Regulation of the Receptor Tyrosine Kinase AXL in Response to Therapy and Its Role in Therapy Resistance in Glioblastoma.

Lea Scherschinski,Anna-Gila Karbe,Julia Sophie Onken,Ingeborg Tinhofer,Irina Kremenetskaia,Markus Prem,Peter Vajkoczy

doi:10.3390/ijms23020982

Abstract

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Highlights

Glioblastoma multiforme (GBM) is the most common and most aggressive brain tumor in adults
Recent efforts in the treatment of glioblastoma multiforme (GBM) have focused on individualized treatment protocols aiming to target certain mutations (e.g., Epidermal Growth Factor Receptor (EGFR) mutation, Neurotrophic Tyrosine Receptor Kinase (NTRK) fusion or multiple receptors using multi-kinase inhibitors e.g., Regorafenib (Stivarga, Bayer, Leverkusen, Germany) and Sunitinib (Sutent, Pfizer, New York, NY, USA), with limited success [3,4,5,6]
We analyzed receptor tyrosine kinase AXL (RTK-AXL) expression on protein levels with Western blot analysis 48 h after TMZ treatment

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common and most aggressive brain tumor in adults. It remains an incurable disease with a markedly diminished life expectancy despite multimodal therapy comprising surgical resection, chemotherapy, and radiation (RTX) [1,2]. RTK-AXL phosphorylation (P-AXL) results in the activation of Phosphoinositide 3-kinases (PI3K) and its downstream target, serine/threonine protein kinase B (Akt) [15]. The Gas6/AXL/PI3K/Akt pathway protects cells from apoptosis via multiple mechanisms. Akt activates ribosomal protein S6 kinase (S6K) of the mechanistic target of the rapamycin (mTOR) pathway and phosphorylates BCL2-associated agonist of cell death (Bad), which is a pro-apoptotic protein [7,14]

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