Benjamin Haley

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd−/− mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd−/− mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

An Integrated Genomic Screen Identifies LDHB as an Essential Gene for Triple-Negative Breast Cancer

Breast cancer has been redefined into three clinically relevant subclasses: (i) estrogen/progesterone receptor positive (ER+/PR+), (ii) HER2/ERRB2 positive, and (iii) those lacking expression of all three markers (triple negative or basal-like). While targeted therapies for ER+/PR+ and HER2+ tumors have revolutionized patient treatment and increased lifespan, an urgent need exists for identifying novel targets for triple-negative breast cancers. Here, we used integrative genomic analysis to identify candidate oncogenes in triple-negative breast tumors and assess their function through loss of function screening. Using this approach, we identify lactate dehydrogenase B (LDHB), a component of glycolytic metabolism, as an essential gene in triple-negative breast cancer. Loss of LDHB abrogated cell proliferation in vitro and arrested tumor growth in fully formed tumors in vivo. We find that LDHB and other related glycolysis genes are specifically upregulated in basal-like/triple-negative breast cancers as compared with other subtypes, suggesting that these tumors are distinctly glycolytic. Consistent with this, triple-negative breast cancer cell lines were more dependent on glycolysis for growth than luminal cell lines. Finally, we find that patients with breast cancer and high LDHB expression in their tumors had a poor clinical outcome. While previous studies have focused on the ubiquitous role of LDHA in tumor metabolism and growth, our data reveal that LDHB is upregulated and required only in certain cancer genotypes. These findings suggest that targeting LDHB or other components of lactate metabolism would be of clinical benefit in triple-negative breast cancer.

CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer

Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

mTORC1-Dependent Metabolic Reprogramming Underlies Escape from Glycolysis Addiction in Cancer Cells.

Although glycolysis is substantially elevated in many tumors, therapeutic targeting of glycolysis in cancer patients has not yet been successful, potentially reflecting the metabolic plasticity of tumor cells. In various cancer cells exposed to a continuous glycolytic block, we identified a recurrent reprogramming mechanism involving sustained mTORC1 signaling that underlies escape from glycolytic addiction. Active mTORC1 directs increased glucose flux via the pentose phosphate pathway back into glycolysis, thereby circumventing a glycolysis block and ensuring adequate ATP and biomass production. Combined inhibition of glycolysis and mTORC1 signaling disrupted metabolic reprogramming in tumor cells and inhibited their growth in vitro and in vivo. These findings reveal novel combinatorial therapeutic strategies to realize the potential benefit from targeting the Warburg effect.

An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth

The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.

Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice

Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/βKlotho Complex

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/βKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/βKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/βKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/βKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/βKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

Identification and Analysis of In Vivo VEGF Downstream Markers Link VEGF Pathway Activity with Efficacy of Anti- VEGF Therapies

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients. Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy. Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies. Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials. Clin Cancer Res; 19(13); 3681–92. ©2013 AACR.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

Background: PILRα is an inhibitory receptor predominantly expressed in myeloid cells. Results: NPDC1 and COLEC12 are novel PILRα ligands. PILRα arginine residues 133 (mouse) and 126 (human) are critical contact residues. Conclusion: PILRα/ligand interactions involve a conserved domain in PILRα and a sialylated protein domain in the ligand. Significance: PILRα interacts with various ligands to alter myeloid cell function. Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Phosphoproteomic Analysis Implicates the mTORC2-FoxO1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases

Pathological angiogenesis mediated by the VEGF receptor could be treated by combining mTOR and PI3K inhibitors. Combining Forces Against Angiogenesis Pathological angiogenesis contributes to the growth of solid tumors, which has led to the development of several antiangiogenic therapies that target the VEGF (vascular endothelial growth factor) signaling pathway, which promotes angiogenesis. Zhuang et al. mapped the signaling networks activated in endothelial cells by VEGF through its receptor tyrosine kinases and found that VEGF promoted the survival of endothelial cells by stimulating the kinase activity of PI3K (phosphatidylinositol 3-kinase) and of mTORC2 (mammalian target of rapamycin complex 2). Because VEGF-activated endothelial cells exposed to mTOR kinase inhibitors exhibited increased activity of multiple receptor tyrosine kinases and PI3K, the cells were less susceptible to the toxic effects of mTOR inhibition. However, combining mTOR and PI3K inhibitors effectively reduced endothelial cell survival. Thus, understanding the signaling pathways that are activated by VEGF can identify potential therapeutic strategies to treat pathological angiogenesis. The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and also represents a major therapeutic target for tumors and intraocular neovascular disorders. The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define the VEGF-regulated phosphoproteome and its temporal dynamics in human umbilical vein endothelial cells and then used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses. The PI3K (phosphatidylinositol 3-kinase)–mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central in activating VEGF-regulated phosphorylation and increasing endothelial cell viability by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1), an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases. This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival but also rendered cells more susceptible to PI3K inhibition. Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and reprogramming of receptor tyrosine kinases in human endothelial cells.