Abstract 4414: Insulin receptor substrate 1 is a key substrate for Pim protein kinases

[BACKGROUND] Proviral integration site for Moloney murine leukemic virus (Pim) proteins are a family of serine/threonine kinases composed of three different isoforms (Pim1, Pim2 and Pim3) that are aberrantly expressed in a wide variety of tumor types, including hematopoietic malignancies. Overexpression of Pim kinases in cells exerts a pleiotropic effect on various signal transduction pathways, including cell cycle progression, cell proliferation and apoptosis. They are now being targeted in human clinical trials by small molecules inhibitors. However, the protein substrates and the mechanism of action of the Pim protein kinases are not known. To understand how Pim is regulating this diverse set of pathways, it is essential to define the substrates for this protein kinase. [MATERIAL and METHOD] To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 serine 1101 (IRS1-S1101) as a potential Pim substrate. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. [RESULT] Our in vitro kinase assay showed that S1101 phosphorylation was markedly induced by incubation of active Pim1 kinase to IRS1 immunoprecipitants, demonstrating that Pim1 is capable of directly phosphorylating IRS1 protein on S1101. Mutation of this residue in IRS1 from serine to alanine (S1101A) completely abolished in vitro phosphorylation by Pim1. Similar results were obtained using the IRS2 protein that contains an identical Pim kinase phosphorylation consensus at 1149. To find whether one or all of the three Pim isoforms are regulating the phosphorylation IRS1, each isoform was transduced into Pim triple knockout (TKO) MEF cells using lentiviruses producing Pim1, Pim2 or Pim3. Each of the three isoforms was sufficient to induce the phosphorylation of IRS1 on S1101. Consistent with these results, the depletion of each Pim kinase isoform individually using siRNA in the various cancer cell lines did not decrease IRS1 phosphorylation, but the knockdown of all three isoforms abolished the phosphorylation of the IRS1 protein. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. [Conclusion] These results demonstrate that IRS1-S1101 is a novel substrate for the Pim kinases and provide the possibility of a novel marker for evaluation of Pim inhibitor therapy. Citation Format: Jin H. Song, Andrew S. Kraft. Insulin receptor substrate 1 is a key substrate for Pim protein kinases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4414.