Inhibition of HSP90 as a Strategy to Radiosensitize Glioblastoma: Targeting the DNA Damage Response and Beyond.

Michael Orth,Na Sun,Kristian Unger,Anna A Friedl,Julia Hess,Valerie Albrecht,Kirsten Lauber,Horst Zitzelsberger,Linda Kinzel,Alexander Nieto,Benjamin Stegen,Karin Seidl,Maximilian Niyazi,Nicolas Winssinger,Claus Belka,Jessica Maas,Axel K Walch,Lisa Kreutzer

doi:10.3389/fonc.2021.612354

Abstract

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damage response factors on the protein level, distinct transcriptomic alterations, impaired DNA damage repair, and reduced clonogenic survival in response to ionizing irradiation in glioblastoma cells in vitro. In vivo, HSP90 inhibition by NW457 improved the therapeutic outcome of fractionated CBCT-based irradiation in an orthotopic, syngeneic GBM mouse model, both in terms of tumor progression and survival. Nevertheless, in view of the promising in vitro results the in vivo efficacy was not as strong as expected, although apart from the radiosensitizing effects HSP90 inhibition also reduced irradiation-induced GBM cell migration and tumor invasiveness. Hence, our findings identify the combination of HSP90 inhibition and radiotherapy in principle as a promising strategy for GBM treatment whose performance needs to be further optimized by improved inhibitor substances, better formulations and/or administration routes, and fine-tuned treatment sequences.

Highlights

Glioblastoma (GBM) is the most aggressive type of primary brain tumor with a highly dismal prognosis and a 5-year overall survival of less than 5% [1]
Initial Quantitative Real-Time PCR (qRT-PCR) profiling confirmed that the human GBM cell lines LN229 and T98G show a broad-range upregulation of diverse DNA damage response (DDR) regulators as compared to normal human astrocytes suggesting that DDR activity is increased—irrespective of the O6methylguanine-DNA-methyltransferase (MGMT) status (Figure 1A and Supplementary Figure 1A) [53]
The highest levels of overexpression were detected for the replication and DDRassociated nucleases FEN1 and EXO1, members of the DNA double-strand break (DSB) detecting MRN complex (MRE11 and NBN), the DNA helicases BLM and PALB2, the singlestrand binding protein RPA1, and members of the XRCC family which are involved in non-homologous end joining (XRCC4 and XRCC6) and alternative non-homologous end joining (XRCC1)

Summary

Introduction

Glioblastoma (GBM) is the most aggressive type of primary brain tumor with a highly dismal prognosis and a 5-year overall survival of less than 5% [1]. HSP90 actively contributes to radioand chemoresistance of GBM and other cancer cells and represents an attractive target for biologically targeted radiosensitization, because HSP90 inhibition (HSP90i) —at least in principle—can affect multiple DDR pathways simultaneously [24,25,26]. This was the focus of the present study for which we made use of the pochoxime-based, second generation HSP90 inhibitor NW457 with documented radiosensitizing potential in other cancer entities [27,28,29,30,31,32]. The invasive morphology of radiotherapytreated tumors was reverted by additional HSP90i, and in vitro migration analyses confirmed that HSP90i does reduce irradiationinduced GBM hypermigration

Methods

Results

Conclusion