G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind G␣ i free of G␤␥ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3⅐ G␣ i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G␣ i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of ␣ 2 -adrenergic receptors or -opioid receptors reduced AGS3-RLuc⅐G␣ i1 -YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by G␤␥ sequestration with the carboxyl terminus of GRK2. G␣ i -dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G␣ i and/or G␣ o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both G␣␤␥ and GPR-G␣ i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Key Points SK2 is overexpressed in myeloma cells and contributes to myeloma cell survival and proliferation. SK2-specific inhibitor promotes proteasome degradation of Mcl-1 and c-Myc and inhibits myeloma growth in vitro and in vivo.

BackgroundPim (proviral insertion in murine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown.MethodsIn the current study, Pim1−/−2−/−3−/− triple knockout (TKO) mice were used to determine the role of Pim kinases in hematopoiesis. Peripheral blood hematological parameters were measured in Pim TKO mice and age-matched wild-type (WT) controls. Primary, secondary, and competitive transplantations were performed to assay the long-term repopulating HSCs in Pim TKO mice. In vivo BrdU incorporation assay and ex vivo Ki67 staining and caspase 3 labeling were performed to evaluate the proliferation and apoptosis of HSCs in Pim TKO mice.ResultsCompared to age-matched WT controls, Pim TKO mice had lower peripheral blood platelet count and exhibited erythrocyte hypochromic microcytosis. The bone marrow cells from Pim TKO mice demonstrated decreased hematopoietic progenitor colony-forming ability. Importantly, Pim TKO bone marrow cells had significantly impaired capacity in rescuing lethally irradiated mice and reconstituting hematopoiesis in primary, secondary and competitive transplant models. In vivo BrdU incorporation in long-term HSCs was reduced in Pim TKO mice. Finally, cultured HSCs from Pim TKO mice showed reduced proliferation evaluated by Ki67 staining and higher rate of apoptosis via caspase 3 activation.ConclusionsPim kinases are not only essential in the hematopoietic lineage cell development, but also important in HSC expansion, self-renewal, and long-term repopulation.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

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