Involvement of reverse signaling via NKG2D ligands in cancer persistence

• Applicant: Professor Dr. Helmut Rainer Salih
• Subject Area: Hematology, Oncology ; Immunology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555977613

Experimental data and clinical evidence like disease relapse after many years of remission indicate that tumor cells often are not eliminated completely after therapeutic intervention. The persisting minimal residual disease (MRD) is enriched for so-called tumor initiating cells (TIC), which mostly are in a state of quiescence that facilitates resistance to treatment and also antitumor immunity. NKG2D is an activating receptor expressed on cytotoxic lymphocytes which potently stimulates antitumor immunity upon recognition of its various MHC class I-related ligands (NKG2DL) that are widely expressed on malignant cells but generally absent on healthy tissue. Notably, despite expression of immunostimulatory NKG2DL, MRD/TIC are frequently not eliminated by antitumor immunity. Recently it was shown that classical MHC I molecules themselves can transduce signals (reverse signaling), which influences important cellular functions of the MHC I expressing cells. Based on (i) the structural relationship between NKG2DL and MHC I as well as (ii) the seemingly counterintuitive fact that residual tumor cells still express immunostimulatory NKG2DL, we reasoned that also NKG2DL could transduce signals. This in turn might induce a state and/or microenvironmental milieu favorable for tumor (initiating) cells, thereby overcompensating the immunostimulatory properties of NKG2DL expression. Preparative analyses revealed that NKG2DL signaling indeed induces release of cytokines which act as pro-survival factors for malignant cells, affects central signaling pathways and mediates chemotherapy resistance in acute myeloid leukemia (AML) cells. In the proposed project, we will analyze the mechanisms underlying NKG2DL signaling and the consequences for cancer stemness as well as resistance to treatment and antitumor immunity. We plan to unravel the molecular mechanisms underlying signaling via NKG2DL using various sophisticated methods like co-immunoprecipitation followed by mass spectrometry, RNASeq/bioinformatic data analysis as well as kinase activation arrays followed by intracellular phospho-flow cytometry and Western blot in the presence or absence of specific inhibitors. Key regulatory elements will further be validated after modifying NKG2DL and/or signaling domains/adaptor molecules using CRISPR-Cas9 technology. In addition, we will study the consequences of NKG2DL signaling for leukemia stemness as well as susceptibility to chemotherapy and (therapeutically induced) antitumor immunity including therapeutic antibodies using AML cells with and without modification by CRISPR-Cas9 in various in vitro analyses and by xenografting (modified) AML cells in NSG mice. Finally, we will evaluate clinically available small molecule inhibitors for targeting the mechanisms underlying NKG2DL by taking advantage of the above described in vitro and in vivo models with the ultimate goal to identify candidate drugs to be evaluated in a clinical study.

• DFG Programme: Research Grants