The role of NFAT2 for susceptibility of tumor cells to NK cell mediated antibody dependent cellular cytotoxicity

• Applicant: Privatdozentin Melanie Märklin, Ph.D.
• Subject Area: Hematology, Oncology ; Immunology ; Clinical Immunology and Allergology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 496349479

In many cancer entities, the introduction of monoclonal antibodies (mAbs) has significantly improved the treatment options for patients. This is exemplified by the anti-CD20 mAb Rituximab and the anti-Her2/neu mAb Trastuzumab, the first antitumor antibodies that became clinically available and meanwhile are a mainstay of therapy in patients with Chronic Lymphocytic Leukemia (CLL) and Her2-positive (Her2+) breast cancer (BC), respectively. Nevertheless, the success of antibodies in cancer treatment has its limitations: many patients do not respond at all, others for limited time only, with occurrence of resistance to mAb treatment contributing to the latter. In preparative work we identified the transcription factor NFAT2 as essential player in resistance to antibody-dependent-cellular-cytotoxicity (ADCC), a major mechanisms contributing to therapeutic efficacy of mAbs. Moreover, we previously identified loss of NFAT2 as a main determinant for CLL transformation from indolent disease to Richter’s syndrome, a highly aggressive form of B cell lymphoma. In line, in Her2+ BC, low NFAT2 expression levels were found to be linked to decreased overall and progression free survival. These findings underline the relevance of NFAT2 in pathophysiology as well as treatment resistance of CLL and BC.Within the proposed project, we aim to elucidate the mechanisms underlying NFAT2 mediated resistance to ADCC in CLL and Her2+ BC. To this end we will utilize a lentiviral CRISPR/Cas9 based model system to delete NFAT2 in CLL and Her2+ BC cell lines and primary malignant cells. We will conduct in vitro cytotoxicity analyses with these knockout cell lines to unravel resistance to ADCC and validate the results with PBMCs of CLL patients, the leukemic Eµ-TCL1 mouse model and patient-derived organoids of Her2+ BC patients. Since perforin and granzymes are major effector molecules underlying ADCC, which in humans is mediated mainly by NK cells, we aim to specifically analyze pore formation by perforin, granzyme-induced apoptosis and endocytosis of both effector molecules. We will use imaging and analysis approaches including immunofluorescence, flow cytometry and proteomics. Candidate genes, mediating the NFAT2 effects, are identified by a prior RNA-Seq analysis. shRNA libraries targeting these genes will be used to perform for RNAi screening to identify potential target genes for development of new treatment strategies. Physiological relevance of the identified candidate genes will be characterized in different in vivo xenograft models of leukemia upon treatment with Rituximab. Disease progression and therapeutic efficacy will be followed by optical bioluminescence imaging (BLI) and flow cytometric analyses. Ultimately, these analyses will unravel the involvement of NFAT2 in resistance of CLL and Her2+ BC to mAb treatment and hold promise to develop optimized immunotherapeutic approaches.

• DFG Programme: Research Grants