The addition of arginine deiminase potentiates Mithramycin A-induced cell death in patient-derived glioblastoma cells via ATF4 and cytochrome C

Abstract

Background Arginine auxotrophy constitutes a shortcoming for ~ 30% of glioblastoma multiforme (GBM). Indeed, arginine-depleting therapy using arginine deiminase from Streptococcus pyogenes (SpyADI) has proven activity against GBM in preclinical studies. The good safety profile of SpyADI renders this agent an ideal combination partner for cytostatic therapy. Methods In this study, we combined the antineoplastic antibiotic Mithramycin A (MitA) with SpyADI to boost single-agent activity and analyzed underlying response mechanisms in-depth. Results MitA monotherapy induced a time- and dose-dependent cytotoxicity in eight patient-derived GBM cell lines and had a radiosensitizing effect in all but one cell line. Combination treatment boosted the effects of the monotherapy in 2D- and 3D models. The simultaneous approach was superior to the sequential application and significantly impaired colony formation after repetitive treatment. MitA monotherapy significantly inhibited GBM invasiveness. However, this effect was not enhanced in the combination. Functional analysis identified SpyADI-triggered senescence induction accompanied by increased mitochondrial membrane polarization upon mono- and combination therapy. In HROG63, induction of lysosomes was seen after both monotherapies, indicative of autophagy. These cells seemed swollen and had a more pronounced cortically formed cytoskeleton. Also, cytochrome C and endoplasmatic reticulum-stress-associated proteins ATF4 and Calnexin were enhanced in the combination, contributing to apoptosis. Notably, no significant increases in glioma-stemness marker were seen. Conclusions Therapeutic utilization of a metabolic defect in GBM along with cytostatic therapy provides a novel combination approach. Whether this SpyADI/MitA regimen will provide a safe alternative to combat GBM, will have to be addressed in subsequent (pre-)clinical trials. Graphical Abstract

Differential Expression of SRY-Related HMG-Box Transcription Factor 2, Oligodendrocyte Lineage Transcription Factor 2, and Zinc Finger E-Box Binding Homeobox 1 in Serum-Derived Extracellular Vesicles: Implications for Mithramycin Sensitivity and Targeted Therapy in High-Grade Glioma

“…The assessment hinges on the results of an ex vivo chemosensitivity assay, which measures alterations in tumor morphology, cell viability through hematoxylin and eosin (H&E) staining, proliferative activity via Ki67 immunostaining, and apoptosis induction as evidenced by cleaved Caspase-3 staining. These observations are congruent with findings reported in the literature. − In the course of our investigation, we discerned two distinct groups of glioma patients differentiated by their response to Mit-A. This bifurcation was facilitated by a proprietary algorithm that computes an M-score, incorporating data from the functional assays described above.…”

“…Interestingly, all Mit-A-responsive patients were observed orienting toward high-grade glioblastomas; this aligns with recent findings which reported that Mit-A inhibits GBM invasiveness in patient-derived high-grade gliomas. 21,22 Moreover, the IDH1 mutation status of the tumor significantly complemented the M-score, suggesting a positive correlation between wild-type IDH1 status and response in the ex vivo setting. This finding contrasts with previous reports that posited Mit-A as a potent inhibitor for IDH mutant high-grade gliomas.…”

“…In a screen of 147 FDA-approved oncology drugs, Mit-A and six other drugs demonstrated promising antiproliferative activity in GSCs of high-grade isocitrate dehydrogenase (IDH) mutant glioma . Another study showed that Mit-A inhibits invasiveness in high-grade gliomas . Mit-A causes severe side effects such as trasaminitis and hepatotoxicity .…”

“…21 Another study showed that Mit-A inhibits invasiveness in highgrade gliomas. 22 Mit-A causes severe side effects such as trasaminitis 23 and hepatotoxicity. 24 Mit-A derivatives have potent antitumor activity, and decreased toxicities will make the compound an attractive drug candidate for glioma treatment due to its ability to cross the blood-brain barrier to modulate the transcriptional targets of GSCs, including SOX2, OLIG2, and ZEB1.…”