G-quadruplex stabilization potentiates anti-tumor immune responses and sensitizes cold tumors for checkpoint therapy

Abstract

Checkpoint inhibitors have revolutionized cancer therapy and improved patient survival in many cancers. However, even in well-responding entities such as melanoma, about 50% of patients are primarily resistant or develop resistance throughout the therapy. Among non-responding tumors, so-called "cold" tumors are found particularly often. These are tumors that contain only low amounts of tumor-infiltrating lymphocytes, especially T cells. Therefore, enhancing T cell infiltration into the tumor microenvironment is a promising approach to increase response rates to checkpoint therapy. <br/> G-quadruplexes, DNA secondary structures that can form in guanine-rich sequences, are important for genome stability, telomere stability, and oncogene expression. Small molecules binding these structures (G4 ligands) were shown to inhibit cancer cell growth by causing apoptosis through DNA damage induction, reducing telomerase activity, and down-regulation of critical oncogenes like KRAS. Recent data further showed that G4 ligands can cause the activation of STING in cancer cells, followed by the induction of a type 1 interferon response <em>in vitro</em>. Additionally, it was shown that G4 ligands can induce markers of immunogenic cell death <em>in vitro</em>. Together these observations led to the hypothesis that G4 ligands, besides their direct apoptotic and anti-proliferative effects, could also harbor the potential to increase the immunogenicity of tumors and thereby sensibilize tumors for checkpoint therapy. <br/> To test this hypothesis the checkpoint therapy-resistant B16OVA melanoma mouse model was used to investigate the effect of intratumoral PDS application on checkpoint therapy.<br/> PDS treatment alone led to a significant reduction in tumor growth and prolonged survival of B16OVA-bearing mice. Additionally, mice receiving PDS responded to checkpoint therapy, leading to further increased survival and a complete response in 20% of double-treated animals. Using single-cell sequencing this study could show that PDS directly and PDS-induced DAMP release activate intratumoral myeloid cells, leading to increased production of pro-inflammatory cytokines and chemokines. In turn, on day nine post-treatment tumor immune cell infiltration is two-fold increased, and especially T cells and NK cells are three-fold enriched compared to vehicle-treated tumors. In addition, cytotoxic T cells and NK cells both produce increased levels of effector molecules, especially granzymes. <br/> Besides the effects on immune cells, this study could also show that PDS increases the immunogenicity of B16OVA cells <em>in vivo</em>. PDS treatment induces the expression of CCL5 and CXCL10, increases the expression of antigen processing and presentation on both MHC I and MHC II, and induces non-silent mutations in actively expressed genes, potentially leading to the formation of neo-antigens.<br/> In sum, PDS has pro-inflammatory effects on both tumor cells as well as different immune cell subsets, transforms the TME towards an immunological hot state, and thereby induces a response to checkpoint blockade in originally resistant B16OVA tumors.