Endocrine Abstracts

Current systemic treatment options for patients with ACCs are far from being satisfactory. DNA damage/repair mechanisms, which involve e.g. ATM/ATR-signalling or RRM1/RRM2 encoded ribonucleotide reductase (RNR) activation commonly contribute to drug resistance. Moreover, also 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 classical chemotherapies (doxorubicin, etoposide, cisplatin, mitotane, gemcitabine, paclitaxel), phytochemicals (9-cis-retinoic-acid, Isoquercitrin) and molecular targeted inhibitors (Erlotinib, Linsitinib, Sorafenib, Sunitinib, XAV-939), we recently provided strong evidence for anti-tumoral efficacy of combined Gemcitabine (G) and Cisplatin (P) treatment against adrenocortical cells (NCI-H295R and MUC-1). However, accompanying induction of RRM1 and RRM2 expression also indicated developing G-resistance, an effect that was partially reversed upon addition of P. Our findings were confirmed for RRM2 protein (NCI-H295R: G: 2.51 ± 0.14, P < 0.001; P: 1.14 ± 0.85, ns; G+P 0.85 ± 0.10, ns vs controls; MUC-1: G: 3.84 ± 0.72, P = 0.002; P: 2.55 ± 0.44, ns; G+P 1.57 ± 0.22, ns vs. controls), RNR-dependent dATP-levels (NCI-H295R: G: 1.64%; P: 83.61%; G+P 124% vs controls; MUC-1: G: 13.33%; P: 101.11%; G+P 102.22% vs. controls) and modulations of related ATM/ATR-signalling (Chk1, Chk2, H2AX). The applied models carry different TP53-mutations from which both enabled significant induction of RRM2b-expression upon specific treatments. However, the P-induced reversal to baseline or below differed in both models. Finally, we screened for complementing inhibitors of the DNA damage/repair system targeting RNR, Wee1, CHK1/2, ATR and ATM applying MTT and BrdU assays. Many tested inhibitors demonstrated already as single agents significant anti-tumour potential (MTT NCI-H295R: untreated 100.0 ± 1.9%; COH29 (dual RRM1/RRM2-complex-inhibitor): 60.2 ± 6.2%, P < 0.001; Adavosertib (Wee1-inhibitor): 84.9 ± 3.0%, P = 0.107;Prexasertib (CHK1/2-inhibitor): 65.61 ± 1.5%, P < 0.001;VE822 (ATR-inhibitor): 101.2 ± 0.7%, P = 0.999; AZD0156 (ATM-inhibitor): 104.3 ± 8.2%, P = 0.995; MUC-1: untreated 100.0 ± 1.0;COH29 50.2 ± 1.5%, P < 0.001;Adavosertib 50.5 ± 2.6%, P < 0.001;Prexasertib 86.5 ± 0.9%, P = 0.002;VE822 89.7 ± 3.1%, P = 0.024;AZD0156 91.9 ± 1.3%, P = 0.119). Of note, the combination G, P and COH29 resulted in previously unreached total cell killing of both, NCI-H295R (untreated 100.0 ± 4.3%; G: 110.7 ± 20.1%, P = 0.196; P: 14.3 ± 0.4%, P < 0.001; G+P: 11.7 ± 1.2%, P < 0.001; G+P+COH29: 0.0 ± 0.1%, P < 0.001) and the commonly highly drug resistant MUC-1 cells (untreated 100.0 ± 1.3; G: 96.4 ± 9.6%, P = 0.996; P: 57.9 ± 6.3%, P < 0.001; G+P: 47.2 ± 4.2%, P < 0.001; G+P+COH29 0.0 ± 0.1%, P < 0.001;). Clinically, our analysis of TCGA and own patient samples revealed for both data sets significantly elevated expression levels of RRM2 (P < 0.001) but not RRM1 in ACC (n = 33/30) compared to normal adrenals (n = 10/21) and adenomas (n = 22/20). Higher RRM2 expression was correlated with larger tumour size (P = 0.02) and strong tendency towards poorer overall survival (P = 0.05). In summary, we provide evidence that RNR-modulating therapies might represent a new therapeutic option for ACC.