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1.  The genomic landscape and evolution of endometrial carcinoma progression and abdominopelvic metastasis 
Nature genetics  2016;48(8):848-855.
Recent studies have detailed the genomic landscape of primary endometrial cancers, but their evolution into metastases has not been characterized. We performed whole-exome sequencing of 98 tumor biopsies including complex atypical hyperplasias, primary tumors, and paired abdominopelvic metastases to survey the evolutionary landscape of endometrial cancer. We expanded and reanalyzed TCGA-data, identifying novel recurrent alterations in primary tumors, including mutations in the estrogen receptor cofactor NRIP1 in 12% of patients. We found that likely driver events tended to be shared by primary and metastatic tissue-samples, with notable exceptions such as ARID1A mutations. Phylogenetic analyses indicated that the sampled metastases typically arose from a common ancestral subclone that was not detected in the primary tumor biopsy. These data demonstrate extensive genetic heterogeneity within endometrial cancers and relative homogeneity across metastatic sites.
PMCID: PMC4963271  PMID: 27348297
Cancer; Metastasis; Precursor; Endometrial cancer; Cancer genomics; Cancer evolution
2.  Long-term benefit of PD-L1 blockade in lung cancer associated with JAK3 activation 
Cancer immunology research  2015;3(8):855-863.
PD-1 immune checkpoint blockade occasionally results in durable clinical responses in advanced metastatic cancers. However, mechanism-based predictors of response to this immunotherapy remain incompletely characterized. We performed comprehensive genomic profiling on a tumor and germline sample from a patient with refractory lung adenocarcinoma who achieved marked long-term clinical benefit from anti-PD-L1 therapy. We discovered activating somatic and germline amino acid variants in JAK3 that promoted PD-L1 induction in lung cancer cells and in the tumor immune microenvironment. These findings suggest that genomic alterations that deregulate cytokine receptor signal transduction could contribute to PD-L1 activation and engagement of the PD-1 immune checkpoint in lung cancer.
PMCID: PMC4527885  PMID: 26014096
3.  Genomic characterization of brain metastases reveals branched evolution and potential therapeutic targets 
Cancer discovery  2015;5(11):1164-1177.
Brain metastases are associated with a dismal prognosis. Whether brain metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. We performed whole-exome sequencing of 86 matched brain metastases, primary tumors and normal tissue. In all clonally related cancer samples, we observed branched evolution, where all metastatic and primary sites shared a common ancestor yet continued to evolve independently. In 53% of cases, we found potentially clinically informative alterations in the brain metastases not detected in the matched primary-tumor sample. In contrast, spatially and temporally separated brain metastasis sites were genetically homogenous. Distal extracranial and regional lymph node metastases were highly divergent from brain metastases. We detected alterations associated with sensitivity to PI3K/AKT/mTOR, CDK, and HER2/EGFR inhibitors in the brain metastases. Genomic analysis of brain metastases provides an opportunity to identify potentially clinically informative alterations not detected in clinically sampled primary tumors, regional lymph nodes, or extracranial metastases.
PMCID: PMC4916970  PMID: 26410082
Genomics; brain metastases; evolutionary patterns; CDK inhibitors; PI3K inhibitors; precision medicine
4.  A Computationally Designed Hemagglutinin Stem-Binding Protein Provides In Vivo Protection from Influenza Independent of a Host Immune Response 
PLoS Pathogens  2016;12(2):e1005409.
Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.
Author Summary
Influenza is a major public health threat, and pandemics, such as the 2009 H1N1 outbreak, are inevitable. Due to low efficacy of seasonal flu vaccines and the increase in drug-resistant strains of influenza viruses, there is a crucial need to develop new antivirals to protect from seasonal and pandemic influenza. Recently, several broadly neutralizing antibodies have been characterized that bind to a highly conserved site on the viral hemagglutinin (HA) stem region. These antibodies are protective against a wide range of diverse influenza viruses, but their efficacy depends on a host immune effector response through the antibody Fc region (ADCC). Here we show that a small engineered protein computationally designed to bind to the same region of the HA stem as broadly neutralizing antibodies mediated protection against diverse strains of influenza in mice by a distinct mechanism that is independent of a host immune response. Protection was superior to that afforded by oseltamivir, a lead marketed antiviral. Furthermore, combination therapy with low doses of the engineered protein and oseltamivir resulted in enhanced and synergistic protection from lethal challenge. Thus, through computational protein engineering, we have designed a new antiviral with strong biopotency in vivo that targets a neutralizing epitope on the hemagglutinin of influenza virus and inhibits its fusion activity. These results have significant implications for the use of computational modeling to design new antivirals against influenza and other viral diseases.
PMCID: PMC4742065  PMID: 26845438
5.  Biogenesis of Influenza A Virus Hemagglutinin Cross-Protective Stem Epitopes 
PLoS Pathogens  2014;10(6):e1004204.
Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that sufficient immune pressure on the HA stem region could select for viral escape mutants with increased steric hindrance from N-linked glycans.
Author Summary
Extensive variation in the IAV HA globular domain severely impedes influenza vaccination. Recent findings demonstrate that StRAbs, specific Abs to the highly conserved stem region of HA, can protect hosts against a broad variety of influenza virus strains. In investigating the binding of StRAbs to HA during its biogenesis in IAV-infected cells, we find that these Abs can bind HA monomers prior to their trimerization in the GC. Binding to HA becomes temperature-dependent, however, as N-linked oligosaccharides mature during transport of trimerized HA through the GC to the cell surface. Our findings support the potential use of monomeric HA stem immunogens to induce broadly neutralizing Abs, but raise the possibility of eventual viral escape from StRAbs, based on structural alterations in the HA that increase steric hindrance of HA stem N-linked glycans on StRAb binding.
PMCID: PMC4055778  PMID: 24945804
6.  A Synthetic Genetic Edge Detection Program 
Cell  2009;137(7):1272-1281.
Edge detection is a signal processing algorithm common in artificial intelligence and image recognition programs. We have constructed a genetically encoded edge detection algorithm that programs an isogenic community of E.coli to sense an image of light, communicate to identify the light-dark edges, and visually present the result of the computation. The algorithm is implemented using multiple genetic circuits. An engineered light sensor enables cells to distinguish between light and dark regions. In the dark, cells produce a diffusible chemical signal that diffuses into light regions. Genetic logic gates are used so that only cells that sense light and the diffusible signal produce a positive output. A mathematical model constructed from first principles and parameterized with experimental measurements of the component circuits predicts the performance of the complete program. Quantitatively accurate models will facilitate the engineering of more complex biological behaviors and inform bottom-up studies of natural genetic regulatory networks.
PMCID: PMC2775486  PMID: 19563759

Results 1-6 (6)