Shuguang Wei

Small molecule–mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer

SUMMARY The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Targeting QseC Signaling and Virulence for Antibiotic Development

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

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.

Small-molecule activation of the TRAIL receptor DR5 in human cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates apoptosis through the death receptors DR4 and DR5. Because of its superior safety profile and high tumor specificity compared to other TNF family members, recombinant soluble TRAIL and agonistic antibodies against its receptors are actively being developed for clinical cancer therapy. Here, we describe the identification and characterization of the small molecules that directly target DR5 to initiate apoptosis in human cancer cells. The activity was initially discovered through a high-throughput chemical screen for compounds that promote cell death in synergy with a small-molecule mimetic of Smac, the antagonist for inhibitor of apoptosis protein. Structure-activity relationship studies yielded a more potent analog called bioymifi, which can act as a single agent to induce DR5 clustering and aggregation, leading to apoptosis. Thus, this study identified potential lead compounds for the development of small-molecule TRAIL mimics targeting DR5 for cancer therapy.

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.

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.

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.

Identification of DNMT1 Selective Antagonists Using a Novel Scintillation Proximity Assay

Background: DNA methylation contributes to the heritable regulation of gene expression. Results: Chemical inhibitors of DNA methyltransferase (DNMT) enzymes were identified. Conclusion: Several inhibitors nonspecifically inhibited multiple methyltransferases through generation of H2O2, but one compound showed selective inhibition of DNMT1 independent of the production of H2O2. Significance: Selective DNMT1 inhibitors will allow for more precise elucidation of the role of this enzyme in gene regulation. A novel scintillation proximity high throughput assay (SPA) to identify inhibitors of DNA methyltransferases was developed and used to screen over 180,000 compounds. The majority of the validated hits shared a quinone core and several were found to generate the reactive oxygen species, H2O2. Inhibition of the production of H2O2 by the addition of catalase blocked the ability of this group of compounds to inhibit DNA methyltransferase (DNMT) activity. However, a related compound, SW155246, was identified that existed in an already reduced form of the quinone. This compound did not generate H2O2, and catalase did not block its ability to inhibit DNA methyltransferase. SW155246 showed a 30-fold preference for inhibition of human DNMT1 versus human or murine DNMT3A or -3B, inhibited global methylation in HeLa cells, and reactivated expression of the tumor suppressor gene RASSF1A in A549 cells. To our knowledge, this work represents the first description of selective chemical inhibitors of the DNMT1 enzyme.

Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation.

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.