Role of calcineurin in affecting tumor growth and the formation of the tumor microenvironment

Mihai Valache1,4, Laura Marongiu1,4, Francesca Mingozzi1, Fabio Facchini1,4, Roberto Spreafico2, Renato Ostuni3, Angelo Lombardo3, Francesca Granucci1,4 1Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2, 20126, Milan, Italy 2Vir Biotechnologies, Inc., San Francisco, California. 3Università Vita-Salute San Raffaele, Milano. 4 National Institute of Molecular Genetics “Romeo ed Enrica Invernizzi”, Milano.

Introduction & Objectives: The purpose of this project is to investigate the role of the Ca2+-dependent phosphatase Calcineurin (CN) in promoting tumor growth and shaping the immune tumor microenvironment. Calcineurin, has been reported to be involved in many different tumors. In this study we aim to characterize the phenotypical and functional changes induced by the constitutive inhibition of calcineurin interaction with substrates.

Method/Description: A series of murine tumor cell lines have been genetically engineered to constitutively block the docking site of calcineurin with substrates. The growth and tumor-formation capacity of these cells have been evaluated in vivo, and the immune infiltrating populations have been analyzed by flow cytometry. Their transcriptional profile has been characterized by qPCR and bulk RNA sequencing.

Results: We found that despite significantly larger precursor exhausted CD8+ T cell infiltrated mutant tumors. By analyzing the RNAseq data, CXCL11/10/9 have been pinpointed as differentially expressed cytokines with CD8+ T cell chemotactic activity. This has been confirmed in vivo in tumor generated by mutant B16 cells. In addition, IL-15 was expressed by the immune infiltrate, suggesting it could be responsible for the expansion of precursor exhausted cells. According with the accumulation of CD8+ T cells, the administration of an anti-immune checkpoint inhibitors resulted in a greatly diminished tumor growth.

Take Home Message/Conclusions: Our studies highlight the role of calcineurin in tumor development and how the modulation of its activity can contribute to the differential recruitment of tumor-infiltrating immune populations. In particular, there is a higher recruitment of CD8+ T cells, possibly due to the overexpression of T cell chemotactic factors. Despite the apparent exhausted phenotype, it can be effectively reversed by immune checkpoint inhibition. Calcineurin emerges as a potential target for novel therapeutic approaches, particularly as a potent aid to already existing immune checkpoint blockade therapies.

• Describe funding body that supported the work (if any) AIRC, Grant n IG2019-23512; FRRB (Fondazione Regionale per la Ricerca Biomedica), IANG-CRC - CP2_12/2018 project

• No conflict of interest