Repressing phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma by microRNA-142-3p restrains the progression of hepatocellular carcinoma.

This paper probes the mechanisms underlying miR-142-3p's modulation of hepatocellular carcinoma (HCC) invasion and apoptosis. Quantitative real-time PCR and Western blot monitored the miR-142-3p profile in HCC tissues and non-tumor tissues. The correlation between miR-142-3p expression and HCC patients' clinicopathological indicators was analyzed. miR-142-3p overexpression and knockdown models were established in HCC cell lines. Cell proliferation was gauged by the colony formation assay and BrdU staining. For measuring apoptosis, flow cytometry and Western blot were implemented. Transwell assay tested cell migration and invasion. miR-142-3p mimics or inhibitors were transfected in Huh7 and HCCLM3 cells. The targeting association between miR-142-3p and PIK3CG was predicted through bioinformatics and further verified by related experiments. The influence of PIK3CG overexpression on miR-142-3p's role in HCC was assayed. A xenografted tumor model was built in mice to validate miR-142-3p knockdown's influence on HCC in vivo. As a result, miR-142-3p exhibited a decreased profile in HCC tissues and cells. Overexpressing miR-142-3p accelerated apoptosis and suppressed the PI3K/AKT/HIF-1α signal. Knocking down miR-142-3p presented opposite effects. PIK3CG overexpression dampened the anti-tumor effect of miR-142-3p. miR-142-3p repressed HCC invasion and intensified apoptosis to restrain HCC by abating the PIK3CG-mediated PI3K/AKT/HIF-1α pathway.

Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ.

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.

Discovery of Potent and Selective PI3Kγ Inhibitors.

The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 µM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.

Ex vivo blockade of PI3K gamma or delta signaling enhances the antitumor potency of adoptively transferred CD8+ T cells.

Adoptive T cell transfer therapy induces objective responses in patients with advanced malignancies. Despite these results, some individuals do not respond due to the generation of terminally differentiated T cells during the expansion protocol. As the gamma and delta catalytic subunits in the PI3K pathway are abundant in leukocytes and involved in cell activation, we posited that blocking both subunits ex vivo with the inhibitor IPI-145 would prevent their differentiation, thereby increasing antitumor activity in vivo. However, IPI-145 treatment generated a product with reduced antitumor activity. Instead, T cells inhibited of PI3Kγ (IPI-549) or PI3Kδ (CAL-101 or TGR-1202) alone were more potent in vivo. While T cells coinhibited of PI3Kγ and PI3Kδ were less differentiated, they were functionally impaired, indicated by reduced production of effector cytokines after antigenic re-encounter and decreased persistence in vivo. Human CAR T cells expanded with either a PI3Kγ or PI3Kδ inhibitor possessed a central memory phenotype compared to vehicle cohorts. We also found that PI3Kδ-inhibited CARs lysed human tumors in vitro more effectively than PI3Kγ-expanded or traditionally expanded CAR T cells. Our data imply that sole blockade of PI3Kγ or PI3Kδ generates T cells with remarkable antitumor properties, a discovery that has substantial clinical implications.

The positive inotropic effect of relaxin-2 in human atrial myocardium is preserved in end-stage heart failure: role of G(i)-phosphoinositide-3 kinase signaling.

BACKGROUND: Relaxin-2, a candidate drug for acute heart failure, has been tested successfully in the first human trials. We investigated relaxin's inotropic effects in human myocardium. METHODS AND RESULTS: In atrial samples from donor (n = 7) and failing (n = 7) hearts, relaxin-2 evoked remarkable positive inotropic effects: showing a half maximum effective concentration of < 1 nmol/L, the maximum peak developed tension (PDT) rose to approximately 270% of baseline, without differences between failing and nonfailing myocardium. The effects critically depended on protein kinase A activation and inhibition of the transient potassium outward current; phosphoinositide-3 kinase inhibition and pertussis toxin pretreatment moderately blunted the effects in nonfailing but markedly suppressed them in failing myocardium. Action potential recordings revealed identical effects of inhibition of the transient potassium outward current and relaxin. In ventricular myocardium, however, relaxin did not show any inotropic effects. The expression of the RXFP1 receptor was moderately decreased in failing compared with nonfailing atrial myocardium but not detectable in any ventricular samples. CONCLUSIONS: Relaxin is a positive inotrope in nonfailing and failing human atria, with critical involvement of protein kinase A and inhibition of the transient potassium outward current and an increasing role for G(i) protein-phosphoinositide-3 kinase signaling in failing myocardium.