Transplanted organoids empower human preclinical assessment of drug candidate for the clinic.

Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.

Global characterization of macrophage polarization mechanisms and identification of M2-type polarization inhibitors.

Macrophages undergoing M1- versus M2-type polarization differ significantly in their cell metabolism and cellular functions. Here, global quantitative time-course proteomics and phosphoproteomics paired with transcriptomics provide a comprehensive characterization of temporal changes in cell metabolism, cellular functions, and signaling pathways that occur during the induction phase of M1- versus M2-type polarization. Significant differences in, especially, metabolic pathways are observed, including changes in glucose metabolism, glycosaminoglycan metabolism, and retinoic acid signaling. Kinase-enrichment analysis shows activation patterns of specific kinases that are distinct in M1- versus M2-type polarization. M2-type polarization inhibitor drug screens identify drugs that selectively block M2- but not M1-type polarization, including mitogen-activated protein kinase kinase (MEK) and histone deacetylase (HDAC) inhibitors. These datasets provide a comprehensive resource to identify specific signaling and metabolic pathways that are critical for macrophage polarization. In a proof-of-principle approach, we use these datasets to show that MEK signaling is required for M2-type polarization by promoting peroxisome proliferator-activated receptor-γ (PPARγ)-induced retinoic acid signaling.

YAP-dependent proliferation by a small molecule targeting annexin A2.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.

Lats1/2 Sustain Intestinal Stem Cells and Wnt Activation through TEAD-Dependent and Independent Transcription.

Intestinal homeostasis is tightly regulated by complex yet poorly understood signaling networks. Here, we demonstrate that Lats1/2, the core Hippo kinases, are essential to maintain Wnt pathway activity and intestinal stem cells. Lats1/2 deletion leads to loss of intestinal stem cells but drives Wnt-uncoupled crypt expansion. To explore the function of downstream transcriptional enhanced associate domain (TEAD) transcription factors, we identified a selective small-molecule reversible inhibitor of TEAD auto-palmitoylation that directly occupies its lipid-binding site and inhibits TEAD-mediated transcription in vivo. Combining this chemical tool with genetic and proteomics approaches, we show that intestinal Wnt inhibition by Lats deletion is Yes-associated protein (YAP)/transcriptional activator with PDZ-binding domain (TAZ) dependent but TEAD independent. Mechanistically, nuclear YAP/TAZ interact with Groucho/Transducin-Like Enhancer of Split (TLE) to block Wnt/T-cell factor (TCF)-mediated transcription, and dual inhibition of TEAD and Lats suppresses Wnt-uncoupled Myc upregulation and epithelial over-proliferation in Adenomatous polyposis coli (APC)-mutated intestine. Our studies highlight a pharmacological approach to inhibit TEAD palmitoylation and have important implications for targeting Wnt and Hippo signaling in human malignancies.

When Small Molecules Are Like Real Estate: It's All about Location, Location, Location.

In this issue of Cell Chemical Biology, Park et al. (2018) demonstrate that targeting apoptazole, an Hsp70 inhibitor, to mitochondria induces apoptosis by a distinct mechanism of action different from unmodified apoptazole, which accumulates in the lysosome. These results highlight the power of subcellular localization in small-molecule selectivity.

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.

Chemical Probe to Identify the Cellular Targets of the Reactive Lipid Metabolite 2- trans-Hexadecenal.

Lipid-derived electrophiles (LDEs) are reactive metabolites, which can covalently modify proteins and DNA and regulate diverse cellular processes. 2- trans-Hexadecenal (2-HD) is a byproduct of sphingolipid metabolism, involved in cytoskeletal reorganization, DNA damage, and apoptosis. In addition, the loss of ALDH3A2, an enzyme removing 2-HD in cells, is responsible for Sjörgen-Larsson Syndrome (SJS), suggesting that accumulation of 2-HD could lead to pathogenesis. However, the targets and the precise mechanisms of 2-HD are not well characterized. Herein, we report an alkyne-2-HD derivative as a bioorthogonal probe to explore the functions of 2-HD. We identified more than 500 potential cellular targets. Among them, the pro-apoptotic protein Bax can be covalently modified by 2-HD directly at the conserved Cys62 residue. Our work provided new chemical tools to explore the cellular functions of LDEs and revealed new mechanistic insights of the deregulation of lipid metabolism in diseases.

Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms.

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. VIDEO ABSTRACT.

ZDHHC7-mediated S-palmitoylation of Scribble regulates cell polarity.

Scribble (SCRIB) is a tumor-suppressor protein, playing critical roles in establishing and maintaining epithelial cell polarity. SCRIB is frequently amplified in human cancers but does not localize properly to cell-cell junctions, suggesting that mislocalization of SCRIB disrupts its tumor-suppressive activities. Using chemical reporters, here we showed that SCRIB localization was regulated by S-palmitoylation at conserved cysteine residues. Palmitoylation-deficient mutants of SCRIB were mislocalized, leading to disruption of cell polarity and loss of their tumor-suppressive activities to oncogenic YAP, MAPK and PI3K/AKT pathways. We further found that ZDHHC7 was the major palmitoyl acyltransferase regulating SCRIB. Knockout of ZDHHC7 led to SCRIB mislocalization and YAP activation, and disruption of SCRIB's suppressive activities in HRas(V12)-induced cell invasion. In summary, we demonstrated that ZDHHC7-mediated SCRIB palmitoylation is critical for SCRIB membrane targeting, cell polarity and tumor suppression, providing new mechanistic insights of how dynamic protein palmitoylation regulates cell polarity and tumorigenesis.

Autopalmitoylation of TEAD proteins regulates transcriptional output of the Hippo pathway.

TEA domain (TEAD) transcription factors bind to the coactivators YAP and TAZ and regulate the transcriptional output of the Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches a fatty acid, palmitate, to cysteine residues and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation did not alter TEAD's localization, but it was required for TEAD's binding to YAP and TAZ and was dispensable for its binding to the Vgll4 tumor suppressor. Moreover, palmitoylation-deficient TEAD mutants impaired TAZ-mediated muscle differentiation in vitro and tissue overgrowth mediated by the Drosophila YAP homolog Yorkie in vivo. Our study directly links autopalmitoylation to the transcriptional regulation of the Hippo pathway.

Energy stress regulates hippo-YAP signaling involving AMPK-mediated regulation of angiomotin-like 1 protein.

Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK) and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1). Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition), mechanical, and metabolic signals to control cellular proliferation and survival.

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/ß-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma.

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/ß-catenin pathway, recombinant WNT3A and stabilized ß-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/ß-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/ß-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/ß-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.

2-Bromopalmitate analogues as activity-based probes to explore palmitoyl acyltransferases.

Reversible S-palmitoylation is an important post-translational modification that regulates the trafficking, localization, and activity of proteins. Cysteine-rich Asp-His-His-Cys (DHHC) domain-containing enzymes are evolutionarily conserved protein palmitoyl acyltransferases (PATs). The human genome encodes 23 DHHC-PATs that regulate diverse cellular functions. Although chemical probes and proteomic methods to detect palmitoylated protein substrates have been reported, no probes for direct detection of the activity of PATs are available. Here we report the synthesis and characterization of 2-bromohexadec-15-ynoic acid and 2-bromooctadec-17-ynoic acid, which are analogues of 2-bromopalmitate (2-BP), as activity-based probes for PATs as well as other palmitoylating and 2-BP-binding enzymes. These probes will serve as new chemical tools for activity-based protein profiling to explore PATs, to dissect the functions of PATs in cell signaling and diseases, and to facilitate the identification of their inhibitors.

Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP.

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser127 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S1P induces YAP nuclear localization through S1P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S1P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S1P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.

Inhibition of proliferation and survival of diffuse large B-cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, remains a partially curable disease. Genetic alterations affecting components of NF-κB signaling pathways occur frequently in DLBCL. Almost all activated B cell-like (ABC) DLBCL, which is the least curable group among the 3 major subtypes of this malignancy, and a substantial fraction of germinal center B cell-like (GCB) DLBCL exhibit constitutive NF-κB pathway activity. It has been demonstrated that ABC-DLBCL cells require such activity for proliferation and survival. Therefore, inhibition of NF-κB activation in DLBCL may provide an efficient and targeted therapy. In screening for small-molecule compounds that may inhibit NF-κB activation in DLBCL cells, we identified a compound, NSC697923, which inhibits the activity of the ubiquitin-conjugating (E2) enzyme Ubc13-Uev1A. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. Importantly, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, suggesting the Ubc13-Uev1A may be crucial for DLBCL growth. Consistently, knockdown of Ubc13 expression also inhibited DLBCL cell survival. The results of the present study indicate that Ubc13-Uev1A may represent a potential therapeutic target in DLBCL. In addition, compound NSC697923 may be exploited for the development of DLBCL therapeutic agents.

Assessing G1-to-S-phase progression after genotoxic stress.

Maintenance of genomic integrity is critical for the survival of organisms. Thus, mammalian cells employ a complex DNA damage response that can sense and repair DNA damage. One important aspect of the cellular DNA damage response is the activation of checkpoints that result in cell cycle arrest. In this chapter we present methods for the induction of genotoxic stress. Additionally, we describe methods for studying the progression of cells from G(1) to S phase after genotoxic stress.

Transcriptional activation of histone genes requires NPAT-dependent recruitment of TRRAP-Tip60 complex to histone promoters during the G1/S phase transition.

Transcriptional activation of histone subtypes is coordinately regulated and tightly coupled with the onset of DNA replication during S-phase entry. The underlying molecular mechanisms for such coordination and coupling are not well understood. The cyclin E-Cdk2 substrate NPAT has been shown to play an essential role in the transcriptional activation of histone genes at the G(1)/S-phase transition. Here, we show that NPAT interacts with components of the Tip60 histone acetyltransferase complex through a novel amino acid motif, which is functionally conserved in E2F and adenovirus E1A proteins. In addition, we demonstrate that transformation/transactivation domain-associated protein (TRRAP) and Tip60, two components of the Tip60 complex, associate with histone gene promoters at the G(1)/S-phase boundary in an NPAT-dependent manner. In correlation with the association of the TRRAP-Tip60 complex, histone H4 acetylation at histone gene promoters increases at the G(1)/S-phase transition, and this increase involves NPAT function. Suppression of TRRAP or Tip60 expression by RNA interference inhibits histone gene activation. Thus, our data support a model in which NPAT recruits the TRRAP-Tip60 complex to histone gene promoters to coordinate the transcriptional activation of multiple histone genes during the G(1)/S-phase transition.