Abstract B02: Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram
Authors: Bista Ranjan , Pepper Oliver , Lee David W. , Azorsa David A. , Arceci Robert J. , Aleem Eiman 
Published: May 2016

Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children. Treatment options for relapsed or refractory AML are limited. Bortezomib, a well-established proteasome inhibitor, is used in AML therapy. It binds to the beta 5 subunit (PSMB5) of the 26S proteasome and inhibits the chymotrypsin (CT)-like activity. Although patients develop resistance to bortezomib, the mechanism is not fully elucidated. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism without major side effects. DS has anticancer cytotoxicity that is in part copper (Cu2+)-dependent (DS/Cu2+). One of the reported mechanisms of action of DS is through proteasome inhibition. We previously demonstrated that both DS/Cu2+ and bortezomib are cytotoxic in a panel of AML cell lines. In the present study, we focused on two acute megakaryoblastic leukemia cell lines derived from patients with Down syndrome (CMY and CMK), one of the cell lines is resistant to cytarabine (CMY), a key drug used in AML treatment. Down syndrome children with leukemia are more prone to suffer from significant toxicity to chemotherapy; therefore, the need for novel treatment options for this category of patients is warranted.

Purpose: The purpose of the present study was to develop an in vitro model of bortezomib resistance in CMY and CMK cell lines, to understand the mechanism of resistance, and to identify drugs that are cytotoxic in the bortezomib-resistant cell lines.

Methods and Results: The bortezomib-resistant cell lines CMY and CMK were generated by exposure to stepwise increasing concentrations of bortezomib over a period of 9 months up to a concentration of 200 nM in CMY, and 100 nM in CMK cells. Viability was determined using Cell Titer Glo assay. CMY B200nM was 70 fold resistant to bortezomib with decreased inhibition of CT-like activity compared to the parent CMY cell line. Similar results were observed in the CMK cell line. An A362C mutation in PSMB5 causing a change from glutamine to proline was found in the bortezomib resistant CMY cell line. In order to confirm that this mutation caused bortezomib resistance, the PSMB5 (A362C) from CMY B200nM was cloned into the pcDNA3.1 vector and transfected into HEK 293A cells. This caused a two-fold higher bortezomib resistance than the corresponding control. The bortezomib-resistant AML cell lines were cross-resistant to the proteasome inhibitors carfilzomib and MG132 compared to the parent cell line, but were sensitive to DS/Cu2+. Moreover, the PSMB5 mRNA levels and the beta 5-subunit protein levels increased with bortezomib resistance , as evaluated by quantitative real-time RT-PCR and western blot, respectively. Although DS/Cu2+ induced ubiquitination similar to bortezomib, no comparable inhibition of CT-like activity was observed, suggesting a different mechanism of proteasome inhibition.

Conclusions: The present results show that (1) the A362C mutation in the PSMB5 may underlie bortezomib-resistance in the cytarabine-resistant CMY cell line. This may provide an explanation for resistance to bortezomib in AML patients, which we hope to assess in the ongoing COG AML trial in which patients are randomized to receive bortezomib plus chemotherapy versus chemotherapy alone, and (2) DS/Cu2+ sensitivity may provide an effective and less toxic treatment approach for AML to be tested in the Down syndrome group of patients with poor outcome following relapse.

Citation Format: Ranjan Bista, Oliver Pepper, David W. Lee, David A. Azorsa, Robert J. Arceci, Eiman Aleem. Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B02.