Abstract PS18-26: Men1611 promotes immune activating myeloid cell polarization
Authors: Capano Stefania , Paoli Alessandro , Fiascarelli Alessio , Tomirotti Andrea Massimiliano , Bigioni Mario , Bressan Alessandro , Bellarosa Daniela , Salerno Massimiliano , Binaschi Monica 
Published: February 2021

Abstract
MEN1611 is a PI3K inhibitor currently in clinical development targeting the p110 α, β and γ isoforms, while sparing the δ subunit. In preclinical models, MEN1611 has demonstrated a long lasting antitumor activity when combined with trastuzumab in HER2+/PiK3CA mutated breast cancer. Aim of this work is to characterize the effects of MEN1611 on the PI3Kγ isoform, highly expressed in tumor-associated macrophages (TAMs), in order to understand whether the anti-tumor activity of MEN1611 might be also mediated by the inflammatory microenvironment. Previous evidences have shown that selective targeting of PI3Kγ by IPI-549 can reshape the inflammatory cells infiltrating tumors towards a less immunosuppressive phenotype and promote cytotoxic T cell-mediated tumor regression. In order to evaluate the effect of MEN1611 on TAMs, we established an in vitro model by differentiating murine and human macrophages from bone marrow-derived monocytes or buffy coats-isolated monocytes respectively. Pro-inflammatory M1 macrophages have been differentiated by treatment with Lipopolysaccharide (LPS) and interferon γ (IFNγ), while pro-tumoral M2 macrophages by interleukin 4 (IL-4) stimulation. The cells, thus obtained, have been incubated with MEN1611 or IPI-549 (positive control) and the effects on their phenotype, gene and protein expression, evaluated through confocal microscopy, RNASeq and flow cytometry respectively. A syngeneic xenograft model of breast cancer based on 4T1 cells has been also established in order to investigate in vivo the activity of MEN1611 on the inflammatory environment (TAMs and T cells). In vitro data showed MEN1611 and IPI-549 ability to repolarize murine and human macrophages towards a pro-inflammatory phenotype. Confocal analysis revealed a shape remodeling from an elongated M2-like towards a round M1-like; gene and protein expression analysis revealed a significant increase of immuno-stimulating factors mRNA and M1 chemokines and cytokines secretion, respectively. A modulation of the inflammatory infiltrate was also observed in vivo where RNASeq and flow cytometry analysis on dissociated treated tumors highlighted an increase of pro-inflammatory markers. The in-silico ingenuity pathway analysis (IPA) performed on genes modulated by MEN1611 revealed the enhancement of some processes related to an immune activating switch, such as the recruitment of myeloid cells or the cytotoxicity of lymphocytes and natural killer cells. In conclusion, we demonstrated in a cellular context that MEN1611 activity on PI3Kγ isoform is responsible for macrophages reprogramming from an immune-suppressive to an immune-activating phenotype. Moreover, we observed that in 4T1 murine breast cancer model, tumor regression induced by MEN1611 was also sustained by a modulation of the inflammatory microenvironment, characterized by an increased recruitment and cytotoxicity of T cells.
Citation Format: Stefania Capano, Alessandro Paoli, Alessio Fiascarelli, Andrea Massimiliano Tomirotti, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Massimiliano Salerno, Monica Binaschi. Men1611 promotes immune activating myeloid cell polarization [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-26.