Small-Molecule Inhibition of Axl Targets Tumor Immune Suppression and Enhances Chemotherapy in Pancreatic Cancer

Abstract

Activation of the receptor tyrosine kinase Axl is associated with poor outcomes in pancreatic cancer (PDAC), where it coordinately mediates immune evasion and drug resistance. Here, we demonstrate that the selective Axl kinase inhibitor BGB324 targets the tumor-immune interface to blunt the aggressive traits of PDAC cells in vitro and enhance gemcitibine efficacy in vivo Axl signaling stimulates the TBK1-NFκB pathway and innate immune suppression in the tumor microenvironment. In tumor cells, BGB324 treatment drove epithelial differentiation, expression of nucleoside transporters affecting gemcitabine response, and an immune stimulatory microenvironment. Our results establish a preclinical mechanistic rationale for the clinical development of Axl inhibitors to improve the treatment of PDAC patients.Significance: These results establish a preclinical mechanistic rationale for the clinical development of AXL inhibitors to improve the treatment of PDAC patients. Cancer Res; 78(1); 246-55. ©2017 AACR.

Figure 1. BGB324 inhibits Axl activity in pancreatic cancer cells in vitro

A) AsPC-1 cells were serum starved in 1% FBS in media overnight and subsequently treated with increasing concentrations of BGB324 (BGB) for 30 minutes. Cell lysates were probed by Western blotting for the indicated targets. B) Panc-1 cells were serum starved overnight as above and subsequently stimulated with DMSO (0.02%) in media (Cntl), Gas6 (200 ng/ml) or Gas6 + BGB324 (2 μM) for 30 minutes followed by Western blot analysis for the indicated targets. C) Pan02 cells were serum starved overnight and subsequently treated with DMSO (0.02%) in media, BGB324 alone (2 μM), AF854 (2.5 nM), an Axl antibody that can stimulate Axl activation (20), or AF854 + BGB324 for 30 minutes. Axl was immunoprecipitated followed by probing for phospho-tyrosine (p-Tyr) and total Axl (upper two rows). Total cell lysates were also probed for the indicated targets. D) Phosphorylated Axl was detected in AsPC-1 and Panc-1 by immunocytochemistry. AsPC-1 cells were serum starved overnight as above (Control) then treated with BGB324 (2 μM) for 30 minutes. Panc-1 cells were serum starved and treated with Gas6 (200 ng/ml) or Gas6 + BGB324 (2 μM). AsPC-1 and Panc-1 cells were fixed permeabilized, stained with anti-phospho-Axl, and subsequently developed by immunofluorescence. Images were analyzed using Elements software; quantification of % area fraction is shown. Data are displayed as mean ± SD and represent 5 images per cell line. *P < 0.05; **P < 0.01; ***P < 0.005. E) The effect of BGB324 on cell migration was assessed by a “scratch” assay. Monolayers of the indicated cells were wounded with a pipet tip. The cells were incubated in serum-free media +/− BGB324 at the indicated concentrations. Wound closure was monitored at 10, 20, and 30 hours and is reported as % wound closure. F) Colony formation for Pan02, KIC1, and 3 human PDA cell lines grown in normal growth media +/− BGB324 at the indicated doses for 14 days. Mean + SD colonies/hpf are shown. G) Cell growth assays were performed in a 96 well format for 5 days using MTS. Drug sensitivity curves for BGB324 are displayed. H) IC₅₀ as determined by MTS assay for gemcitabine alone, gemcitabine+BGB324, and fold reduction in presence of BGB324 are displayed.

Figure 2. Axl inhibition in mice with advanced PDA improves survival

KIC mice (A) and Pan02 tumor-bearing mice (B) were enrolled in a survival study and randomized to vehicle (0.5% [w/w] hydroxypropyl methylcellulose/0.1% [w/w] Tween 80 in water, control, PO BID), BGB324 (50 mg/kg PO BID), gemcitabine (25 mg/kg IP twice a week), and gemcitabine + BGB324. Therapy was initiated at day 49 (KIC) or day 17 post tumor cell injection (Pan02) and maintained until sacrifice. C) In vivo assessment of treatment response of subcutaneously (SC) implanted pancreatic PDX. Panc 281 and Panc 163 were implanted SC on the flanks of athymic mice (n = 8–10/group). When tumors were established, mice were treated with vehicle (control, PO BID), BGB324 (50 mg/kg PO BID), gemcitabine (25 mg/kg IP twice a week), or the combination. Effects on tumor growth are shown after 4 weeks of treatment. D) Panc 265 was orthotopically implanted into nu/nu athymic mice. After tumor establishment, mice were treated as in (C) (n = 6–7/group). Total gross metastasis was determined by evaluation of liver, diaphragm, GI lymph nodes, and lung at the time of sacrifice. BGB324 alone or in combination with gemcitabine significantly reduced the rate of metastasis (p=0.001 vs control, Fisher’s exact test)

Figure 3. BGB324 in combination with gemcitabine inhibits cell survival and epithelial plasticity

A) Tumor tissue from KIC mice treated with BGB324 +/− gemcitabine was evaluated by immunofluorescence for cleaved-caspase 3, phospho-histone-3, endomucin, E-cadherin, and vimentin. Images were analyzed using Elements software; quantification of % area fraction is shown. Data are displayed as mean ± SD and represent 5 images per tumor with 4 animals per group analyzed. *P < 0.05; **P < 0.01; ***P < 0.005; by ANOVA with Tukey’s MCT. B) Lysates of tumors from KIC animals treated with BGB324 +/− gemcitabine were probed for the indicated targets by Western blotting. C) Tumor tissue from KIC animals treated with BGB324 +/− gemcitabine was evaluated by immunohistochemistry for the expression level of equilibrative nucleoside transporter 1 (ENT1). Quantification of % area fraction is shown. Scale bar, 100 μM.

Figure 4. Inhibition of Axl with BGB324 alters the immune landscape of PDA

A) Tumor lysates from KIC mice treated with vehicle (Cntl), gemcitabine (Gem), BGB324 (BGB) or the combination (Combo) were analyzed for cytokine context by Milliplex assay. The levels of Ccl11, Il-7, Il-1β, and Il-6 are shown as mean ± SD (n = 2/group). B) Tumor lysates from KIC animals treated as above were probed for the level of active TBK1 and NF-κB by Western blotting for the indicated targets. C, D) The effect of Axl on TBK1 signaling was probed in AsPC-1 (C) and Panc-1 (D) cells by Western blot analysis. AsPC-1 cells were serum starved overnight and subsequently treated with increasing concentrations of BGB324 for 30 minutes. Panc-1 cells were serum starved and subsequently stimulated with control (0.02% DMSO in media), Gas6 (200 ng/ml), or Gas6 + BGB324 (2 μM) for 30 minutes. Tumor cell lysates were probed for phosphorylated TBK1 and downstream targets. E) Tumor tissue from KIC mice treated with BGB324 +/− gemcitabine were evaluated by immunofluorescence for macrophage (F4/80) and Arginase 1 (Arg1) and counterstained with DAPI. Images were analyzed using Elements software; quantification of % area fraction is shown. Data are displayed as mean ± SD and represent 5 images per tumor with 4 animals per group analyzed. **P < 0.01; ***P < 0.005; ****P < 0.001; by ANOVA with Tukey’s MCT. F) Flow cytometry of KPC-M09 subcutaneous tumors treated with vehicle (control) or BGB324 (50 mg/kg, PO, BID) for two weeks as described in the supplementary methods. BGB324 reduced monocytic MDSCs (CD11b⁺ Ly6G^- Ly6C⁺), PD-L1⁺ M-MDSCs, tumor associated macrophages (TAMs, CD11b⁺ Ly6G^- Ly6C^- F4/80⁺ CD11c⁺ MHCII⁺) and Arg1⁺ TAMs. *, P < 0.05; **, P < 0.01 by t-test.