Abstract

Introduction: Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of acute myeloid leukemia (AML), however, c-Myc has been an elusive therapeutic target for drug development. In this study, we sought to target aberrant c-Myc expression by inhibition of the PAM signaling pathway in conjunction with all-trans retinoic acid (ATRA) as a strategy to reduce stem cell potential as well as to induce growth arrest, differentiation and apoptosis in AML cells. Methods: Small molecule inhibitors of PI3K (ZSTK474), mTOR (WYE- 125132) and PI3K/mTOR (NVP-BEZ235, dactolisib) where used alone or in combination with ATRA to treat AML cell lines and primary patient samples. Colony formation potential, cell growth, cell cycle arrest, differentiation and apoptosis were analyzed. c-Myc levels were monitored by qPCR and immunoblotting. Global gene expression profiling was conducted using Affymetrix GeneChip® ST Arrays.
Results: PI3K and mTOR inhibition by ZSTK474 or WYE-125132 respectively, induced cell cycle arrest at low nanomolar concentrations in AML cell lines and primary patient samples. The dual PI3K/mTOR inhibitor NVP-BEZ235 was the most potent PAM inhibitor tested. Addition of ATRA further augmented the anti-leukemic effects of PAM inhibitors in vitro. Co-treatment lead to reduced colony formation capacity as well as cell cycle arrest, differentiation and programmed cell death. We observed a rapid down regulation of c-Myc following treatment with NVP-BEZ235 via impaired translation, destabilization and enhanced degradation. ATRA treatment caused transcriptional and translational repression. NVP-BEZ235 combined with ATRA produced maximal c-Myc suppression and the greatest anti-leukemic effects. Detailed analysis of transcriptome changes in MV4-11 cells following treatment with NVP-BEZ235 and ATRA, revealed that both agents regulated the same biologic pathways, but acted on different gene sets within these pathways. Conclusions: Here we present a novel multi-target approach to suppress aberrant c-Myc levels in AML. While inhibition of the PAM pathway leads to inhibition of c-Myc translation, destabilization and degradation, supplementary treatment with ATRA can further decrease c-Myc levels by inhibition of its transcription as well as translation. These data support the clinical investigation of ATRA combined with PAM inhibitors especially in AML patient with high c-Myc activity.