Suppression of the gene encoding PDZ domain-containing protein decreases cold tolerance and overwintering survival of the mosquito, Culex pipiens (Culicidae: Diptera).

In diapausing mosquitoes, cold tolerance and prolonged lifespan are important features that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that PDZ domain-containing protein (PDZ) (post synaptic density protein [PSD95], drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 protein [zo-1]) domain-containing protein is involved with these diapause features for overwintering survival in Culex mosquitoes. The expression level of pdz was significantly higher in diapausing adult females in the early stage in comparison to their nondiapausing counterparts. Suppression of the gene that encodes PDZ by RNA interference significantly decreased actin accumulation in the midgut of early-stage adult diapausing females. Inhibition of pdz also significantly reduced the survivability of diapausing females which indicates that this protein could play a key role in preserving the midgut tissues during early diapause.

Structural characterization of dicyanopyridine containing DNMT1-selective, non-nucleoside inhibitors.

DNMT1 maintains the parental DNA methylation pattern on newly replicated hemimethylated DNA. The failure of this maintenance process causes aberrant DNA methylation that affects transcription and contributes to the development and progression of cancers such as acute myeloid leukemia. Here, we structurally characterized a set of newly discovered DNMT1-selective, reversible, non-nucleoside inhibitors that bear a core 3,5-dicyanopyridine moiety, as exemplified by GSK3735967, to better understand their mechanism of inhibition. All of the dicyanopydridine-containing inhibitors examined intercalate into the hemimethylated DNA between two CpG base pairs through the DNA minor groove, resulting in conformational movement of the DNMT1 active-site loop. In addition, GSK3735967 introduces two new binding sites, where it interacts with and stabilizes the displaced DNMT1 active-site loop and it occupies an open aromatic cage in which trimethylated histone H4 lysine 20 is expected to bind. Our work represents a substantial step in generating potent, selective, and non-nucleoside inhibitors of DNMT1.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo.

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss-of-function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

Translation in amino-acid-poor environments is limited by tRNAGln charging.

An inadequate supply of amino acids leads to accumulation of uncharged tRNAs, which can bind and activate GCN2 kinase to reduce translation. Here, we show that glutamine-specific tRNAs selectively become uncharged when extracellular amino acid availability is compromised. In contrast, all other tRNAs retain charging of their cognate amino acids in a manner that is dependent upon intact lysosomal function. In addition to GCN2 activation and reduced total translation, the reduced charging of tRNAGln in amino-acid-deprived cells also leads to specific depletion of proteins containing polyglutamine tracts including core-binding factor α1, mediator subunit 12, transcriptional coactivator CBP and TATA-box binding protein. Treating amino-acid-deprived cells with exogenous glutamine or glutaminase inhibitors restores tRNAGln charging and the levels of polyglutamine-containing proteins. Together, these results demonstrate that the activation of GCN2 and the translation of polyglutamine-encoding transcripts serve as key sensors of glutamine availability in mammalian cells.

Yap/Taz promote the scavenging of extracellular nutrients through macropinocytosis.

The uptake of macromolecules and cellular debris through macropinocytosis has emerged as an important nutrient acquisition strategy of cancer cells. Genetic alterations commonly found in human cancers (e.g. mutations in KRAS or loss of PTEN) have been shown to increase macropinocytosis. To identify additional effectors that enable cell growth dependent on the uptake of extracellular proteins, pancreatic ductal adenocarcinoma (PDA) cells were selected for growth in medium where extracellular albumin was the obligate source of the essential amino acid leucine. Analysis of global changes in chromatin availability and gene expression revealed that PDA cells selected under these conditions exhibited elevated activity of the transcriptional activators Yap/Taz. Knockout of Yap/Taz prevented growth of PDA cells in leucine-deficient medium, but not in complete medium. Furthermore, constitutively active forms of Yap or Taz were sufficient to stimulate macropinocytosis of extracellular protein. In addition to promoting the uptake of plasma proteins, Yap/Taz also promoted the scavenging of apoptotic cell bodies and necrotic debris by PDA cells. The Yap/Taz transcriptional target Axl was found to be essential for cell growth dependent on the uptake of dead cells and cell debris. Together, these studies suggest that the Hippo pathway effectors Yap and Taz are important transcriptional regulators of endocytic nutrient uptake.

Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen.

The majority of tumor-infiltrating T cells exhibit a terminally exhausted phenotype, marked by a loss of self-renewal capacity. How repetitive antigenic stimulation impairs T cell self-renewal remains poorly defined. Here, we show that persistent antigenic stimulation impaired ADP-coupled oxidative phosphorylation. The resultant bioenergetic compromise blocked proliferation by limiting nucleotide triphosphate synthesis. Inhibition of mitochondrial oxidative phosphorylation in activated T cells was sufficient to suppress proliferation and upregulate genes linked to T cell exhaustion. Conversely, prevention of mitochondrial oxidative stress during chronic T cell stimulation allowed sustained T cell proliferation and induced genes associated with stem-like progenitor T cells. As a result, antioxidant treatment enhanced the anti-tumor efficacy of chronically stimulated T cells. These data reveal that loss of ATP production through oxidative phosphorylation limits T cell proliferation and effector function during chronic antigenic stimulation. Furthermore, treatments that maintain redox balance promote T cell self-renewal and enhance anti-tumor immunity.

High-Resolution In Vivo Identification of miRNA Targets by Halo-Enhanced Ago2 Pull-Down.

The identification of microRNA (miRNA) targets by Ago2 crosslinking-immunoprecipitation (CLIP) methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily applicable to an in vivo setting. To overcome these limitations and facilitate the investigation of miRNA functions in vivo, we have developed a method based on a genetically engineered mouse harboring a conditional Halo-Ago2 allele expressed from the endogenous Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be purified from cells and tissues expressing the endogenous Halo-Ago2 allele. We demonstrate the reproducibility and sensitivity of this method in mouse embryonic stem cells, developing embryos, adult tissues, and autochthonous mouse models of human brain and lung cancers. This method and the datasets we have generated will facilitate the characterization of miRNA-mRNA networks in vivo under physiological and pathological conditions.

Promoter demethylation of the asparagine synthetase gene is required for ATF4-dependent adaptation to asparagine depletion.

Tumor cells adapt to nutrient-limited environments by inducing gene expression that ensures adequate nutrients to sustain metabolic demands. For example, during amino acid limitations, ATF4 in the amino acid response induces expression of asparagine synthetase (ASNS), which provides for asparagine biosynthesis. Acute lymphoblastic leukemia (ALL) cells are sensitive to asparagine depletion, and administration of the asparagine depletion enzyme l-asparaginase is an important therapy option. ASNS expression can counterbalance l-asparaginase treatment by mitigating nutrient stress. Therefore, understanding the mechanisms regulating ASNS expression is important to define the adaptive processes underlying tumor progression and treatment. Here we show that DNA hypermethylation at the ASNS promoter prevents its transcriptional expression following asparagine depletion. Insufficient expression of ASNS leads to asparagine deficiency, which facilitates ATF4-independent induction of CCAAT-enhancer-binding protein homologous protein (CHOP), which triggers apoptosis. We conclude that chromatin accessibility is critical for ATF4 activity at the ASNS promoter, which can switch ALL cells from an ATF4-dependent adaptive response to ATF4-independent apoptosis during asparagine depletion. This work may also help explain why ALL cells are most sensitive to l-asparaginase treatment compared with other cancers.

Impaired Expression of Rearranged Immunoglobulin Genes and Premature p53 Activation Block B Cell Development in BMI1 Null Mice.

B cell development is a highly regulated process that requires stepwise rearrangement of immunoglobulin genes to generate a functional B cell receptor (BCR). The polycomb group protein BMI1 is required for B cell development, but its function in developing B cells remains poorly defined. We demonstrate that BMI1 functions in a cell-autonomous manner at two stages during early B cell development. First, loss of BMI1 results in a differentiation block at the pro-B cell to pre-B cell transition due to the inability of BMI1-deficient cells to transcribe newly rearranged Igh genes. Accordingly, introduction of a pre-rearranged Igh allele partially restored B cell development in Bmi1-/- mice. In addition, BMI1 is required to prevent premature p53 signaling, and as a consequence, Bmi1-/- large pre-B cells fail to properly proliferate. Altogether, our results clarify the role of BMI1 in early B cell development and uncover an unexpected function of BMI1 during VDJ recombination.

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

Critical role for PI3-kinase in regulating the use of proteins as an amino acid source.

Ras-transformed cells can grow in amino acid-poor environments by recovering amino acids through macropinocytosis and lysosomal catabolism of extracellular proteins. However, when studying nontransformed fibroblasts, we found that Ras GTPases are dispensable for growth-factor-stimulated macropinocytosis and lysosomal catabolism of extracellular proteins. Instead, we establish a critical role for phosphatidylinositol 3-kinase (PI3-kinase) signaling in cell proliferation that is supported by protein macropinocytosis. Downstream of PI3-kinase, distinct effectors have opposing roles in regulating uptake and catabolism of extracellular proteins. Rac1 and PLC are required for nutritional use of extracellular proteins. In contrast, Akt suppresses lysosomal catabolism of ingested proteins when free amino acids are abundant. The interplay between these pathways allows cells with oncogenic PIK3CA mutations or PTEN deletion to grow using diverse amino acid sources. Thus, the prevalence of PI3-kinase and PTEN mutations in cancer may result in part because they allow cells to cope with fluctuating nutrient availability.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. We identified the ubiquitin ligase Huwe1 as a crucial regulator of HSC function via its post-translational control of the oncoprotein N-myc (encoded by Mycn). We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid-fate specification in mice. Through the use of a fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc expression was upregulated in response to stress or following loss of Huwe1, which led to increased proliferation and stem-cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, which suggested that the attenuation of N-myc by Huwe1 is essential for reestablishing homeostasis following stress.

Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10(-8)) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.

DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2.

Mammalian cells possess two amino acid-sensing kinases: general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). Their combined effects orchestrate cellular adaptation to amino acid levels, but how their activities are coordinated remains poorly understood. Here, we demonstrate an important link between GCN2 and mTORC1 signaling. Upon deprivation of various amino acids, activated GCN2 up-regulates ATF4 to induce expression of the stress response protein Sestrin2, which is required to sustain repression of mTORC1 by blocking its lysosomal localization. Moreover, Sestrin2 induction is necessary for cell survival during glutamine deprivation, indicating that Sestrin2 is a critical effector of GCN2 signaling that regulates amino acid homeostasis through mTORC1 suppression.

Crystal structures of human RIP2 kinase catalytic domain complexed with ATP-competitive inhibitors: Foundations for understanding inhibitor selectivity.

Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (ß,γ-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase.

CXCL12-Producing Vascular Endothelial Niches Control Acute T Cell Leukemia Maintenance.

The role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of Cxcr4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.