Abstract LB-42: PDK1 knockdown selectively inhibits growth of cancer cells harboring activating mutations involved in MAPK signaling

Phosphoinositide-dependent protein kinase-1 (PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway which is the most commonly deregulated signaling pathway across all cancers. Increased PDK1 activity confers chemoresistance in tumor cell lines, and reduced PDK1 expression prevents the onset of tumorigenesis in mouse models. To demonstrate that PDK1 is a promising target for cancer therapy, we used a siRNA-based approach to determine the effects of PDK1 knockdown in cancer cells and primary early-passage normal cells. Loss of PDK1 protein caused a significant reduction in phosphorylation of two PDK1 substrates, AKT (T308) and RSK1 (S221). Interestingly, knockdown of PDK1 protein had preferential growth inhibitory effects in cancer cells containing activating mutations of KRAS or BRAF, compared to cells with wild-type KRAS or BRAF. In KRAS/BRAF-mutant cells, apoptosis was evidenced by PARP-1 cleavage and an increase in the number of sub-G1 DNA content. Overexpression of mouse PDK1 rescued these cells from apoptosis, confirming the specificity of the siRNA-mediated effects. Consistent with these observations, stable expression of mutant KRAS (G12V) in NL-20 immortalized normal lung epithelial cells sensitized these cells to apoptosis upon knockdown of the PDK1 protein. Importantly, overexpression of a kinase-dead mouse PDK1 mutant did not rescue cells from apoptosis, suggesting that the kinase activity is required for these PDK1 functions. Based on these results, we sought to identify inhibitors of the kinase activity of PDK1 for development as anticancer agents. GSK2334470 was identified as a potent inhibitor of PDK1 kinase (IC50 0.5 nmol/l) that demonstrated a high level of specificity to PDK1 in an in vitro kinase panel. In cell-based mechanistic assays, GSK2334470 effectively inhibited phosphorylation of the PDK1-dependent phosphorylation sites [AKT T308 (IC50: 100 nmol/L) and RSK S221 (IC50: 291 nmol/L)], but not the PDK1-independent phosphorylation site on AKT, S473 (IC50: >30 umol/L). Antiproliferative effects were seen in several cell lines using low micromolar concentrations of inhibitor; however, no correlation between growth inhibition and KRAS/BRAF mutations was observed. Further optimization may be necessary for improved potency and KRAS/BRAF selectivity. Currently, efforts are underway to better understand the disconnect between siRNA data and compound data in several experimental cancer models, particularly in those where KRAS or BRAF mutations predominate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-42.