Immuno-oncology agent IPI-549 is a modulator of P-glycoprotein (P-gp, MDR1, ABCB1)-mediated multidrug resistance (MDR) in cancer: In vitro and in vivo

Abstract

Phosphoinositide 3-kinase gamma isoform (PI3Kγ) plays a critical role in myeloid-derived cells of the immunosuppressive tumor microenvironment. IPI-549, a recently discovered small molecule selective PI3Kγ inhibitor, is currently under immuno-oncology clinical trials in combination with nivolumab, an anti-PD-1 monoclonal antibody immune checkpoint blocker. The purpose of this study is to investigate whether IPI-549 could reverse P-glycoprotein (P-gp)-mediated MDR when combined with chemotherapeutic substrates of P-gp. Cytotoxicity assays showed that IPI-549 reverses P-gp-mediated MDR in SW620/Ad300 and LLC-PK-MDR1 cells. IPI-549 increases the amount of intracellular paclitaxel and inhibits the efflux of paclitaxel out of SW620/Ad300 cells. ABCB1-ATPase assay showed that IPI-549 stimulates the activity of ABCB1-ATPase. IPI-549 does not alter the expression and does not affect the subcellular localization of P-gp in SW620/Ad300 cells. The combination of IPI-549 with paclitaxel showed that IPI-549 potentiates the anti-tumor effects of paclitaxel in P-gp-overexpressing MDR SW620/Ad300 xenograft tumors. With clinical trials beginning to add newly approved immune checkpoint-based immunotherapy into standard-of-care immunogenic chemotherapy to improve patient outcomes, our findings support the rationale of adding IPI-549 to both the chemotherapeutic and immunotherapeutic aspects of cancer combination treatment strategies.

Keywords: ABCB1 transporter; Combination chemotherapy; IPI-549; Immune checkpoint; Multidrug resistance; P-glycoprotein.

Figure 1.. Chemical structure of IPI-549 and cytotoxicity of IPI-549 on parental and drugresistant cells.

Chemical structure of IPI-549 (A). Cell survival (%) percentage was measured after treatment with IPI-549 for 72 h on parental and drug-resistant cells: SW620 and SW620/AD300 cells (B), LLC-PK1 and LLC-PK-MDR1 cells (C), S1 and S1-M1–80 cells (D). Points with error bars represent the mean ± SD for independent determinations in triplicate. The above figures are representative of three independent experiments.

Figure 2.. Effect of IPI-549 on the accumulation and efflux of [³H]-paclitaxel and the effect of IPI-549 on ABCB1-ATPase activity.

The effect of IPI-549 on accumulation of [³H]-paclitaxel in SW620 and SW620/AD300 cells (A) and effect of IPI-549 on the efflux of [³H]-paclitaxel on SW620 (B) and SW620/Ad300 cells (C). A time course (0, 30, 60, 120 min) versus percentage of intracellular [³H]-paclitaxel remaining (%) was plotted. Columns are the mean of triplicate determinations; the error bars represent the SD, ** P<0.01, * P<0.05 versus the control group without the reversal agent. Verapamil 10 μM was used as positive control. Effect of IPI-549 on ABCB1-ATPase activity (D). Crude membranes (10 μg protein/reaction) from High Five cells expressing ABCB1 were incubated with increasing concentrations of IPI-549 (0–40 μM).The inset shows stimulation of ATP hydrolysis at concentration of 0–10 μM IPI-549. The mean values are plotted and error bars depict SD.

Figure 3.. Effect of IPI-549 on the expression and subcellular localization of ABCB1 on MDR SW620/Ad300 cells.

SW620/Ad300 cells were treated with 10 μM IPI-549 for 0, 24, 48, and 72 h. Equal amounts (80 μg) of cell lysate were loaded into each well and subjected to western blot analysis (A). Bar graphs of ABCB1 blot analysis were performed with ImageJ software of the National Institutes of Health (B). The differences were not statistically significant (P > 0.05). The effect of 10 μM IPI-549 on the subcellular localization of ABCB1 on SW620/Ad300 (C). Nuclear staining was performed with propidium iodide (PI); ABCB1 staining was performed with green fluorescence protein (GFP)-labeled ABCB1 antibody.

Figure 4.. Induced Fit Docking (IFD) analysis of IPI-549 to ABCB1.

The best-scored binding pose of IPI-549 within human homology ABCB1 (Glide gscore: −14.602 kcal/mol) predicted by IFD computation is shown in (A). The location of the IPI-549 molecule as a ball and stick model is shown within the ABCB1 internal cavity, with the atoms colored as carbon–orange, hydrogen– white, oxygen–red, nitrogen–blue. Amino acids that have hydrogen bonding or π-π stacking interactions with IPI-549 are shown and depicted as sticks with the same color scheme as above except that carbon atoms are represented in grey. Only polar hydrogens are shown. Dotted yellow lines indicate hydrogen-bonding interactions, while dotted blue lines indicate π -π stacking interactions. Values of the relevant distances are given in Å. (B) The two-dimensional ligand−receptor interaction diagram of IPI-549 and human ABCB1. The amino acids within 4 Å are shown as colored bubbles, blue indicates polar residues, and green indicates hydrophobic residues. Grey circles indicate solvent exposure. Hydrogen bonds are shown by the purple arrow, and π-π stacking aromatic interactions are shown by green lines.

Figure 5.. Effect of IPI-549 and paclitaxel on the growth of parental SW620 tumors in athymic mice.

The images of excised SW620 tumors implanted subcutaneously in athymic NCR mice (n=6) treated with vehicle, IPI-549, paclitaxel, and the combination of IPI-549 and paclitaxel (A). Changes in tumor volume over time following the implantation (B). Data points represent the mean tumor volumes for each treatment group. The mean weight of the excised SW620 tumors from the mice treated with vehicle, IPI-549, paclitaxel, and the combination of IPI-549 and paclitaxel at the end of the 15-day treatment period (C). Error bars, SD. * p < 0.05 versus the vehicle group.

Figure 6.. Effect of IPI-549 and paclitaxel on the growth of MDR SW620/Ad300 tumors in athymic mice.

The images of excised SW620/Ad300 tumors implanted subcutaneously in athymic NCR mice (n=6) treated with vehicle, IPI-549, paclitaxel, and the combination of IPI-549 and paclitaxel (A). Changes in tumor volume over time following the implantation (B). Data points represent the mean tumor volumes for each treatment group. The mean weight of the excised SW620/Ad300 tumors from the mice treated with vehicle, IPI-549, paclitaxel, and the combination of IPI-549 and paclitaxel at the end of the 15-day treatment period (C). Error bars, SD. * p < 0.05 versus the vehicle group. # p < 0.05 versus the paclitaxel group

Figure 7.

The effect of vehicle, IPI-549, paclitaxel, and combination (IPI-549 + paclitaxel) on the body weight (A), white blood cell count (B) and platelet count (C) in athymic mice at the end of the 15-day treatment period. The differences were not statistically significant (P > 05).