Lipid and Protein Substrates of PI3K in Cytokine Receptor Survival Signalling: Deregulation in Leukemia

New therapeutic approaches to acute myeloid leukemia (AML) must ultimately target cell survival pathways in leukemic cells in order to be effective. We have identified a serine residue (Ser585) in the cytoplasmic domain of the common GM-CSF and IL-3 receptor beta subunit which is phosphorylated in response to sub-picomolar concentrations of growth factor and is involved in signalling cytokine-mediated survival via 14-3-3 zeta phosphoserine adaptor. While Serine 585 is tightly controlled in non-transformed haematopoietic cells from normal donors, Serine 585 is constitutively phosphorylated in AML blasts suggesting a role in AML cell survival and a novel target for anti-leukaemic therapy. We attempted to isolate Ser585 kinase activity from leukemic blasts and characterise this activity in response to serine/threonine kinase inhibitors in biochemical and biological assays.

Results: Cell extracts from primary AML blasts (>99% blasts by flow/morphology) obtained from adult patients were fractionated and assayed for intrinsic serine 585 peptide (13-mer) kinase activity via 32P gamma-ATP in vitro kinase assay. A single peak of Ser585 kinase activity was isolated and tested against a panel of serine/threonine kinase inhibitors. Kinase activity was selectively sensitive to LY294002, wortmannin and quercelin suggesting a role for the PI3K family of kinases in activating this residue. Ser585 kinase activity was also directly present in both p85 and p110 alpha PI3K immunoprecipitates from AML blasts and leukemic cell lines tested on both Ser585 peptide and recombinant beta cytoplasmic domain protein substrates. Serine 585 phosphorylation induced by sub-picomolar concentrations of GM-CSF in TF1.8 cells was inhibited by three different isoform selective p110 alpha inhibitors used at low nanomolar ranges consistent with reported IC50s. These results suggest a novel role for protein kinase rather than lipid kinase activity of PI3K alpha subunit in low dose cytokine signalling. We also show induction of serine phosphorylation of p85 PI3K regulatory subunit on Ser608 by GM-CSF, a previously reported protein substrate of PI3K. Furthermore, p110 alpha and delta inhibitors abrogate GM-CSF dependent survival of murine lineage negative bone marrow progenitor cells and also exert apoptotic activity on flow-sorted CD34+CD38−CD123+ sub-populations of primary AML blasts.

Conclusions: Inhibition of Ser585 phosphorylation by targetting PI3K protein kinase activity by isoform selective inhibitors represents a novel approach toward the eradication of residual leukemic stem cells.