Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Characterization of the antitumor activity of the PI3K δ-sparing inhibitor MEN1611 mediated by the PIK3γ inhibition on macrophages and tumour immune infiltrate

PhD Student: Alessandro Paoli Tutor: Dott. Alessandro Bressan, Prof. Rocco Palermo PhD Abstract: Characterization of the antitumor activity of the PI3K δ-sparing inhibitor MEN1611 mediated by the PIK3γ inhibition on macrophages and tumour immune infiltrate The tumour microenvironment (TME) is a complex multicellular environment. The TME typically comprises immune cells, including tumour-associated macrophages (TAM), fibroblasts, blood vessels, lymphatics and cancer cells that regulates cancer progression and may influence therapeutic and clinical outcome. The phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) signalling pathway plays an important role in the regulation of signal transduction and biological processes such as cell cycle, growth and proliferation and its aberrant activation is one of the most frequent events in human cancer (Yang, J. et Al. Mol Cancer 18, 26 2019). Literature data have shown that PI3Kγ inhibition can reshape the inflammatory cells infiltrating the tumours towards a less immunosuppressive phenotype (Megan M. Kaneda et Al. Nature 2016). MEN1611 is a PI3K inhibitor currently in clinical development targeting the p110 α, β, and γ isoforms, while sparing the δ subunit (J. Ohwada et Al. Bioorg Med Chem Lett. 2011). MEN1611 showed a long-lasting antitumor activity when combined with Trastuzumab (H. Tanaka et Al. Clin Cancer Res. 2011). This project aim is to characterize the effects of MEN1611 inhibition of the PI3Kγ isoform, highly expressed in tumour-associated macrophages (TAMs), in order to investigate the possible anti-tumour activity of MEN1611 mediated by the inflammatory microenvironment modulation. In-vitro and in-vivo models were established in order to evaluate the effect of MEN1611 on TAMs. Murine macrophages were differentiated from bone marrow-derived monocytes (A. Sica and A. Mantovani J Clin Invest. 2012). Obtained cells, were treated with MEN1611 or IPI-549 (as positive control). In this project we characterized the effects of MEN1611 and IPI-549 on the gene expression of M1/M2 macrophages using RNA-Seq technology in order to understand similar and independent pathways modulated from the two compounds using Ingenuity Pathway Analysis (IPA). A syngeneic xenograft model of breast cancer based on 4T1 cells was implemented to investigate the modulation of the inflammatory infiltrate mediated by MEN1611 and IPI-549. RNA-Seq analysis was performed on dissociated treated tumours. Subsequently, an in-silico IPA was done on genes modulated by MEN1611/IPI-549 to investigate the processes related to the immune-activating switch. All these data were used to generate a gene signature associated with MEN1611 activity on the tumour immune-infiltrate that were used to investigate a subpopulation that could be targeted by MEN1611 using public databases. In vivo and in vitro results together suggest that MEN1611 anti-tumoral activity might be also mediated by inflammatory cells infiltrating tumours through PI3Kγ inhibition. Moreover, in silico results suggest that the modulation of TAM induced by MEN1611 could improve overall survival of breast cancer patients.