescence emission of tryptophan residues, indicating a direct interaction between CD38 and cGAMP. Further, time-course measurements of fluorescence recovery suggest that natural cGAMP is actively transformed by the enzyme. Finally, reverse phase HPLC and ESI-TOF mass spectrometry show the complete disappearance of cGAMP after incubation with recombinant CD38, indicating that the enzyme can degrade cGAMP into different chemical species.