Bruce A. Posner

The structure of the G protein heterotrimer Giα1β1γ2

The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

Autosis is a Na+,K+-ATPase–regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia–ischemia

Significance We show that the selective overactivation of autophagy can cause cell death with unique morphological features distinct from apoptosis or necrosis. This unique type of autophagic cell death, termed “autosis,” occurs not only in vitro but also in vivo in cerebral hypoxia–ischemia. Moreover, autosis is inhibited both in vitro and in vivo by cardiac glycosides, which are Na+,K+-ATPase antagonists used in clinical medicine. Our findings contribute to the basic understanding of cell-death mechanisms and suggest strategies for protecting cells against stresses such as hypoxia–ischemia. A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed “autosis,” has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia–ischemia in vivo. A chemical screen of ∼5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na+,K+-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na+,K+-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na+,K+-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

Regulators of G Protein Signaling 6 and 7 PURIFICATION OF COMPLEXES WITH Gβ5 AND ASSESSMENT OF THEIR EFFECTS ON G PROTEIN-MEDIATED SIGNALING PATHWAYS

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10,pleckstrin) and GGL (G protein γsubunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein β subunits, and certain biochemical properties of RGS6/β5 and RGS7/β5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gβ5 subunit (but not Gβs 1–4) are found in the cytosol. When purified, these complexes are similar to RGS11/β5 in that they act as GTPase-activating proteins specifically toward Gαo. Unlike conventional Gβγ complexes, RGS6/β5 and RGS7/β5 do not form heterotrimeric complexes with either Gαo-GDP or Gαq-GDP. Neither RGS6/β5 nor RGS7/β5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-β1 or -β2. However, the RGS/β5 complexes inhibited β1γ2-mediated activation of phospholipase C-β2. RGS/β5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to Gi and Go by binding to phospholipase C and stimulating the GTPase activity of Gαo.

Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.

Suberoylanilide Hydroxamic Acid (Vorinostat) Up-regulates Progranulin Transcription RATIONAL THERAPEUTIC APPROACH TO FRONTOTEMPORAL DEMENTIA

Progranulin (GRN) haploinsufficiency is a frequent cause of familial frontotemporal dementia, a currently untreatable progressive neurodegenerative disease. By chemical library screening, we identified suberoylanilide hydroxamic acid (SAHA), a Food and Drug Administration-approved histone deacetylase inhibitor, as an enhancer of GRN expression. SAHA dose-dependently increased GRN mRNA and protein levels in cultured cells and restored near-normal GRN expression in haploinsufficient cells from human subjects. Although elevation of secreted progranulin levels through a post-transcriptional mechanism has recently been reported, this is, to the best of our knowledge, the first report of a small molecule enhancer of progranulin transcription. SAHA has demonstrated therapeutic potential in other neurodegenerative diseases and thus holds promise as a first generation drug for the prevention and treatment of frontotemporal dementia.

Structural basis of activity and subunit recognition in G protein heterotrimers

BACKGROUND Inactive heterotrimeric G proteins are composed of a GDP-bound alpha subunit (Galpha) and a stable heterodimer of Gbeta and Ggamma subunits. Upon stimulation by a receptor, Galpha subunits exchange GDP for GTP and dissociate from Gbetagamma, both Galpha and Gbetagamma then interact with downstream effectors. Isoforms of Galpha, Gbeta and Ggamma potentially give rise to many heterotrimeric combinations, limited in part by amino acid sequence differences that lead to selective interactions. The mechanism by which GTP promotes Gbetagamma dissociation is incompletely understood. The Gly203-->Ala mutant of Gialpha1 binds and hydrolyzes GTP normally but does not dissociate from Gbetagamma, demonstrating that GTP binding and activation can be uncoupled. Structural data are therefore important for understanding activation and subunit recognition in G protein heterotrimers. RESULTS The structures of the native (Gialpha1beta1gamma2) heterotrimer and that formed with Gly203-->AlaGialpha1 have been determined to resolutions of 2.3 A and 2.4 A, respectively, and reveal previously unobserved segments at the Ggamma2 C terminus. The Gly203-->Ala mutation alters the conformation of the N terminus of the switch II region (Val201-Ala203), but not the global structure of the heterotrimer. The N termini of Gbeta and Ggamma form a rigid coiled coil that packs at varying angles against the beta propeller of Gbeta. Conformational differences in the CD loop of beta blade 2 of Gbeta mediate isoform-specific contacts with Galpha. CONCLUSIONS The Gly203-->Ala mutation in Gialpha1 blocks the conformational changes in switch II that are required to release Gbetagamma upon binding GTP. The interface between the ras-like domain of Galpha and the beta propeller of Gbeta appears to be conserved in all G protein heterotrimers. Sequence variation at the Gbeta-Galpha interface between the N-terminal helix of Galpha and the CD loop of beta blade 2 of Gbeta1 (residues 127-135) could mediate isoform-specific contacts. The specificity of Gbeta and Ggamma interactions is largely determined by sequence variation in the contact region between helix 2 of Ggamma and the surface of Gbeta.

Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration

A shot in the arm for damaged tissue Tissue damage can be caused by injury, disease, and even certain medical treatments. There is great interest in identifying drugs that accelerate tissue regeneration and recovery, especially drugs that might benefit multiple organ systems. Zhang et al. describe a compound with this desired activity, at least in mice (see the Perspective by FitzGerald). SW033291 promotes recovery of the hematopoietic system after bone marrow transplantation, prevents the development of ulcerative colitis in the intestine, and accelerates liver regeneration after hepatic surgery. It acts by inhibiting an enzyme that degrades prostaglandins, lipid signaling molecules that have been implicated in tissue stem cell maintenance. Science, this issue 10.1126/science.aaa2340; see also p. 1208 A compound that inhibits prostaglandin degradation enhances tissue regeneration in multiple organs in mice. [Also see Perspective by FitzGerald] INTRODUCTION Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. To date, therapeutic interventions have largely focused on targeting two PGE2 biosynthetic enzymes, cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), with the aim of reducing PGE2 production. In this study, we take the converse approach: We examine the role of a prostaglandin-degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as a negative regulator of tissue repair, and we explore whether inhibition of this enzyme can potentiate tissue regeneration in mouse models. RATIONALE We used 15-PGDH knockout mice to elucidate the role of 15-PGDH in regulating tissue levels of PGE2 and tissue repair capacity in multiple organs. We then developed SW033291, a potent small-molecule inhibitor of 15-PGDH with activity in vivo. We used SW033291 to investigate the therapeutic potential of 15-PGDH inhibitors in tissue regeneration and to identify a 15-PGDH–regulated hematopoietic pathway within the bone marrow niche. RESULTS We found that in comparison with wild-type mice, 15-PGDH–deficient mice display a twofold increase in PGE2 levels across multiple tissues—including bone marrow, colon, and liver—and that they show increased fitness of these tissues in response to damage. The mutant mice also show enhanced hematopoietic capacity, with increased neutrophils, increased bone marrow SKL (Sca-1+ C-kit+ Lin−) cells (enriched for stem cells), and greater capacity to generate erythroid and myeloid colonies in cell culture. The 15-PGDH–deficient mice respond to colon injury from dextran sulfate sodium (DSS) with a twofold increase in cell proliferation in colon crypts, which confers resistance to DSS-induced colitis. The mutant mice also respond to partial hepatectomy with a greater than twofold increase in hepatocyte proliferation, which leads to accelerated and more extensive liver regeneration. SW033291, a potent small-molecule inhibitor of 15-PGDH (inhibitor dissociation constant Ki ~0.1 nM), recapitulates in mice the phenotypes of 15-PGDH gene knockout, inducing increased hematopoiesis, resistance to DSS colitis, and more rapid liver regeneration after partial hepatectomy. Moreover, SW033291-treated mice show a 6-day-faster reconstitution of hematopoiesis after bone marrow transplantation, with accelerated recovery of neutrophils, platelets, and erythrocytes, and greater recovery of bone marrow SKL cells. This effect is mediated by bone marrow CD45– cells, which respond to increased PGE2 with a fourfold increase in production of CXCL12 and SCF, two cytokines that play key roles in hematopoietic stem cell homing and maintenance. CONCLUSIONS Studying mouse models, we have shown that 15-PGDH negatively regulates tissue regeneration and repair in the bone marrow, colon, and liver. Of most direct utility, our observations identify 15-PGDH as a therapeutic target and provide a chemical formulation, SW033291, that is an active 15-PGDH inhibitor in vivo and that potentiates repair in multiple tissues. SW033291 or related compounds may merit clinical investigation as a strategy to accelerate recovery after bone marrow transplantation and other tissue injuries. Inhibiting 15-PGDH accelerates tissue repair. (A) The enzyme 15-PGDH degrades and negatively regulates PGE2. (B) SW033291 inhibits 15-PGDH, increases tissue levels of PGE2, and induces CXCL12 and SCF expression from CD45– bone marrow cells. This in turn accelerates homing of transplanted hematopoietic stem cells (HSC), generation of mature blood elements, and post-transplant recovery of normal blood counts. Inhibiting 15-PGDH similarly stimulates cell proliferation after injury to colon or liver, accelerating repair of these tissues. Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state.

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approximation of the receptor-α subunit complex, we have considered the properties of A326S Giα1, a mutation discovered originally in Gsα (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164–168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the αi1β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Giα1 for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Giα1, βγ prevents irreversible inactivation of the α subunit at 30 °C. However, the mutation accelerates irreversible inactivation of α at 37 °C despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Giα1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Giα1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Gα subunit in the GDP-bound state.

Towards patient-based cancer therapeutics

A new approach to the discovery of cancer therapeutics is emerging that begins with the cancer patient. Genomic analysis of primary tumors is providing an unprecedented molecular characterization of the disease. The next step requires relating the genetic features of cancers to acquired gene and pathway dependencies and identifying small-molecule therapeutics that target them.

XPO1-dependent nuclear export is a druggable vulnerability in KRAS-mutant lung cancer

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.