Dianhydrogalactitol synergizes with topoisomerase poisons to overcome DNA repair activity in tumor cells

Beibei Zhai,Jeffrey Bacha,Dennis Brown,Mads Daugaard,Sudha Sravanti Kotapalli,Yue Li,Anne Steinø

doi:10.1038/s41419-020-02780-8

Beibei Zhai, Jeffrey Bacha + Show 5 more

Open Access

https://doi.org/10.1038/s41419-020-02780-8

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Abstract

1,2:5,6-Dianhydrogalactitol (DAG) is a bi-functional DNA-targeting agent currently in phase II clinical trial for treatment of temozolomide-resistant glioblastoma (GBM). In the present study, we investigated the cytotoxic activity of DAG alone or in combination with common chemotherapy agents in GBM and prostate cancer (PCa) cells, and determined the impact of DNA repair pathways on DAG-induced cytotoxicity. We found that DAG produced replication-dependent DNA lesions decorated with RPA32, RAD51, and γH2AX foci. DAG-induced cytotoxicity was unaffected by MLH1, MSH2, and DNA-PK expression, but was enhanced by knockdown of BRCA1. Acting in S phase, DAG displayed selective synergy with topoisomerase I (camptothecin and irinotecan) and topoisomerase II (etoposide) poisons in GBM, PCa, and lung cancer cells with no synergy observed for docetaxel. Importantly, DAG combined with irinotecan treatment enhanced tumor responses and prolonged survival of tumor-bearing mice. This work provides mechanistic insight into DAG cytotoxicity in GBM and PCa cells and offers a rational for exploring combination regimens with topoisomerase I/II poisons in future clinical trials.

Highlights

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults[1]
Compared with untreated cells that progressed with a normal cell cycle profile after culturing in complete medium, DAG-treated cells displayed a strong timedependent S/G2-phase cell cycle arrest starting at 19 h post serum addition (Fig. 2a, b), indicating that DAGmediated cytotoxicity depends on DNA replication in these cells
As cyclin A is exclusively expressed in S and G2 cell cycle phases[27] and phospho-histone H2AX is extensively used as a surrogate marker for DNA DSBs28, we examined cyclin A2 and ɣH2AX expression using immunofluorescent (IF) staining in serum-starved PC-3 cells with or without DAG pulse treatment (50 μM DAG for 1 h) in complete medium

Summary

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults[1]. Current standard-of-care for newly diagnosed GBM patients include surgical resection, concomitant radiation with alkylating agent temozolomide (TMZ), and adjuvant TMZ therapy afterwards[2]. These treatments improve overall survival, most patients experience TMZ resistance and tumor recurrence, and the prognosis of GBM patients remains poor with a median survival time of 12–15 months. 1,2:5,6-Dianhydrogalactitol (DAG) is a bi-functional DNA-targeting agent causing N7-guanine alkylation and inter-strand crosslinks[4]. A detailed understanding of DAG cytotoxicity and cellular survival mechanisms is needed in order to further improve the outcomes of GBM therapeutic strategies in clinic

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