Abstract 4352: Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine.

Abstract

Erufosine is a new antineoplastic agent from the class of alkylphosphocholines, which interferes with AKT phosphorylation, the related signal transduction and confers apoptosis. An improved understanding of its mode of action is important for its potential clinical application. The present study demonstrates that erufosine's antileukemic efficacy is related to the level of cellular retinoblastoma (Rb) protein expression. Stable Rb-knockdown in SKW-3 leukemia T-cells was induced by lentiviral delivery of constructs expressing short-hairpin-RNA species with 21 or 27 bp lengths. The cytotoxicity of erufosine was tested via MTT proliferation test on thus engineered cell populations with reduced Rb expression levels. Likewise, the clonogenicity of these engineered cell populations was determined by CFU assay before and after treatment with erufosine. The effects on cell cycle were investigated by FACS analysis. The expression of signal transduction factors was examined by RT-PCR and immunoblot. The Rb protein expression levels in the shRNA-transduced cells ranged from 1%- 36% of wild type or nonsense control. Low Rb expression correlated with significantly diminished antiproliferative and anticlonogenic activities of erufosine (p<0.05) at concentrations near and above the IC50. Erufosine induced a G2 arrest, and complete Rb-deficiency aggravated this condition. With regard to proteins involved in cell cycle and apoptosis induction, Rb-deficiency reduced the expression of cyclin D3, that of the subsequently regulated kinase Cdk 4 and activated caspases 3 and 9, followed by PARP-cleavage. In partial variance, erufosine reduced cyclin D3 at RNA and protein levels, but affected not or only slightly the expression of its partner kinase Cdk 4. Rb-knockdown impaired the cytotoxic effect of erufosine significantly and contributed to the formation of a resistant cell fraction. It is concluded that Rb is a main mediator of erufosine's antileukemic activity and this suggests that the effect of the drug in patients might be predicted by determining the Rb expression status. Citation Format: Maya M. Zaharieva, Milen Kirilov, Minquang Chai, Stefan M. Berger, Spiro M. Konstantinov, Martin R. Berger. Down regulation of retinoblastoma protein expressionimpedes the antileukemic activity of erufosine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2013-4352