Abstract LBA010: Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors
Authors: Brägelmann Johannes , Lorenz Carina , Borchmann Sven , Nishii Kazuya , Wegner Julia , Ostendorp Jenny , Ast David , Heimsoeth Alena , Lohneis Philipp , Zillinger Thomas , Ullrich Roland , Ohashi Kadoaki , Schlee Martin , Sos Martin 
Published: December 2021

Abstract
Introduction: A major challenge in the treatment of kinase driven tumors is the inevitable emergence of resistance and the limited efficacy of subsequent treatments including immunotherapy. Methods: To gain deeper insights into the adaptive processes of cells surviving kinase inhibition we studied the mechanisms enabling cell persistence despite targeted treatment across various in vitro and in vivo tumor models. This included transcriptomic profiles of EGFR-, BRAF-, HER2- or ALK-driven colon, melanoma and lung cancer cells and of melanoma patients during kinase inhibition. To investigate underlying mechanisms we employed CRISPR/Cas9 and overexpression experiments and stimulation of nucleic acid receptors (NARs) TLR3, cGAS and RIG-I with synthetic agonists to assess combination therapies. Finally, we used humanized tumor xenografts and a syngeneic EGFR-mutant lung cancer model to validate the inflammatory effects of kinase inhibition and the efficacy of adding RIG-I agonist IVT4 to kinase inhibition in vivo. Results: Analysis of dynamic transcriptomic changes during kinase inhibition by time-series RNA-seq of oncogene-dependent cells displayed rapid induction of an interferon gene signature and suppression of MYC- and E2F-target genes. This was accompanied by induction of p27 protein levels, cell cycle arrest and an increase of b-galactosidase positive cells, indicating a senescence-associated inflammatory response that occurred in a cell autonomous manner in vitro and in vivo. In vivo, EGFR inhibition also increased T-cells, but anti-PD-1 therapy following EGFR inhibitor pre-treatment did not prevent tumor recurrence. Interestingly, the kinase inhibitor induced interferon signature was independent of caspase activation and of the nucleic acid sensing machinery. Instead, functional experiments indicated that induction of interferon signaling was dependent on inhibition of MAPK signaling and subsequent IRF1 activation, providing a potential mechanism of action. Numerous innate immune pathway members including NARs MDA-5, TLR3 and RIG-I were upregulated, which we aimed to exploit therapeutically. Evaluation of NAR agonists showed that only stimulation of RIG-I with its agonist IVT4 evoked cytokine secretion and synergistically enhanced cell death in vitro. In humanized xenografts and syngenic immunocompetent mouse models adding IVT4 led to additional tumor shrinkage after EGFR-inhibition and decreased T cell exhaustion. Effects of IVT4 on tumor volumes were lower in immunodeficient xenografts and after NK- or CD8-cell depletion in the syngeneic model indicating that immunological influences in addition to tumor cell intrinsic effects contribute to the efficacy in vivo. Conclusion: Taken together, our findings show that kinase inhibition may promote tumor cell-autonomous, senescence-associated inflammatory signaling and may sensitize oncogene-driven tumors to therapeutic intervention using RIG-I agonist IVT4. Our results thus provide a novel treatment strategy and a basis to explore combining immunotherapy with kinase inhibition in the future.
Citation Format: Johannes Brägelmann, Carina Lorenz, Sven Borchmann, Kazuya Nishii, Julia Wegner, Jenny Ostendorp, David Ast, Alena Heimsoeth, Philipp Lohneis, Thomas Zillinger, Roland Ullrich, Kadoaki Ohashi, Martin Schlee, Martin Sos. Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA010.