Trabectedin and olaparib combination primes cellular immunotherapy with HLA-independent killer lymphocytes against sarcoma preclinical models through the cGAS-STING pathway

Background. Advanced sarcomas have a severe prognosis, limited therapeutic options, and are seldom sensitive to immunotherapy with checkpoint inhibitors. Novel strategies are urgently needed. Methods. We set up a preclinical approach for testing the combination of chemotherapy (trabectedin) and PARP1 inhibitor (olaparib) to potentiate the HLA-independent cellular immunotherapy with natural killer (NK) or cytokine-induced killer (CIK) lymphocytes. The NK/CIK activators NKG2DLs were studied by qRT-PCR and flow cytometry. Drug-induced transcriptomic events were studied by RNA sequencing and the activation of the cytosolic DNA sensing pathway (cGAS-STING-IRF3-IFNβ) was explored. Focusing on the putative cancer stem cell (CSCs) subpopulation, OCT4 marker was evaluated. IRF3 silencing experiments were done to investigate the role of the cGAS-STING pathway in the control of NKG2DLs. NK/CIK killing activity on both bulk and OCT4-positive cells was tested by viability assay and flow cytometry, respectively. Results. We obtained the enhancement of NK/CIK activity against sarcoma cell lines through an integrated approach based on the combination of trabectedin and olaparib. These drugs upregulated the NKG2DL expression at both mRNA and protein level by inducing the genotoxicity pathway (WP4286) and repressing the cellular response to DNA damage stimulus (GO:0006974). The drug treatment significantly increased the cytosolic DNA content and consequently activated the cGAS-STING-IRF3 pathway and IFNβ production. These molecular events significantly enhanced the antitumor activity of NK and CIK against drug-pretreated sarcoma cells. The OCT4-positive cell fraction was enriched after chemotherapy. Noteworthy, NK/CIK cells efficiently killed both the drug-surviving bulk and CSC subpopulation. Conclusions. Trabectedin+olaparib treatment followed by NK/CIK-based immunotherapy is a promising therapeutic strategy against sarcomas, deserving further investigations.