MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment

Abstract

Background: Programmed death-1 (PD-1) immune checkpoint blockade has achieved clinical successes in cancer therapy. However, the response rate of anti-PD-1 agents remains low. Additionally, a subpopulation of patients developed hyperprogressive disease upon PD-1 blockade therapy. Combination therapy with targeted agents may improve immunotherapy. Recent studies show that p53 activation in the myeloid linage suppresses alternative (M2) macrophage polarization, and attenuates tumor development and invasion, leading to the hypothesis that p53 activation may augment antitumor immunity elicited by anti-PD-1 therapy.

Method: Using APG-115 that is a MDM2 antagonist in clinical development as a pharmacological p53 activator, we investigated the role of p53 in immune modulation and combination therapy with PD-1 blockade.

Results: In vitro treatment of bone marrow-derived macrophages with APG-115 resulted in activation of p53 and p21, and a decrease in immunosuppressive M2 macrophage population through downregulation of c-Myc and c-Maf. Increased proinflammatory M1 macrophage polarization was observed in the spleen from mice treated with APG-115. Additionally, APG-115 has co-stimulatory activity in T cells and increases PD-L1 expression in tumor cells. In vivo, APG-115 plus anti-PD-1 combination therapy resulted in enhanced antitumor activity in Trp53^wt, Trp53^mut, and Trp53-deficient (Trp53^-/-) syngeneic tumor models. Importantly, such enhanced activity was abolished in a syngeneic tumor model established in Trp53 knockout mice. Despite differential changes in tumor-infiltrating leukocytes (TILs), including the increases in infiltrated cytotoxic CD8⁺ T cells in Trp53^wt tumors and M1 macrophages in Trp53^mut tumors, a decrease in the proportion of M2 macrophages consistently occurred in both Trp53^wt and Trp53^mut tumors upon combination treatment.

Conclusion: Our results demonstrate that p53 activation mediated by APG-115 promotes antitumor immunity in the tumor microenvironment (TME) regardless of the Trp53 status of tumors per se. Instead, such an effect depends on p53 activation in Trp53 wild-type immune cells in the TME. Based on the data, a phase 1b clinical trial has been launched for the evaluation of APG-115 in combination with pembrolizumab in solid tumor patients including those with TP53^mut tumors.

Keywords: APG-115; Anti-PD-1; Immuno-oncology; MDM2 inhibitor; Macrophage; Tumor microenvironment; p53.

Fig. 1

APG-115 suppresses alternative M2 macrophages polarization in vitro and increases M1 macrophages in vivo through activation of p53 pathway. a BMDMs were generated under the treatment with m-CSF for 7 days and then treated with IL-4 (20 ng/mL) to induce alternative macrophage polarization (M2) for 24 h in the absence or presence of APG-115. Cells were then harvested for detection of M2 macrophages (CD206⁺MHC-II^low) by flow cytometry. b the mRNA expression levels of Arg-1 and Retnla in the above BMDMs induced by the treatment with IL-4 (20 ng/mL) with or without APG-115 were analyzed by RT-qPCR. Duplicated samples were tested. c Western blot analysis of p53, p21, c-Myc and c-Maf total proteins in BMDMs treated with IL-4 (20 ng/mL) with or without APG-115 (1 μM) for 0, 4, or 24 h, or sequentially treated with IL-4 and then APG-115 for 24 h each (24 h + 24 h). d Quantification of C. 0 (black bars), 4 (blue bars), or 24 h (green bars), or sequentially treated with each agent for 24 h (24 h + 24 h, red bars). e naïve BALB/c mice were treated with APG-115 (10 mg/kg, Q2D × 2 doses; n = 5). Two days after the last dose, spleens were collected, dissociated into single-cell suspensions, and stained with macrophage markers for flow cytometry analysis. Macrophages were defined as CD11b⁺ F4/80^hi, and further analyzed for M1 macrophages by expression of MHC-II. Pooled data of percentages of macrophages gated on CD45⁺CD3⁻ live cells (f) and percentages of M1 macrophages gated on macrophages (g) from five mice were plotted

Fig. 2

APG-115 increase mouse T cell proliferation and enhances mouse CD4⁺ T cell activation. a CD4⁺ T and CD8⁺ T cells were positively selected from mouse spleens using magnetic beads and then stimulated with indicated concentrations of plate-bound anti-CD3 and 2 μg/mL anti-CD28 in the presence of 250 nM APG-115 or DMSO. After 72 h, relative cell numbers were determined using CellTiter-Glo luminescent cell viability assay (Promega) and normalized to unstimulated cultures treated with DMSO control. * P < 0.05. b immunoblots for the expression of caspase 3, cleaved caspase 3, and Zap-70 (loading control) in total cell lysates of anti-CD3/CD28-stimulated CD4⁺ T cells exposed to APG-115 or solvent control DMSO for 3, 6, or 24 h (h). c CD4⁺ T cells were positively selected from mouse spleens using magnetic beads and then stimulated with 10 μg/mL plate-bound anti-CD3 and 2 μg/mL anti-CD28 in the presence of 250 nM APG-115 or DMSO for the indicated periods of time. T cell activation markers (CD25 and CD62L) were determined by flow cytometry. CD25^high CD62L^low T cells represented an activated population. d an increase in cell size was shown after APG-115 treatment

Fig. 3

APG-115 upregulates PD-L1 expression on MH-22A tumor cells. MH-22A mouse tumor cells were treated with indicated concentrations of APG-115 for 72 h in vitro. a expression levels of MDM2, p53, total STAT3 (t-STAT3), phosphorylated STAT3 (p-STAT3), PD-L1, and β-actin (loading control) were determined by Western blotting. b PD-L1 expression levels which were reflected by fluorescence intensity were determined by flow cytometry and the same results were shown as a bar chart (c)

Fig. 4

APG-115 enhances anti-PD-1 antibody mediated tumor suppression in Trp53^wt, Trp53^mut and Trp53^−/− syngeneic mouse tumor models. APG-115 was tested alone and in combination with anti-PD-1 antibody in mice subcutaneously implanted with Trp53^wt MH-22A (a-d; n = 8), Trp53^mut MC38 (e-g; n = 10), or Trp53^−/− MH-22A (h-j; n = 10) tumor cells. APG-115 was orally administered every day in Trp53^wt MH-22A models or every other day in both Trp53^mut MC38 and Trp53^−/− MH-22A models. Anti-PD-1 antibody was administered intraperitoneally BIW. Treatments were conducted for 3 weeks in Trp53^wt MH-22A and Trp53^mut MC38 models, and for 12 days in Trp53^−/− MH-22A model. Data representing at least two independent experiments were presented as the mean of tumor volumes of mice in each group (A, E, H) or tumor volumes for individual mice (B, C, D, F, G, I and J). The control groups were treated with APG-115 vehicle (A) or isotype antibody plus vehicle (I + V; E and H)

Fig. 5

APG-115-enhanced antitumor activity in combination with anti-PD-1 blockade is abolished in Trp53 knockout mice implanted with Trp53^mut MC38 tumor cells. The effect of APG-115 was evaluated in combination with anti-PD-1 antibody in a subcutaneous MC38 model established in Trp53 knockout C57BL/6 J mice (n = 12/group). APG-115 was orally administered every other day and anti-PD-1 antibody was administered intraperitoneally BIW

Fig. 6

Flow cytometry analysis of TILs in the TME of syngeneic tumors with wild-type (a) or mutant (b) Trp53. Mice with established MH-22A or MC38 tumors were treated with 10 mg/kg APG-115 (a and b), 10 mg/kg (a) or 5 mg/kg (b) anti-PD-1 antibody, or the combination as described in the legend of Fig. 4. The control group was treated with isotype control antibody and APG-115 vehicle (I + V). On day 14, syngeneic tumors were harvested, dissociated into single-cell suspensions, and stained for flow cytometry analysis. Percentages of CD45⁺, CD3⁺ T cells, CD8⁺ T cells, M1 and M2 macrophages in the tumors under the different treatments were assessed. Data were representative of two (a) or three (b) independent experiments and shown as dot plots (n = 5 or 10). ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05, by one-way ANOVA with Bonferroni post-test. I + V indicates isotype control and vehicle of APG-115