Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (i) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3'UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative regulators of this phosphorylation, PTEN and PDK1, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC 50 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to smallmolecule FLT3 inhibitors such as PKC412. IntroductionFLT3, a class 3 receptor tyrosine kinase, is targeted and activated by somatic mutation in acute myeloid leukemia (AML). Internal tandem duplication (ITD) mutations of the FLT3 juxtamembrane (JM) occur in approximately 24% of patients with AML and in 15% of patients with secondary AML 1,2 and are associated with shortened disease-free survival. 3 Mutations in the activation loop (AL), typically D835Y, occur in approximately 7% of patients with AML and 3% of patients with myelodysplastic syndromes (MDS) 4,5 and in patients with T-cell ALL (T-ALL). 6 An increased frequency of FLT3 mutations has also been associated with mutations involving the mixed-lineage leukemia (MLL) gene. 7 ITD and AL mutations result in constitutive FLT3 kinase activity. When FLT3 receptors harboring such mutations are introduced into mammalian cells, downstream signaling pathways are activated that lead to factor-independent growth in vitro and to leukemogenesis in vivo. 8,9 For example, the production of FLT3-ITD mutant proteins in primary murine bone marrow cells results in a lethal myeloproliferative phenotype. 10 Thus, FLT3 is a leukemia oncogene, and activating FLT3 mutations likely contribute significantly to the development of leukemia in humans.Several small-molecule inhibitors block the kinase activity of FLT3 with high potency 10-13 (eg, PKC412, MLN518, SU11248) and can prolong the lifespans of mice harboring leukemias expressing mutant FLT3 receptors. 10,14 In phase-1 and -2 clinical trials, FLT3 inhibitors have reduced FLT3 phosphorylation 15-17 and leukemia...