Abstract 2642: Small molecule inhibitor borrelidin induces apoptosis in acute lymphoblastic leukemia cell lines in association with GCN2 kinase pathway activation

Abstract

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children. L-asparaginase (L-ASNase), an important agent in the treatment of ALL, catalyzes the hydrolysis of asparagine (Asn), leading to activation of the general control nonderepressible-2(GCN2) kinase stress-responsive pathway, inhibition of protein synthesis, and apoptotic cell death. Unfortunately, allergic hypersensitivity reactions can occur in patients after repeated administrations of ASNase and are the main reason for discontinuation of ASNase treatment. Borrelidin, a small molecule nitrile-containing macrolide, is a known inhibitor of bacterial and eukaryal threonyl-tRNA synthetase (ThrRS). As both ASNase and borrelidin increase the levels of uncharged tRNA, we hypothesize that ASNase may be replaced by borrelidin. Thus, the aim of the present study was to investigate if borrelidin could have a potential role in targeting ALL cell lines and, whether the GCN2 kinase pathway activation is involved as one of the downstream mechanisms by which borrelidin affects lymphoblastic cells. Methods: In order to mediate inhibition of threonyl-tRNA synthesis and to induce nutritional stress in ALL cell lines, Jurkat and CEM cells, cells were treated with borrelidin. Proliferation assay was also performed and the growth rate of these cells was studied. Propidium iodide staining and flow cytometry were performed to study the apoptotic effects of borrelidin on these cells as well as to study the effects of borrelidin on cell cycle progression. The levels of phosphorylated GCN2, total GCN2, phosphorylated eIF2α, total eIF2α, CHOP and cleaved PARP were evaluated by Western blot analysis. Results: Borrelidin was able to potently inhibit the proliferation and growth of ALL cell lines in low concentrations. Borrelidin showed a greater apoptotic effect on T cells compared to primary fibroblasts (half maximal inhibitory concentration (IC50) of 50 ng/ml and 375 ng/ml respectively). Flow cytometry and Western blot analysis indicated that borrelidin was able to increase the level of apoptosis in treated Jurkat cells as compared with non-treated control groups (3.9 ± 1.26 and 0.77 ± 0.06, respectively; p<0.01, n=3). Flow cytometry results showed a significant dose-dependent G1 arrest in Jurkat cells treated with borrelidin (10.88 ± 0.63) compared to that of non-treated control group (4.18 ± 0.24; p<0.001, n=3). Activation of the GCN2 kinase pathway as well as induction of the proapoptotic CHOP protein was significantly higher in ALL cell lines treated with borrelidin relative to the control non-treated cells. Conclusions: These findings collectively suggest for the first time that borrelidin may have promising potentials in treating ALL by activating the GCN2 kinase pathway, inducing apoptosis and mediating G1 arrest in T-cells. 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 2642. doi:10.1158/1538-7445.AM2011-2642