Abstract The prodrug Laromustine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino) carbonyl] hydrazine; Cloretazine; VNP40101M) yields methyl isocyanate and a chloroethylating species (90CE) upon decomposition in situ. Laromustine's cytotoxicity is principally attributed to 90CE via a proposed mechanism that involves interstrand DNA cross-linking. However, the role of methyl isocyanate in the antineoplastic function has not been fully defined. A derivative of Laromustine that generates only methyl isocyanate and not the chloroethylating species, 1,2-bis[methylsulfonyl]-1-[(methylamino) carbonyl] hydrazine (101MDCE), is reported here to induce endothelial cell (EC) death through a non-apoptotic pathway at both relatively low (50 μM) and relatively high (200 μM) concentrations. 90CE, which lacks carbamoylating activity, did not induce this cell fate. 101MDCE, but not 90CE, also inhibited EC tube formation in vitro, a process involved in angiogenesis, which is critical for tumor growth and metastases. To understand the molecular mechanism for 101MDCE-induced EC death, we examined signaling pathways activated by 101DMCE in ECs. We found that 101DMCE, but not 90CE, activated JNK/p38 pathways and their upstream apoptosis activator signal-regulating kinase 1 (ASK1) using Western blot analyses. In resting ECs, ASK1 forms a complex with the reduced form of thioredoxin (Trx). The single cysteine residue in ASK1 (C250) and C32 or C35 in Trx are critical for this interaction. 101MDCE effected ASK1 dissociation from Trx, but not from the phosphoserine-binding inhibitor 14-3-3, in vitro and in vivo, consistent with the known reactivity of methyl isocyanate with the sulfhydryl groups of cysteine residues. We previously reported that the carbamoylating activity of Laromustine inhibits the enzyme Trx reductase from leukemia cell extracts. We conclude that carbamoylating agents induce ASK1-dependent EC death through dissociation of ASK1 from Trx either directly by targeting the critical Cys groups in the ASK1-Trx complex or indirectly by inhibiting Trx reductase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4469. doi:10.1158/1538-7445.AM2011-4469