AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model

Abstract

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti-AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786-0-Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786-0-Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786-0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi-target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

Keywords: bemcentinib; orthotopic RCC; tilvestamab.

FIGURE 1

AXL expression in 786‐0 Luc cells at the mRNA and protein levels. (a) AXL expression in 786‐0‐Luc cells is detected at the mRNA level by RT‐qPCR. (b) AXL protein expression is confirmed by western blot analysis in both 786‐0 and 786‐0 Luc cells. DNA ladder; Thermo Scientific™ SM1173, Product Code. 11803983

FIGURE 2

In vitro effects of specific inhibitors on AXL and AKT phosphorylation in 786‐0 cells. The pAXL levels, as measured in ELISA, are significantly reduced in tilvestamab (a) and bemcentinib (b)‐treated 786‐0 cells compared to their respective controls. Tilvestamab and bemcentinib results are reported as ratio over different controls. (c) Accordingly, pAKT levels are significantly reduced when cells are treated for 1 h with tilvestamab prior to Gas6 stimulation. ANOVA test with Tukey's post hoc is performed for statistical analysis. Bars presented with mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.01

FIGURE 3

Schematic overview of the design of the two in vivo studies I (a) and II (b). 786‐0 Luc cells are implanted orthotopically in mice, and randomized into different treatment groups after initial tumor growth. (a) The effects of the treatment are measured by bioluminescence and 3D‐USG at days 10, 17, 24, and 34 after start of therapy in study. (b) Tumor growth in study II is measured by bioluminescence at days 20 and 30 after start of therapy. Tumor tissue is harvested at the time of sacrifice for immunohistochemistry analyses, as detailed in “Section 2”

FIGURE 4

Bemcentinib inhibits progression of orthotopically implanted RCC 786‐0 tumors. The 786‐0‐Luc cells are orthotopically implanted in the kidneys, and treatment is initiated following detectable tumor growth. Antitumor effects of bemcentinib are measured and documented after 34 days administration by bioluminescence (a) and 3D USG (b), as detailed in “Section 2.” Graph made in IBM SPSS Statistics Version 26, and graph presented with mean ± SE. One‐way ANOVA test. **p < 0.05, ***p < 0.001

FIGURE 5

Comparative effects of AXL‐specific and multi‐targeted tyrosine kinase inhibitors, alone or in combination on the progression of orthotopically implanted 786‐0 cells. The 786‐0 cells are implanted orthotopically, and the indicated treatments are initiated following initial tumor growth. After a 34‐day administration, the effects of treatments in tumor growth are measured by bioluminescence, as detailed in “Section 2.” We obtained a clear tumor inhibitory effect of all treatment groups compared to the two controls. GraphPad Prism version 9.0.1 is used to generate the graph. ANOVA test with Dunnett's post hoc is applied. **p < 0.002