MerTK as a therapeutic target in glioblastoma

Abstract

Background: Glioma-associated macrophages and microglia (GAMs) are components of the glioblastoma (GBM) microenvironment that express MerTK, a receptor tyrosine kinase that triggers efferocytosis and can suppress innate immune responses. The aim of the study was to define MerTK as a therapeutic target using an orally bioavailable inhibitor, UNC2025.

Methods: We examined MerTK expression in tumor cells and macrophages in matched patient GBM samples by double-label immunohistochemistry. UNC2025-induced MerTK inhibition was studied in vitro and in vivo.

Results: MerTK/CD68+ macrophages increased in recurrent tumors while MerTK/glial fibrillary acidic protein-positive tumor cells did not. Pharmacokinetic studies showed high tumor exposures of UNC2025 in a syngeneic orthotopic allograft mouse GBM model. The same model mice were randomized to receive vehicle, daily UNC2025, fractionated external beam radiotherapy (XRT), or UNC2025/XRT. Although median survival (21, 22, 35, and 35 days, respectively) was equivalent with or without UNC2025, bioluminescence imaging (BLI) showed significant growth delay with XRT/UNC2025 treatment and complete responses in 19%. The responders remained alive for 60 days and showed regression to 1%-10% of pretreatment BLI tumor burden; 5 of 6 were tumor free by histology. In contrast, only 2% of 98 GBM mice of the same model treated with XRT survived 50 days and none survived 60 days. UNC2025 also reduced CD206+ macrophages in mouse tumor samples.

Conclusions: These results suggest that MerTK inhibition combined with XRT has a therapeutic effect in a subset of GBM. Further mechanistic studies are warranted.

Keywords: MerTK; glioblastoma; macrophage; microenvironment.

Fig. 1

MerTK expression in GBM. (A) MerTK is expressed in both GFAP+ tumor cells and CD68+ macrophages in a representative, matched pair of GBMs taken at initial diagnosis (left panel) and recurrence (right panel). Scale bar = 100 microns. (B) Western blot showed MerTK expression in human GBM (U87, U251, GSC20, GSC11) and mouse cells, including TRP cells, J774 macrophage, and EOC 2 microglia. Human 293T cells served as positive controls. Brain tissues from mertk^WT and mertk^−/− mice were used as positive and negative controls, respectively. (C) Dual staining for MerTK in CD68+ and GFAP+ cells at initial diagnosis and recurrence was quantified in 15 pairs of GBM. Percentage of MerTK+/CD68+ cells had a 6-fold increase of mean at disease recurrence (N = 15, P < 0.001) (a), while no consistent change of MerTK+/GFAP+ tumor cells was observed (N = 15, P = 0.14) (b).

Fig. 2

UNC2025 exerts antiproliferative effects in vitro. (A) UNC2025 inhibited growth of a group of human and mouse cell lines by MTS assay. (B) UNC2025 synergized with radiation in TRP cells by colony formation assay.

Fig. 3

UNC2025 regulates MerTK and downstream signaling. (A) Representative western blots of UNC2025-treated TRP cells (3 h) showed decreased MerTK signaling (pMer) and increased apoptosis (cleaved caspase-3), but no change in Axl signaling. (B) Quantification of expression relative to untreated control (mean ± SEM) from 3 independent experiments is shown. (C) Representative western blots of UNC2025-treated TRP cells (1 h) showed decreased signaling of mitogen-activated protein kinase (pERK) and phosphatidylinositol-3 kinase (pAkt). (D) Quantification of expression relative to untreated control (mean ± SEM) from 3 independent experiments is shown. Representative western blots of MerTK signaling in UNC2025-treated GSC20 at 3 h (E) and 1 h (F). *P < 0.05, **P < 0.01, ***P < 0.0001.

Fig. 4

UNC2025 pharmacokinetics. (A) Mean concentrations of UNC2025 in plasma, normal brain, peritumoral brain, and brain tumor in TRP allograft mice. The lower limits of quantitation (LLOQ) were 58.4 ng/mL (plasma) and 88.0 ng/g for brain tumor and tissues. (B) Summary of pharmacokinetic parameters.

Fig. 5

UNC2025 induces long-term survival in a subset of TRP allograft mice treated with concomitant radiation. UNC2025 plus radiotherapy results in complete pathological response in a subset of TRP allograft mice. (A) Pooled data from 3 independent experiments showed median survivals of 21, 22, 35, and 35 days for control (N = 29), UNC2025 (N = 32), XRT (N = 19), and UNC2025 plus XRT (N = 36) treatments, respectively. (B) Kaplan–Meier analysis of data in (A) plus historical data from 6 previously published TRP control and XRT-treated allografts showed similar median survivals of 21 (N = 98) and 32 days (N = 98) in control and XRT-treated TRP allografts. Only 2 of 98 mice (2%) that received XRT survived beyond 50 days. (C) Longitudinal BLI of mean tumor growth relative to time of treatment randomization and allocation (day 7) shows growth inhibition by XRT with or without UNC2025. (D) However, a significant reduction in mean BLI was evident in mice that responded to XRT plus UNC2025 (N = 6) compared with nonresponders (N = 26). (E) BLI of 6 nonresponders (A to F) and 6 responders (G to L) to XRT plus UNC2025 (see Supplementary Table S1).

Fig. 6

UNC2025 modulates macrophage phenotype. (A) Percentage (mean ± SEM) of F4-80+ GAM was decreased by UNC2025 (N = 12) and XRT (N = 11) in TRP allografts, but these effects did not reach statistical significance. (B) UNC2025 plus XRT (N = 13) significantly (P = 0.02) decreased percentage (mean ± SEM) of GAMs with M2 phenotype (CD206+). *P < 0.05.