Abstract 2238: Role of phosphoinositide-dependent protein kinase-1 (PDK1) in leukemogenesis of acute leukemia
2012-04-01

Abstract The Phosphoinositide-dependent Protein Kinase-1 (PDK1) is a key regulator of AKT signaling by phosphorylation of a threonine residue at codon 308. PDK1 itself is activated by PI3kinases and plays an important role in growth factor signaling. We previously demonstrated that AKT is globally activated in acute leukemia, e.g. via gain-of function tyrosine kinase mutations. Consecutively we have shown that tyrosine kinase inhibitors suppress codon 473 phosphorylation - but leave Thr308 activated, hypothetically via activation by growth factors. Accordingly, we show that phosphorylation status of AKT in bone marrow leukemia blasts is higher compared to circulating blasts, which favors the hypothesis of a protective bone marrow microenvironment due to the vast occurrence of growth factors and chemokines, which activate PI3K/AKT signaling. We aimed to investigate whether activation of AKT(Thr308) is crucial for the maintenance of leukemia cells and leukemogenesis and silenced PDK1 by retrovirally transducing different short hairpin RNA constructs cloned into a mammalian vector into AML cell lines. Proliferation capacity of the PDK1-silenced cells in comparison to an empty vector control was studied using an XTT-based assay. Cell lines with suppressed transcription of PDK1 revealed a significantly diminished proliferation capacity compared to the empty vector controls. To evaluate potency of tumor engraftment and aggressiveness of disease in vivo, we transplanted stably transfected PDK1-shRNA silenced acute leukemia cells into NOD/SCID mice. Isogenic cells transfected with an empty vector were used as the control group. Mice were regularly monitored for disease symptoms and leukemic blast count. Already on day 8, the peripheral blast count in the control mice revealed a faster increase with higher cell counts (∼ 4 x 10ex3/microL) compared to the PDK1-silenced group (∼ 0,5 x 10ex3/microL). Over time, all mice finally developed leukemia, with the control mice to be the first. In addition, we tested two novel PI3K/mTOR inhibitors, NVP-BEZ235 and NVP-BGT226, with regard to the antiproliferative capacity in ex vivo leukemia blasts. Both agents revealed potent antiproliferation with NVP-BGT226 the more potent agent. NVP-BGT226 induced apoptosis in ex vivo blasts in a subset of patients, with an IC50 in the low nanomolar range. In summary, we here identify the PDK1-AKT signal transduction pathway to be crucial for aggressiveness of disease. Of note, it was not possible to suppress development of leukemia completely, arguing for a multistep mechanism of leukemogenesis. Our data supports descriptive expression analysis of PDK1 or AKT(Thr308) that link high expression with unfavorable clinical outcome. Therefore, targeting the PI3K/AKT axis which activates PDK1 in acute leukemia may be beneficial to improve therapy outcome in a subset of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2238. doi:1538-7445.AM2012-2238