Hypermethylation of O6-Methylguanine-DNA Methyltransferase (MGMT) Predicts Response to Laromustine (Cloretazine®) in Patients with Acute Myelogenous Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)

Abstract

Laromustine (Cloretazine®) (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl] hydrazine), a sulfonylhydrazine prodrug producing chlorethylating and carbamoylating subspecies, has demonstrated clinical activity in patients with hematologic disorders (Giles et al., J Clin Oncol, 2007). The effect of laromustine is modulated primarily through the formation of hard chloroethylating electrophiles with preferential alkylating activity for the O6 position of guanine, ultimately resulting in the formation of interstrand cross-links which prevent DNA replication and transcription, leading to cell death. The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing O6-chloroethylguanine alkylations, critical to the formation of interstrand crosslinks. Epigenetic silencing of the MGMT encoding gene related to DNA hypermethylation has been shown to participate in the pathogenesis of neoplastic disease (Hegi et al., NEJM 2005). Since the alkylating properties of laromustine target DNA sites normally repaired by MGMT, the absence of the enzyme may represent a unique cellular environment for specific susceptibility to laromustine (Ishiguro et al., Mol Cancer Ther, 2005). In vitro findings have supported the hypothesis that cellular content of MGMT may predict response. In vivo correlation between clinical response in patients treated with laromustine and the cellular evidence of epigenetic silencing of the encoding MGMT gene has yet to be demonstrated (Giles et al., Clin Cacner Res, 2004). Our research aimed to determine the DNA methylation status of MGMT isolated from the peripheral blood or bone marrow of patients with AML or high-risk MDS enrolled to a phase II, single-agent study of laromustine (600 mg/m2 IV over 30 minutes) (Giles et al. J Clin Oncol, 2007). We also aimed to establish a correlation between hypermethylation of MGMT and clinical response to laromustine. Combined bisulfite restriction analysis (COBRA) was used to determine the MGMT gene methylation status of patients treated with laromustine. Bone marrow or peripheral blood leukocyte samples from 76 patients enrolled in a phase II, single agent study of laromustine were coded and blinded to investigators. DNA from each sample was extracted and bisulfite treated. PCR was used to amplify the MGMT CpG Island promoter region (REF/NT_008818.15/Hs10_8975) from 58 patients, prior to methylation specific restriction enzyme digestion. Results were correlated with clinical data of response to laromustine. The DNA methylation status of MGMT was determined in 58 of the enrolled patients. DNA hypermethylation was found in 3 of the 58 patients (5%). Two of these 3 patients achieved a complete response (CR) (66%), compared to 11 of the 55 patients who achieved a CR, CR with platelet recovery < 100,000/mm3 (CRp) or a partial response (PR) and did not exhibit MGMT hypermethylation (20%). Hypermethylation of the MGMT gene promoter is a rare event in AML (5%), however, epigenetic inactivation of MGMT may predict a subgroup of patients with a higher likelihood of response to laromustine (Odds Ratio=8). Clinical investigation of laromustine in patients with AML, high-risk MDS and other cancers, specifically those with a higher methylation frequency of the MGMT encoding gene is warranted.