Repurposing Disulfiram for Targeting of Glioblastoma Stem Cells: An In Vitro Study.

Lisa Zirjacks,Lukas Klumpp,Daniel Zips,Stephan M Huber,Nicolai Stransky,Franziska Eckert,Lukas Prause,Katrin Ganser,Sabine Schleicher,Rupert Handgretinger

doi:10.3390/biom11111561

Lisa Zirjacks, Lukas Klumpp + Show 8 more

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https://doi.org/10.3390/biom11111561

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Abstract

Mesenchymal glioblastoma stem cells (GSCs), a subpopulation in glioblastoma that are responsible for therapy resistance and tumor spreading in the brain, reportedly upregulate aldehyde dehydrogenase isoform-1A3 (ALDH1A3) which can be inhibited by disulfiram (DSF), an FDA-approved drug formerly prescribed in alcohol use disorder. Reportedly, DSF in combination with Cu2+ ions exerts multiple tumoricidal, chemo- and radio-therapy-sensitizing effects in several tumor entities. The present study aimed to quantify these DSF effects in glioblastoma stem cells in vitro, regarding dependence on ALDH1A3 expression. To this end, two patient-derived GSC cultures with differing ALDH1A3 expression were pretreated (in the presence of CuSO4, 100 nM) with DSF (0 or 100 nM) and the DNA-alkylating agent temozolomide (0 or 30 µM) and then cells were irradiated with a single dose of 0–8 Gy. As read-outs, cell cycle distribution and clonogenic survival were determined by flow cytometry and limited dilution assay, respectively. As a result, DSF modulated cell cycle distribution in both GSC cultures and dramatically decreased clonogenic survival independently of ALDH1A3 expression. This effect was additive to the impairment of clonogenic survival by radiation, but not associated with radiosensitization. Of note, cotreatment with temozolomide blunted the DSF inhibition of clonogenic survival. In conclusion, DSF targets GSCs independent of ALDH1A3 expression, suggesting a therapeutic efficacy also in glioblastomas with low mesenchymal GSC populations. As temozolomide somehow antagonized the DSF effects, strategies for future combination of DSF with the adjuvant standard therapy (fractionated radiotherapy and concomitant temozolomide chemotherapy followed by temozolomide maintenance therapy) are not supported by the present study.

Highlights

IntroductionPreclinical in vitro and animal studies demonstrated a tumoricidal, chemo- and/or radio-therapy-sensitizing (for review see [1]) as well as antitumor immune-response boosting activity [2,3] of disulfiram in several
Primary cultures of glioma stem cells show different growth phenotypes ranging from free-floating spheroids to adherent monolayers [53]
LK7 Primary LK7 and LK17 glioblastoma stem cells (pGSC) grew in complete NeuroCult stem cell (NSC) medium as an attached monolayer while LK17 pGSCs formed adherent spheroids (Figure 1A) with doubling times of about 1.0 (LK17) and 1.7 (LK7) days (Figure 1B,C)

Summary

Introduction

Preclinical in vitro and animal studies demonstrated a tumoricidal, chemo- and/or radio-therapy-sensitizing (for review see [1]) as well as antitumor immune-response boosting activity [2,3] of disulfiram in several. Among those are melanoma [4], non-small-cell lung cancer (NSCLC) [5], liver cancer [6], prostate cancer [7], pancreatic cancer [8], breast cancer [9], head and neck squamous cell carcinoma (HNSCC) [10], atypical teratoid/rhabdoid tumors [11], and glioblastoma [12,13]. Due to the preclinical evidence for an antitumor effect of disulfiram, several clinical trials with glioblastoma patients (ClinicalTrials.gov identifiers NCT03363659, NCT01777919, NCT01907165, NCT02715609, NCT03151772, NCT03034135, NCT02678975, NCT02770378) have been initiated, are ongoing or finalized (e.g., [14])

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