Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment

SIMPLE SUMMARY: The current clinical gold standard etoposide, doxorubicin, cisplatin, and mitotane (EDP-M) is not satisfying for the treatment of adrenocortical carcinoma (ACC). However, clinical translation of novel, preclinically promising therapies were unfortunately disappointing in recent years, indicating that utilized tumor models inadequately predicted clinical applicability of novel pharmacological approaches. In an attempt to optimize the current preclinical armamentarium, our workgroup initiated a comparative drug screen of clinically relevant chemotherapies and therapies targeting IGF, EGF, and Wnt signaling pathways in the classical NCI-H295R cell line and, for the first time, in the recently developed highly drug-resistant MUC-1 cell line. These testings revealed gemcitabine and cisplatin as a promising combination, but further investigations also indicated developing drug resistance mechanisms on the molecular level. We aimed to decipher underlying resistance mechanisms, identified ribonucleotide reductase as an important player, and successfully targeted the involved DNA damage/repair mechanism. ABSTRACT: Current systemic treatment options for patients with adrenocortical carcinomas (ACCs) are far from being satisfactory. DNA damage/repair mechanisms, which involve, e.g., ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia/Rad3-related (ATR) protein signaling or ribonucleotide reductase subunits M1/M2 (RRM1/RRM2)-encoded ribonucleotide reductase (RNR) activation, commonly contribute to drug resistance. Moreover, the regulation of RRM2b, the p53-induced alternative to RRM2, is of unclear importance for ACC. Upon extensive drug screening, including a large panel of chemotherapies and molecular targeted inhibitors, we provide strong evidence for the anti-tumoral efficacy of combined gemcitabine (G) and cisplatin (C) treatment against the adrenocortical cell lines NCI-H295R and MUC-1. However, accompanying induction of RRM1, RRM2, and RRM2b expression also indicated developing G resistance, a frequent side effect in clinical patient care. Interestingly, this effect was partially reversed upon addition of C. We confirmed our findings for RRM2 protein, RNR-dependent dATP levels, and modulations of related ATM/ATR signaling. Finally, we screened for complementing inhibitors of the DNA damage/repair system targeting RNR, Wee1, CHK1/2, ATR, and ATM. Notably, the combination of G, C, and the dual RRM1/RRM2 inhibitor COH29 resulted in previously unreached total cell killing. In summary, we provide evidence that RNR-modulating therapies might represent a new therapeutic option for ACC.