The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

Abstract

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

Keywords: PI3/AKT/mTOR inhibition; Phosphoinositide 3-kinase delta (PI3K-δ); apoptosis; combinatorial therapy; malignant pleural mesothelioma; tumor induced-immunosuppression.

Fig. 1

PI3K-δ protein expression: characteristics and clinical impact on the survival of MPM patients. (A) PI3K-δ protein expression in 89 primary MPM specimens was assessed by evaluating the intensity of cytoplasmic immunostaining: 0/no staining; 1+/faint; 2+/moderate; 3+/strong staining in at least 10% tumor cells. Scale, 100 µm. (B) Patients with moderate/strong PI3K-δ expression had a shorter overall survival (OAS) compared to those with negative/weak expression (12 vs. 25 months, respectively; Log-rank (Mantel-Cox) test, P=0.0452). Survival data were available for 51 patients (n=14 no/low, n=37 moderate/high PI3K-δ). Survival was calculated from time of diagnosis.

Fig. 2

Roginolisib impairs mesothelioma cell viability and induces apoptotic cell death. (A) MPM cell lines PXF698, PXF1118 and PXF1752 were exposed to the indicated concentrations of roginolisib. The viability of the cells was measured after 72 h of treatment by a MTT cytotoxicity assay. Data points, mean ± SD of independent triplicate experiments. Immunohistochemistry analysis of PI3K-δ protein expression in PXF698, PXF1118 and PXF1752 cells. Scale, 50 µm. (B) PXF698 cells were exposed to roginolisib (100 µM), paclitaxel (400 nM) or DMSO in the presence of the RealTime-Glo Annexin V Apoptosis and Necrosis Assay. Luminescence (phosphatidylsprrerine:annexin V binding – apoptosis; solid line) and fluorescence (loss of membrane integrity – necrosis; dashed line) were recorded for 40 h at the indicated time points. A clear temporal lag between phosphatidylserine exposure and lack of membrane integrity is indicative of apoptotic cell death leading to secondary necrosis. Data points, mean of duplicate samples, representative of independent duplicate experiments. (C) Caspase-3/7 activity after treatment of cells with 100 µM roginolisib or 0.1% DMSO for 24 h. Data points, mean ± SD of independent triplicate experiments. 2way ANOVA test (Dunnett‘s multiple comparison); ****, p<0.0001; ***, p=0.0002. (D) Apoptotic membrane blebbing after treatment of PXF698 cells with 100 µM roginolisib for 24 h, detected by phase-contrast microscopy. (E) PARP cleavage and levels of Mcl-1 and BIM in PXF698 and PXF1752 cells after 100 µM roginolisib treatment at 0, 4, 24 h. (F) Immunoblotting shows the levels of p-AKT (S473) and p-ERK1/2 (T202/204) in PXF1118 and PXF1752 cells after 100 µM roginolisib treatment at 0, 4, 24 h. Bar graphs represent semi-quantitative analysis of phospho-protein expression relative to total protein, normalized to protein levels at 0 h. (G) Schematic illustration of PI3K/AKT/mTOR and ERK signaling.

Fig. 3

Synergistic cytotoxicity of PI3K-δ inhibitors and sapanisertib (mTORC1/2 inhibitor) or ipatasertib (AKT inhibitor). (A) MPM cells were treated with PI3K-δ inhibitors roginolisib (Rog) or idelalisib (Idel) as single agents and in combination with sapanisertib (Sapa) (PXF698) or ipatasertib (Ipa) (PXF1118) for 72 h, after which cell viability was assessed using an MTT cytotoxicity assay. Data points, mean of quintuplets of two independent experiments. (B) Schematic illustration of PI3K/AKT/mTOR and ERK signaling. Growth factors and hormones activate PI3 kinase catalyzing the production of PIP3, which in turn coordinates cell proliferation, metabolism and survival via activating AKT and mTOR signaling pathways. mTOR kinase serves as core component of two distinct protein complexes, mTORC1 and mTORC2. Activated Ras triggers both PI3K/AKT and ERK signaling, regulating cell proliferation and survival. (C) Molecular effects of simultaneous PI3K-δ/mTOR and PI3K-δ/AKT inhibition. Immunoblotting shows the levels of p-AKT (S473), p-PRAS40 p-PRAS40 (T246), p-RPS6 (S235/236) and p-ERK1/2 (T202/204) in PXF1118 cells treated with 100 µM roginolisib (Rog), 100 µM idelalisib (Idel), 30 nM sapanisertib (Sapa), 10 µM ipatasertib (Ipa), or their combinations as indicated for 4 h.

Fig. 4

PIK3CD gene expression correlates with the abundance of CD8⁺ T cells and M2 macrophages in MPM tumors. PIK3CD gene expression data from TCGA data sets (n=87 MPM samples) were plotted against an immune profile for Tregs (A), CD8⁺ T-cells (B), NK cells (C), M2 macrophages (D) and M1 macrophages (E). r, correlation coefficient; p, p-value.

Figure 5

Roginolisib enhances the cytotoxicity of cisplatin and nivolumab in patient-derived tumor cells in the presence of stromal cells. (A) BAP1-wild-type epithelioid (BAP⁺ epi) and BAP1-null sarcomatoid (BAP⁻ sar) MPM cells were co-cultured with peripheral mononuclear cells (PBMCs) and exposed to the indicated concentrations of roginolisib in the absence (-MRC5) or presence of fibroblasts (+MRC5). The viability of the cells was measured after 72 hours by WST-1 staining. Data points, mean of independent quadruplicate experiments. (B) MPM cells were treated with 5 µM cisplatin (Pt) and 1 µg/ml nivolumab (Nivo) for 72 h in the absence and presence of MRC5 cells. (C, D) The combination of roginolisib with 5 µM cisplatin plus 1 µg/ml nivolumab, either as combined/concomitant treatment (C) or in sequential treatment (D), was investigated in the absence (-MRC5) and presence of fibroblasts (+MRC5). 0: untreated cells. Nonparametric Kruskal–Wallis test followed by Dunn's multiple comparison test; ***p<0.001: vs. untreated cells; °p<0.05, °°°p<0.001: MRC5+ vs MRC5-.

Figure 6

Roginolisib exerts immunomodulatory activity. BAP1-wild-type epithelioid (A) and BAP1-null sarcomatoid (B) MPM cells were co-cultured with peripheral mononuclear cells (PBMCs) in the absence or presence of MRC5 fibroblasts and treated with roginolisib alone, co-incubated with cisplatin/nivolumab or in sequential treatment (cisplatin/nivolumab followed by roginolisib). The immunophenotype of immune cells was analyzed by flow cytometry (n = 2, two independent experiments). *p<0.05, **p<0.01, ***p<0.001.