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The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling., Competing Interests: Declaration of interests N.V. reports consultant and advisory board activities for Novartis, Petra Pharmaceuticals, Reactive Biosciences, and Magnet Biomedicine. E.A.K. is a shareholder of Eli Lilly and Company, and E.A.K. and K.K. are employees of Loxo Oncology at Lilly. L.C.C. is a founder and member of the board of directors of Agios Pharmaceuticals; is a founder and receives research support from Petra Pharmaceuticals; has equity in and consults for Cell Signaling Technologies, Volastra, Larkspur, and 1 Base Pharmaceuticals; and consults for Loxo-Lilly. J.L.J. has received consulting fees from Scorpion Therapeutics and Volastra Therapeutics. J.L.J. reports consultant activities for Scorpion Therapeutics and Volastra Therapeutics. M.D.G. reports personal fees from Novartis AG, Pfizer, Inc., and Scorpion Therapeutics. L.C.C., B.D.H., and M.D.G. are inventors on patents for Combination Therapy for PI3K-associated Disease or Disorder and The Identification of Therapeutic Interventions to Improve Response to PI3K Inhibitors for Cancer Treatment. B.D.H., L.C.C., and M.D.G. are co-founders and shareholders in Faeth Therapeutics. R.E.S. is on the sponsored advisory board for Miromatrix, Inc., and Lime Therapeutics and is a consultant and speaker for Alnylam, Inc. T.M.Y. is a stockholder and on the board of directors of DESTROKE, Inc., an early-stage start-up developing mobile technology for automated clinical stroke detection., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders., Competing Interests: Declaration of Interests J.A.M. is a member of the scientific advisory board (SAB) of 908 Devices. L.C.C. is a founder and member of the Board of Directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals. These companies are developing novel therapies for cancer. N.S.G. is a founder, SAB member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. N.S.G., T.Z., and N.P.K. are inventors on a patent application covering chemical matter in this publication owned by the Dana-Farber Cancer Institute., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Insulin stimulates the conversion of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) to phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ), which mediates downstream cellular responses. PI(4,5)P 2 is produced by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and by phosphatidylinositol-5-phosphate 4-kinases (PIP4Ks). Here, we show that the loss of PIP4Ks (PIP4K2A, PIP4K2B, and PIP4K2C) in vitro results in a paradoxical increase in PI(4,5)P 2 and a concomitant increase in insulin-stimulated production of PI(3,4,5)P 3 . The reintroduction of either wild-type or kinase-dead mutants of the PIP4Ks restored cellular PI(4,5)P 2 levels and insulin stimulation of the PI3K pathway, suggesting a catalytic-independent role of PIP4Ks in regulating PI(4,5)P 2 levels. These effects are explained by an increase in PIP5K activity upon the deletion of PIP4Ks, which normally suppresses PIP5K activity through a direct binding interaction mediated by the N-terminal motif VMLΦPDD of PIP4K. Our work uncovers an allosteric function of PIP4Ks in suppressing PIP5K-mediated PI(4,5)P 2 synthesis and insulin-dependent conversion to PI(3,4,5)P 3 and suggests that the pharmacological depletion of PIP4K enzymes could represent a strategy for enhancing insulin signaling., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate., (Copyright © 2018. Published by Elsevier Inc.)