PKC412 Directly Inhibits the Serine/Threonine Protein Kinase Pim-1 in Cell Lines and Acute Myeloid Leukemia Cells as a Novel Mechanism of Inhibition of Multidrug Resistance

The serine/threonine protein kinase Pim-1, encoded by a proto-oncogene originally identified as the proviral insertion site in Moloney murine leukemia virus lymphomagenesis, is frequently overexpressed in acute myeloid leukemia (AML) and other malignancies and has been implicated in regulation of both cell cycle and apoptosis. We recently demonstrated that Pim-1 also regulates drug resistance. Pim-1 phosphorylates and activates the ATP-binding cassette (ABC) transporters P-glycoprotein (Pgp; MDR1; ABCB1) and breast cancer resistance protein (BCRP; ABCG2), which are strongly associated with clinical drug resistance in AML and other malignancies, and Pim-1 knockdown results in inhibition of drug resistance mediated by both of these proteins. Efforts are in progress to identify and characterize clinically applicable Pim-1 inhibitors. PKC412 (n-benzoyl-staurosporine; midostaurin), a staurosporine analog originally characterized as a protein kinase C (PKC) inhibitor at nanomolar concentrations, is currently being tested as an inhibitor of fms-like tyrosine kinase 3 (FLT3) signaling in AML, with 10 micromolar trough concentrations achieved in clinical trials. Staurosporine has been shown to complex with Pim-1, and we hypothesized that PKC412 might be a clinically applicable inhibitor of Pim-1 kinase and that PKC412 inhibition of Pim-1 kinase would result in inhibition of multidrug resistance mediated by both Pgp and BCRP. Pim-1 activity was measured by an in vitro kinase assay of BAD phosphorylation, and Pim-1 expression, phosphorylation and co-immunoprecipitation were measured by Western blot analysis. Transport by Pgp and by BCRP was measured by comparing uptake of the Pgp- and BCRP-specific fluorescent substrates 3,3′-diethyloxacarbocyanine iodide [DiOC₂(3)] and pheophorbide A, respectively, measured by flow cytometry, in the presence and absence of the Pgp- and BCRP-specific inhibitors PSC-833 and fumitremorgin C. PKC412 at a 5 micromolar concentration inhibited Pim-1 kinase activity in Pim-1-overexpressing multidrug resistant cell lines including HL60/VCR and 8226/Dox6, which overexpress Pgp, and 8226/MR20, which overexpresses BCRP, as well as in other cell lines with Pim-1 overexpression, including K562 and MOLM14 leukemia cells. Direct inhibition of Pim-1 kinase by PKC412 was demonstrated by the in vitro kinase assay. Moreover, PKC412 inhibition of Pim-1 auto-phosphorylation in vitro provided additional evidence that PKC412 directly inhibits Pim-1. PKC412 at a 5 micromolar concentration inhibited Pim-1 binding to both Pgp and BCRP, and markedly decreased phoshorylation of both of these proteins. PKC412 inhibited drug transport by Pgp at a range of concentrations, with maximum effect at 5 micromolar, and inhibited drug transport by BCRP at concentrations of 5 micromolar and higher. We also looked at the effect of PKC412 on Pim-1 activity in pretreatment blasts from AML patients. Pim-1 was overexpressed in 6 of 7 AML samples studied, including 3 of 3 with FLT3 internal tandem duplication, and PKC412 at a 5 micromolar concentration inhibited Pim-1 activity, as demonstrated by decreased BAD phosphorylation, in all 6 AML samples with Pim-1 overexpression. Thus PKC412 directly inhibits activity of the serine/threonine protein kinase Pim-1 at a clinically applicable concentration, and PKC412 inhibition of Pim-1 decreases its binding to, and phosphorylation of, both Pgp and BCRP and decreases drug transport by both of these proteins. Inhibition of Pim-1 by PKC412 at a 5 micromolar concentration is a novel mechanism of inhibition of multidrug resistance mediated by Pgp, in addition to its known inhibition of PKC at nanomolar concentrations, and is also a mechanism of inhibition of multidrug resistance mediated by BCRP. PKC412 is a clinically applicable inhibitor of Pim-1 kinase and PKC412 and other future Pim-1 inhibitors represent novel chemosensitizing agents, as well as novel anti-leukemic agents.