Abstract 1384: Selective PI3Kγ inhibitor ZX-4081 reprograms the tumor microenvironment to facilitate anti-cancer immunity
Authors: Li Ying-Ying , Guo Kun , Peng Zhiyuan , Kong Deming , Hao Xiaolin , Yang Jinfu , Qin Xiaoli (Shelley) 
Published: July 2021

Abstract
PI3Kγ is highly expressed in leukocytes and also known to play a critical role in activation of myeloid cells at sites of inflammation and tumorigenesis, T cell recruitment into tumor microenvironment, and chemokine-mediated chemotaxis as well as growth factor signaling. Previous studies reported that blockade of PI3Kγ-AKT signaling is able to inhibit tumor growth and metastasis by increasing CD8+T cell activation and M1(anti-tumoral)-like macrophage appearance in the tumor milieu, and thereby overcome resistance to immune checkpoint inhibitors (e.g., anti-PD1 antibody). In this report, we present a highly selective PI3Kγ inhibitor ZX-4081 with an IC50 of 1.5 nM for PI3Kγ- and over 1000-fold selectivity over the other isoform (α, β, and δ) in biochemical assay. To confirm inhibitory effect of ZX-4081 on PI3Kγ-AKT activation, LPS-stimulated THP-1 cells (monocytic cells) were treated with 10 nM or 500 nM ZX-4081, and their AKT phosphorylation (at T308 and S473 sites) were examined by immunoblotting. The immunoblot result indicated that both T308 and S473 phosphorylation was down-regulated by ZX-4081 in a time-dependent and dose-dependent manner. Next, to verify whether ZX-4081 can alter macrophage polarization, M-CSF-stimulated human peripheral blood mononuclear cells (PBMCs) were subsequently either induced into M1-like macrophages with LPS and IFN-γ or induced into M2-like macrophages with IL-4 and IL-13 in the absence (vehicle) or presence of ZX4081 (from 1.52 to1000 nM). The flow cytometry result exhibited that ZX-4081 effectively facilitated macrophage polarization toward M1-like phenotype, evidenced by an increase (a 2.5-fold increase at 10 nM compared to vehicle) in iNOS and CD86 expressions of M1-like macrophages; however, CD206 expression (M2-like phenotype) was not changed by ZX-4081 treatment in M2-like macrophages. Additionally, ZX-4081 could reduce both IL-10 protein and mRNA levels in M2-like macrophages (2-5 times lower than vehicle). The cell proliferation assessment showed that ZX-4081(&lt; 5μM) did not exert potent growth inhibitory effects on solid tumor cell lines including HT-29, HT-1197, 4T1, B16-F10, and CT-26 cells. In the 4T1 syngeneic mouse model, ZX-4081 treatment at 25 mg/kg (p.o, BID), 50 mg/kg (p.o., QD), and 50 mg/kg (p.o., BID) for 28 days gave rise to a reduction in tumor growth by 34.6%, 21.2%, and 44.1% in size, respectively (p&lt; 0.0001). Therefore, the in vivo inhibitory effect of ZX-4081 on 4T1 tumor growth was attributed to its modulation on the mouse anti-tumor immunity. Overall, our data suggest that ZX-4081 possesses ability of tumor growth inhibition by reprogramming tumor microenvironment via impairing PI3Kγ-AKT-mediated macrophage polarization to enhance anti-cancer immunity.
Citation Format: Ying-Ying Li, Kun Guo, Zhiyuan Peng, Deming Kong, Xiaolin Hao, Jinfu Yang, Xiaoli (Shelley) Qin. Selective PI3Kγ inhibitor ZX-4081 reprograms the tumor microenvironment to facilitate anti-cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1384.