POSTDOC RESEARCH DAY 2016
WEDNESDAY
September 21st
1:00pm-5:00pm
SECTION I
ORAL PRESENTERS
Abstract Number 1
Doua Azzouz
Medicine
Does Dysbiosis Within the Intestinal Microbiome Contribute to SLE
Pathogenesis?
Doua F. Azzouz, Lelise Getu, Adriana Heguy, Jill P. Buyon, Gregg J. Silverman*
Background: Systemic lupus erythematosus (SLE) is the archetypic systemic
autoimmune disease, caused by a combination of genetic and environmental
factors. Animal models in other inflammatory and autoimmune diseases have
provided ample evidence of a role for intestinal bacteria in development of
the systemic immune autoreactivity. In this study we shed light on the
composition of the gut microbiome in SLE patients and its potential impact
on the disease pathogenesis. Methods: DNA was directly extracted from 61
female SLE patients and 19 female controls fecal samples; as well as from the
endogenous IgA coated and non-coated bacteria. All DNAs were then
amplified for the16S bacterial rRNA gene and sequenced using Illumina
technology. Results: Alpha diversity showed that SLE microbiome exhibited
less diversity than healthy subjects (p= 0.002). These patterns were seen in
both treated and untreated patients and more significant in patient with high
SLEDAI. SLE patients displayed a significant decrease in Firmicutes compared
to controls. In addition, SLE patients had increased representation of specific
operational taxonomic units (OTUs). Sorted IgA-coated bacteria also
identified differential representation of certain OTUs in SLE. Interestingly,
Prevotella copri, recently linked to new-onset RA, was expanded in only SLE
patients with high SLEDAI. Conclusion: We provide the first demonstration
that clinical SLE disease is associated with gut microbiome imbalances with
decreases in taxonomic diversity and expansion of specific OTUs. This
imbalance is more significant in patients with active disease or in flare. Studies
of IgA -coated bacteria highlighted that microbes of certain OTUs are
immunologically recognized by SLE hosts, which differ from controls. These
candidate pathobionts may trigger disease initiation and flares.
Abstract Number 2
Nicole Fehrenbacher
NYU Perlmutter Cancer Center
Identification of G-Protein Coupled Receptor 31 (GPR31) as a Novel Mediator
of Kras4B Membrane Association
Nicole Fehrenbacher1, Israel Tojal da Silva2, Kwang-Jin Cho3, Craig Ramirez1,
Shafi Kuchay1,4,5, Jie Shi1, Susan Thomas1, Michele Pagano1,4,5, John F Hancock3,
Dafna Bar-Sagi1, and Mark R. Philips1
Most Ras-dependent cancers are driven by activation of the KRAS gene.
Targeting Kras4B, the most common splice variant of the KRAS locus, has
been a top priority for anti-cancer therapy. Attachment of oncogenic Ras to
cellular membranes is absolutely required to sustain transformation,
suggesting that interfering with membrane association of Ras is one path to
anti-Ras therapeutics. We have developed a novel assay to quantify Kras4B
membrane association and applied it to a human whole-genome siRNA screen
as well as a small compound screen. We identified thirteen candidate genes
required for efficient Kras4B membrane association. Among these is a Gprotein coupled receptor, GPR31. Confocal microscopy revealed colocalization of GFP-GPR31 and Tomato-Kras4B and HA-GPR31 could be coimmunoprecipitated with Kras4B, suggesting a protein-protein interaction.
This interaction was dependent on prenylation of Kras4B. Silencing of GPR31
with RNAi reduced the membrane localization of GFP-Kras4B and inhibited
fluid-phase macropinocytosis, cell number and survival of various cancer cell
lines. The macropinocytosis defect could be rescued by ectopic expression of
GFP-GPR31. RNAi-induced GPR31 silencing affected the survival of both KRAS
mutant and wild-type cancer cell lines. These data suggest that GPR31 may be
a novel target for anti-Kras therapy.
1 Perlmutter Cancer Center, New York University School of Medicine, NY 10016, USA.
2 Laboratory of Molecular Immunology, The Rockefeller University, NY 10065, USA
and present Laboratory of Computational Biology, A. C. Camargo Cancer Center, São
Paulo 01509-010, Brazil.
3 Department of Integrative Biology and Pharmacology, The University of Texas
Medical School at Houston, TX 770301501, USA.
4 Department of Biochemistry and Molecular Pharmacology, New York University
School of Medicine, 522 First Avenue, SRB 1107, New York, NY 10016, USA
5 Howard Hughes Medical Institute, 522 First Avenue, SRB 1107, New York, NY 10016,
USA
Abstract Number 3
Carmen Hurtado Del Pozo
Medicine
Inhibition of Rage Promotes Lipolysis and Thermogenesis in Beige and Brown
Adipocytes
Carmen Hurtado del Pozo, Juan Francisco Aranda Gomez, Evelyn Litwinoff,
Fei Song, Ann Marie Schmidt*
Cold-induced adaptive thermogenesis has gained considerable interest due to
the 'rediscovery' of brown adipose tissue in adult humans in 2009. In response
to these findings, activation of human BAT has been proposed as a potential
treatment for obesity and diabetes type 2. We recently showed that
deficiency in Receptor for Advanced Glycation Endproducts (RAGE) protects
mice against diet-induced obesity by increasing energy expenditure. Using
microarray analysis we found that adipocytes retrieved from visceral adipose
tissue of Ager-/- mice fed High Fat Diet (HFD) display higher expression of
genes related to fatty acid oxidation, thermogenesis and lipolysis than
adipocytes from WT mice. Accordingly, we found that primary brown and
beige adipocytes from Ager-/- mice display a higher 3-adrenergic response.
To demonstrate the autonomous role of RAGE in brown adipose tissue, we
performed a transplantation of Ager -/- brown adipose tissue into wt mice
and brown adipose tissue from wt to wt mice. After 16 weeks on HFD, mice
bearing Ager-/- BAT displayed significantly greater protection against weight
gain and insulin resistance compared to WT mice that received BAT from WT
mice. Our novel results suggest that RAGE is a novel target for the treatment
of obesity.
Abstract Number 4
Michael Peled
Medicine
Combinatorial Proteomic Analysis of the Receptor Programmed Cell Death-1
Uncovers New Checkpoint Modulators
Michael Peled, Adam Mor*
Programmed cell death (PD)-1 is one of the most important inhibitory
receptors in T cells. Antibodies targeting PD-1 elicited clinical responses in
multiple tumors. Nevertheless, response is limited to a fraction of patients and
thus, a more complete understanding of this pathway should provide novel
biomarkers for responders and new therapeutic targets. Here we used mass
spectrometry to uncover proteins that either interact with the cytoplasmic tail
of PD-1 or are dephosphorylated downstream of the PD-1. PD-1 ligation is
known to induce the binding of the phosphatase SHP2 to the cytoplasmic tail
of PD-1, which dephosphorylates signaling components of the T cell receptor,
hence preventing T cell activation. We identified SHP2 as a PD-1 interacting
protein, and in addition we found 17 novel PD-1-interacting proteins, and
approximately 60 proteins that their phosphorylation status was changed
upon PD-1 signaling. Two of these proteins, EFHD2 (EF-Hand-domain-familymember-D2) and SH2D1A (SH2-Domain-containing-1A), were analyzed for
their contribution to PD-1 inhibitory responses. Silencing EFHD2 abrogated
PD-1 effects, including elimination of the ability of PD-1 to inhibit cytokine
secretion and cell proliferation. EFHD2 co-localized with PD-1 in the
immunological synapse where it contributed to PD-1 clustering. In contrast,
SH2D1A over-expression blocked PD-1 effects. SH2D1A physically competes
with SHP2 and prevents its binding to PD-1. Thus, we have identified proteins
that can modulate PD-1 function in opposite directions, and should be further
explored as potential therapeutic targets.
Abstract Number 5
Volkan Sayin
Pathology and Perlmutter Cancer Center
Keap1 is a Bona Fide Tumor Suppressor with Genotype Specific theraputic
Vulnerabilities in Lung Cancer
Volkan Sayin, Rodrigo Romero, Shawn Davidson, Simranjit Singh, Sarah
Leboeuf, Matthew Bauer, Matthew Vander Heiden, Tyler Jacks, Thales
Papagiannakopoulos*
Despite recent advances, lung cancer remains the leading cause of cancer
death. Keap1, the negative regulator of the anti-oxidant transcription factor
Nrf2, has been recently shown to be mutated in over 20% of lung
adenocarcinoma. Here, we use a CRISPR/Cas9-based platform that utilizes a
genetically engineered mouse models (GEMM) of Kras-driven lung
adenocarcinoma for the functional characterization of Keap1 mutations in lung
cancer. We show that cell autonomous loss of Keap1 or Nrf2 mutations
abolishing Keap1 binding both accelerate the progression and metastasis of
lung adenocarcinoma through hyper-activation of the Nrf2–antioxidant
pathway. Following the functional characterization of driver mutations in
cancer, the goal of precision medicine is to identify genotype-specific
vulnerabilities. Combining genetic screens and metabolomics we have
identified novel mechanisms underlying the role of the Nrf2/Keap1 pathway in
lung cancer. We report unique metabolic dependencies in Keap1 mutant lung
cancer cells which we validate both genetically and with small molecule
inhibitors. The CRISPR/Cas9-based approach used in this study provided an
accelerated discovery platform to characterize the importance of the
Nrf2/Keap1 pathway in lung adenocarcinoma. We conclude that Keap1 is a
bona fide tumor suppressor and that Nrf2/Keap1 mutant lung cancers have
genotype-specific vulnerabilities that could be therapeutically exploited.
SECTION II
POSTER SESSION
Abstract Number 6
Jaume Amengual
Cardiology
Beta-Carotene as a Source of Vitamin A in Immune Cells
Jaume Amengual, Tessa Barrett, Felix Zhou, Edward A. Fisher*
β-carotene (BC) is the natural precursor of vitamin A, a potent gene regulator
involved in cellular homeostasis. Whereas considerable amounts of ingested
BC are present in human plasma and tissues under regular dietary conditions,
mice completely metabolize BC to vitamin A in the intestine and liver, even
when treated with pharmacological doses of this compound. To determine
the role of BC in vitamin A production in immune cells in vivo, we used a
knockout mouse model lacking the enzyme BC oxygenase 1 (BCO1), which
converts BC to vitamin A. We irradiated Bco1-/- mice (bone marrow recipient
mice) and performed bone marrow transplant experiments using wild-type
and Bco1-/- mice as bone marrow donors. Then, mice were fed a diet
containing BC as the only source of vitamin A for 8 weeks before drawing
blood to isolate monocytes, T-cells, B-cells, and neutrophils for mRNA
analysis. We observed changes in key proteins involved in cell differentiation
and inflammation, indicating that the absence of BCO1 in immune cells
impaired vitamin A formation and its downstream effects on gene expression.
Abstract Number 7
Francesco Boccalatte
Pathology
N-myc ubiquitylation is Essential for Hematopoietic Stem Cell Maintenance
and Lymphoid Specification
Bryan King, Francesco Boccalatte, Kelly Crusio, Jingjing Wang, Clarisse
Kayembe and Iannis Aifantis*
Hematopoietic stem cells (HSC) are normally quiescent in the bone marrow
(BM) of adults but respond dynamically to injury or infection to replenish the
blood system. The ubiquitin-ligase system is known to attenuate signals that
instruct HSC to proliferate, mainly by controlling Myc proteins. We found that
loss of the ubiquitin-ligase Huwe1 results in significant stabilization of N-myc
leading to loss of self-renewal and decreased lymphoid specification.
Abstract Number 8
Vivian Bradaschia Correa
Orthopaedic Surgery
The Selective Serotonin Re-Uptake Inhibitor Fluoxetine Directly Inhibits
Osteoblast Differentiation and Mineralization During Fracture Healing in Mice
Vivian Bradaschia-Correa, Anne M Josephson, Matthew M Mizrahi, Shane S
Neibart, Chao Liu, Devan Mehta, Oran Kennedy, Alesha B Castillo, Philipp
Leucht*
Chronic use of serotonin re-uptake inhibitors (SSRI) has been linked to
osteoporosis. We investigated the effect of fluoxetine on fracture repair in
mice, and on the in vitro mineralization capacity of primary osteoprogenitor
cells (OPCs). Twelve week-old C57BL/6J mice received 10 mg/kg/day
fluoxetine, bone fracture repair was analyzed on created femur fractures and
monocortical tibial defects. Samples were subjected to µCT analysis,
histomorphometry and IHC. Bone marrow OPCs were cultured in osteogenic
media alone or in presence of 5, 10 or 20 μM fluoxetine, with and without
50mM serotonin. Mineralization activity was analyzed by alizarin red staining
and ALP activity and the expression of osteogenic markers was evaluated by
qRT-PCR. At 28 days, the fluoxetine-treated animals demonstrated a
significantly smaller and biomechanically weaker bony callus. The
monocortical tibial defects, which heal exclusively through an
intramembranous process, confirmed a direct effect of fluoxetine on
osteoblast differentiation and mineralization. In vitro studies established that
fluoxetine promotes a serotonin-independent decreasesed osteogenic
differentiation and mineralization. Surgeons taking care of patients on SSRIs,
who undergo treatment for delayed unions or non-unions may consider
converting them to an antidepressant of another class in order to avoid the
negative effects outlined in this study.
Abstract Number 9
Axel Concepcion
Pathology
Store-Operated Ca2+ Entry Regulates Ca2+-Activated Chloride Channels and
Eccrine Sweat Gland Function
Axel R. Concepcion, Martin Vaeth, Larry E. Wagner II, Miriam Eckstein, Lee
Hecht, Jun Yang, Hyosup P. Shin, Carl Weidinger, Rodrigo S. Lacruz, David E.
Yule and Stefan Feske*
Eccrine sweat glands are essential for sweating and thermoregulation in
humans. Patients with loss-of-function mutations in the Ca2+ releaseactivated Ca2+ (CRAC) channel genes ORAI1 and STIM1 that abolish storeoperated Ca2+ entry (SOCE) suffer from anhidrosis and hyperthermia at high
ambient temperature. Here we show that CRAC channel-deficient patients
and mice with conditional deletion of Orai1 (Orai1K14Cre) or Stim1 and Stim2
(Stim1/2K14Cre) genes in ectodermal tissues failed to sweat despite normal
sweat gland development. SOCE was absent in agonist-stimulated sweat
glands from Orai1K14Cre and Stim1/2K14Cre mice and human sweat gland
cells lacking ORAI1 or STIM1 expression. Abolished SOCE was associated with
impaired chloride secretion by primary murine sweat glands from Orai1K14Cre
mice. In human sweat gland cells, SOCE mediated by ORAI1 was necessary for
agonist-induced chloride secretion and activation of the Ca2+-activated
chloride channel (CaCC) Anoctamin 1 (ANO1, TMEM16A). By contrast, the
expression of TMEM16A, the water channel aquaporin 5 (AQP5) and other
regulators of sweat gland function was normal in the absence of SOCE. Our
findings demonstrate that Ca2+ influx via store-operated CRAC channels is
essential for CaCC activation, chloride secretion and sweat production in
humans and mice.
Abstract Number 10
Thomas Coughlin
Orthopedic Surgery
Subchondral Osteocyte Apoptosis and Bone Remodeling in a Model of Post
Traumatic Osteoarthritis
Thomas R. Coughlin, Anthony Scarallo, Matin Lendhey, Youjin Lee, Jessica
Lavery, Oran D. Kennedy*
Post Traumatic Osteoarthritis (PTOA) occurs subsequent to traumatic joint
injury, such as ACL rupture, and makes up 12% of the overall OA disease
burden. This injury affects multiple joint tissues, yet the contribution of each in
chronic PTOA progression is unknown. Interestingly, microscopic cracking in
the subchondral bone occurs during injury, and may be related to Bone
Marrow Lesions that are observed in clinical MRIs. This kind of damage, in
other skeletal systems, is repaired by osteoclast-mediated bone remodeling
via osteocyte apoptosis. In this study, we hypothesized that ACL rupture
causes increased osteocyte apoptosis via microdamage, and that this
response would correlate spatially and temporally with subchondral bone
remodeling. Thus, we induced ACL rupture in the left knee of C57BL/6 mice
(INJ) using a noninvasive ACL rupture technique, where contralateral limbs
were controls (CNT). We assessed levels of osteocyte apoptosis, bone
formation, and resorption. We found apoptotic osteocytes in INJ and CNT,
which were associated with increased bone resorption and decreased
formation after injury. Taken together, this suggests that microdamage
induced osteocyte apoptosis may induce pro-osteoclastogenic events in early
PTOA. Identifying acute changes following ACL rupture may lead to novel
early intervention strategies to prevent PTOA progression.
Abstract Number 11
Omar El Shahawy
Population Health
Physicians’ Knowledge, Beliefs and Practices Regarding E-cigarettes: Results
from a National Survey of US Primary Care Physicians in 2015
Omar El Shahawy, Scott Sherman*
Introduction: Physicians are recommending e-cigarettes despite limited
guidance from clinical practice organizations. Methods: We used a modified
Dillman approach to administer a mailed survey to a national random sample
(N=1430) of primary care physicians (PCPs) in 2015. Survey content was
informed by existing literature and qualitative research. Multiple logistic
regression identified the factors associated with PCPs’ e-cigarette
recommendation. Results: The response rate was 26%. 59% (n=166) reported
previously recommending e-cigarettes to their patients who smoke for either
smoking cessation only (17%) or harm reduction only (10%) or both (72%).
PCPs’ knowledge regarding e-cigarettes was low, but beliefs regarding ecigarettes ability to help in quitting and offer relative harm reduction was
high. Patients’ interest in using e-cigarettes (OR=1.35, 1.12-1.63) and the PCP
having favorable beliefs regarding e-cigarettes ability to help in quitting
(OR=1.84, 1.48-2.28), to reduce harm compared to other tobacco (OR=1.11,
1.05-1.16), and deter patients from using cessation medications (OR=0.78,
0.63-0.96) were associated with PCPs’ reports of previously recommending
e-cigs to their patients who smoke. Conclusions: Results illustrate an
opportunity to improve PCPs’ e-cigarette-related knowledge while their
practice is still developing. The impact of improving PCPs’ knowledge is
unknown; however, once their practice is established it is difficult to change.
Abstract Number 12
Nikki Evensen
Pediatrics
MSH6 Haploinsufficiency at Relapse Contributes to the Development of
Thiopurine Resistance in Pediatric B-Lymphoblastic Leukemia
Nikki Evensen, PhD; P.Pallavi Madhusoodhan, MD; Julia Meyer, PhD; Jason
Saliba, PhD; Ashfiyah Chowdhury, BSc; David Araten, MD; Jacob Nersting,
PhD; Kjeld Schmiegelow, MD; William L. Carroll, MD*
The outcome for children with relapsed acute lymphoblastic leukemia (ALL) is
poor, therefore understanding biological mechanisms underlying drug
resistance is essential in developing therapies. We noted relapse-specific
deletions in MSH6 in 8% of patients. MSH6, a crucial part of the DNA Mismatch
Repair (MMR) pathway, helps maintain genomic integrity. Our aim was to
determine whether MSH6 deletion results in a hypermutator phenotype
associated with secondary mutations involved in resistance and/or failure to
initiate apoptosis directly in response to chemotherapeutics. Stable
knockdown of MSH6 using shRNA resulted in a significant increase in IC50
and TG incorporation compared to non-targeting (NT) control cells upon
thiopurine treatment. This coincided with activation of the cell cycle regulator
Chk1 and an S phase arrest in NT cells. The DNA damage marker ɣH2AX and
the apoptosis marker p53 were also observed in NT cells but not MSH6-KD
cells following treatment, indicative of DNA repair. MSH6 did not lead to
microsatellite instability (MSI) or increased mutation rate, measured by
detection of cell surface proteins, suggesting it does not lead to a
hypermutator phenotype. MSH6 deletions can be added to the list of genetic
alterations associated with resistance to purine analogues highlighting the
critical role of maintenance therapy.
Abstract Number 13
Gaurav Jain
Basic Science and Craniofacial Biology, College of Dentistry
Mollusk Shell Engineering - Role of Nacre Associated Protein AP7 in Early
Nucleation Events
Gaurav Jain, Martin Pendola, John Spencer Evans*
Nacre layer of a sea shell exhibit superior strength and fracture-resistant
properties, partly due to the presence of protein phase. AP7 — a nacreassociated protein of the Pacific Red abalone (Haliotis rufescens), is known to
self-assemble under in vitro mineralization conditions. However, its role in
early nucleation, deposition and organization of minerals towards toughness
properties remains unclear. AP7 protein forms large mineral aggregates and
protein phases in the presence of Ca(II) ions. In this study, we investigated the
role of AP7 in early nucleation events in the presence of Mg(II) ions, a known
stabilizer that forms aragonite in vitro. Electron microscopy data and Raman
analysis show that AP7 inhibits the formation of the aragonite mineral crystals
when compared to control. Using atomic force microscopy, we find that the
protein self-assembles to form large aggregates and mineral nanoparticles on
mica in the presence of Mg(II) and Ca(II). Potentiometric titration analysis
show delayed nucleation events in the presence of AP7 as well as Mg(II)
indicating prolonged pre-nucleation cluster formation along with amorphous
calcium carbonate (ACC). Our results indicate that AP7 protein hydrogels
may be sequestering Ca(II) and Mg(II), slowing pre-nucleation cluster and
subsequent phase formation. We conclude that the AP7 plays a role during
the early nucleation and organizational events of the mineral growth phase in
the nacre layer of the sea shell. Further study of AP7’s molecular mechanism,
its coordination with other shell proteins, will contribute towards
development of fracture tolerant materials.
Abstract Number 14
Olga Katsara
Microbiology
Increased Activity of Chondrocyte Translational Apparatus Accompanies
Osteoarthritis
Olga Katsara, Mukundan Attur, Rachel Ruoff, Steven B. Abramson, and
Victoria Kolupaeva*
Degeneration of articular cartilage is central to osteoarthritis (OA) pathology;
however, the molecular mechanisms leading to these irreversible changes are
still poorly understood. Here, we investigated how changes in the
chondrocytes translational apparatus may contribute to OA. Normal and OA
human knee cartilage was used to analyze the activity of the translational
machinery. In addition, chondrocytes isolated from lesional and non-lesional
areas of OA cartilage were used to estimate relative rate of protein synthesis.
Furthermore, experimental OA was induced by ACL-t in rats to investigate
changes in the translational apparatus associated with the development of
OA. Last, the role of IL-1 β signaling, using rat articular chondrocytes, was
assessed in vitro. We determined several novel traits of human OA
chondrocytes, including upregulation of AKT2 and AKT3 on the posttranscriptional level and increased rate of total protein synthesis, likely caused
by 4E-BP1 inactivation- a known repressor of cap-dependent translation.
Moreover, in IL-1β treatment in vitro and in OA chondrocytes this inactivation
was mTOR-dependent and crucial for up-regulating protein synthesis in
general and in particular for MMP13 and ADAMTS5. In summary, our data
revealed the contribution of translational and non-translational effectors to
dysregulated chondrocyte homeostasis and offer a novel niche in the search
for OA therapeutic targets.
Abstract Number 15
Shan Liu
Neurology
Targeting the apoE/Aβ Interaction as a Therapeutic Strategy for AD
Shan Liu*, Grant Allington, Yanjie Sun, Frances Prelli, Bernard Brown, Yong-Uk
Kwon and Thomas Wisniewski
Inheritance of the apoE4 allele is the strongest genetic risk factor for the most
common, late-onset AD. However, there is no consensus on how different
apoE isotypes contribute to AD pathogenesis. It has been hypothesized that
apoE and apoE4 in particular is an amyloid catalyst or “pathological
chaperone”. Alternatively it has been posited that apoE is an Aβ clearance
factor, with apoE4 been worse at this function compared to apoE3 or E2. We
suggest that these seemingly opposing views can be reconciled and that the
optimal therapeutic target may be to specifically prevent the interaction of
apoE with Aβ. We show that blocking the Aβ/apoE interaction by Aβ12-28P
peptides constitutes a novel therapeutic approach for AD by reducing Aβ
parenchymal deposition in APP/PS1 Tg mice and vascular amyloid deposits in
TgSwDI mice as well as tau related pathology in aged 3xTg mice. In addition,
we have developed effective peptidomimetic inhibitors of the Aβ/apoE
interaction that are derived from the Aβ12-28P sequence. Peptoid compounds
due to their inherent resistance to degradation are known to have more
favorable pharmacokinetic properties. Preliminary in vivo data with these
peptidomimetic using APPSwe/PS1L166P mice with pronounced amyloid
associated pathologies and robust gliosis will be presented.
Abstract Number 16
Denise Minton
Pathology
SHMT2 and Mitochondrial One Carbon Metabolism are Required for Proper
Mitochondrial respiration
Denise R. Minton, Daniel J. McLaughlin, David M. Sabatini, Kivanc Birsoy, and
Richard L. Possemato*
Cancer cells must adapt their metabolism to facilitate a high proliferation rate
in a tumor microenvironment that has low levels of nutrients, including
glucose. A better understanding of these adaptations might reveal cancer cell
liabilities that can be exploited for therapeutic benefit. Here we used a
CRISPR-based genetic screen to identify genes whose loss sensitizes human
cancer cells to low glucose conditions. The screen yielded SHMT2, the
mitochondrial serine hydroxymethyltransferase, loss of which greatly reduces
proliferation in low glucose. SHMT2 normally catalyzes the conversion of
serine to glycine and methylene tetrahydrofolate to support mitochondrial
one carbon metabolism. We found that loss of SHMT2 leads to a reduction in
the translation of mitochondrial encoded proteins and reduced mitochondrial
respiration. These defects are likely due to a reduction in N-formylmethioninetRNA(Met), which receives its formyl group from one carbon metabolism and
is used for translation initiation specifically in the mitochondria. Moreover, the
defects from SHMT2 loss can be rescued by treatment with formate. Thus, we
conclude that SHMT2 and mitochondrial one carbon metabolism are required
for proper mitochondrial respiration in cancer cells.
Abstract Number 17
Jonathan Platkiewicz
Neuroscience Institute
Inferring Microcircuit Connectivity Dynamics from Spike Data
Jonathan Platkiewicz and Asohan Amarasingham*
There has been recently a great deal of interest in "mapping the brain",
namely in establishing the precise structural organization of neural
microcircuits. High-density extracellular recordings offer the unique
opportunity to observe simultaneously the activity of hundreds of neurons
with millisecond precision in the behaving mammal. Neural connectivity is
typically inferred from this recording type by extracting the spikes from the
extracellular potentials and seeking the cell pairs that exhibit finely-timed
spike correlation. Alternative statistical methods, such as the generalized
linear model, have also been applied, but these methods still rely on the
assumption that a correlation, of some type, between two spike trains reflects
a direct synaptic coupling. There is, however, no widely-accepted biophysical
justification for this assumption, nor is there much "ground truth" data that
might provide validation. Numerical simulations of biophysical models of
monosynaptic spike transfer, which are biologically faithful to some degree,
are natural candidates with which to approach this issue, given current
experimental limitations. First, we show that a millisecond spike correlation
can be observed between monosynaptically connected neurons. Second,
using the developed biophysical models combined with nonparametric
statistical tools, we show how connectivity can be inferred from large-scale
spike recordings, even in settings of extreme firing rate nonstationarities.
Abstract Number 18
Rosa Rubio
Microbiology
Suppression of Autophagy by HSV1 Us3 via ULK1-Repression and Beclin
Phosphorylation
Rosa Rubio, Ian Mohr*
Besides stimulating anabolic processes including protein synthesis, mTORC1
activation antagonizes catabolic processes like autophagy. While the HSV1
Us3 ser/thr kinase activates mTORC1, its role in controlling autophagy has not
been investigated. Here, we establish that viruses lacking the Us3 ORF or
encoding a kinase-deficient Us3 protein are unable to induce the
phosphorylation of ULK1, a major upstream autophagy regulator whose
activity is suppressed by mTORC1, and inhibit autophagy. While differences in
overall levels of HSV1 ICP34.5, which controls autophagy by binding to beclin,
another critical autophagy pathway component, were not detected in cells
infected with Us3-deficient HSV1, elevated autophagy was observed in cells
infected with ICP34.5-deficient HSV1 despite the presence of Us3. This
suggests that the capacity of Us3 to suppress autophagy in HSV1-infected
cells is ICP34.5-dependent, and the ability of ICP34.5 to suppress autophagy
is Us3-dependent. In addition to influencing processes downstream of
mTORC1, by stimulating it, Us3 unexpectedly acted in an mTORC1independent manner, similarly to Akt, and directly phosphorylated beclin on
S295. Apart from defining two new roles for Us3 in α-herpesvirus biology, this
establishes the host autophagy regulator beclin is a new, direct Us3 substrate,
illustrating another example of overlapping substrate specificity shared by
Us3 and the host kinase Akt.
Abstract Number 19
Maite Sabalza
Department of Basic Sciences, NYU College of Dentistry
Development of a ZIKV Specific, Rapid and Simultaneous Molecular and
Serological Diagnostic Assay
Sabalza M, Barber C, Yasmin R, Montagna RA, Abrams WR and Malamud D*
In the current Zika virus (ZIKV) outbreak in Brazil, ZIKV infection during
pregnancy has been associated with microcephaly and other severe birth
defects. Currently there is no vaccine available. This is why diagnosis is critical
for disease control. The current diagnostic tools rely on molecular and
serological diagnosis tests but it is being challenging because of the low
viremia and cross-reactivity of ZIKV antibodies with other flaviviruses. Our
goal is to develop a rapid diagnostic for ZIKV that will simultaneously detect
the presence of viral RNA and a host antibody response to specific ZIKV
antigens in a single, small sample of saliva or blood. Currently, I am working
on the discovery of conserved ZIKV epitope recognized by host type specific
IgM and IgG for identifying diagnostic peptides. So far, I have identified three
unique ZIKV peptides using a ZIKV high-density peptide microarray. I am also
optimizing a loop-mediated isothermal amplification (LAMP) assay to detect
ZIKV RNA in saliva or blood. Subsequent experiments will focus on screening
and validation of the three unique ZIKV peptides to be incorporated in a
serological diagnostic assay to run in parallel with a LAMP optimized assay for
ZIKV detection.
Abstract Number 20
Kosuke Tamura
Department of Population Health
Are Neighborhood Food Environments Associated with Body Mass Index and
Blood Pressure? Application of GPS Technology Among Low-Income Housing
Residents in New York City
Kosuke Tamura, Jessica Athens, Brian Elbel, Basile Chaix, Seann D. Regan,
Dustin T. Duncan*
Purpose: The objective was to examine the associations of food environments
with body mass index (BMI) and blood pressure (BP) in New York City (NYC)
neighborhoods using global positioning system (GPS) data. Methods: Data
came from the NYC Low-Income Housing, Neighborhoods and Health Study,
including objectively measured BMI and BP data (n=102, mean age=39.3
years), and one week of GPS data. Five food environment variables include:
fast-foods, wait-service restaurants, bodegas, supermarkets, and grocery
stores around home and GPS-defined neighborhoods. We examined
associations of the food environment with BMI, systolic and diastolic BP,
controlling for demographics and neighborhood demographic characteristics.
Results: In fully-adjusted models, there were inverse associations of fast-food
outlets, wait-service restaurants, grocery stores with BMI (B=-0.36; B=-0.26;
B=-0.18, all p<0.01) and diastolic BP (B=-0.53; B=-0.41; B=-0.347, all p<0.05)
around home. Food environments were not associated with systolic BP. With
GPS-defined neighborhoods, most associations were insignificant. Conclusion:
Examining how the food environment is associated with CVD risk may have
the potential to shed light on determinants of CVD risk. Inconsistent
relationships between the food environments and CVD risk factors were
found. Further research is needed to investigate these relationships, including
by level of urbanicity and in different geographic areas.
Abstract Number 21
Ryohei Tsutsumi
Perlmutter Cancer Center
Spatio-Temporal Regulation of SHP2 Action by Endosomal ROS Production
Ryouhei Tsutsumi, Jana Harizanova, Rabea Stockert, Katrin Schröder, Philippe
I. H. Bastiaens, Benjamin G. Neel*
Reactive oxygen species (ROS) are produced transiently in response to
various cell stimuli, including cytokines and growth factors, and function as
second messengers that directly oxidize and regulate target proteins.
Interleukin 1 (IL-1) or tumor necrosis factor alpha (TNF alpha) signaling
requires compartmentalized ROS generation at specialized endosomes,
termed “redoxosomes”. However, whether redoxosomes function in other
types of signaling pathways and the specific signaling proteins oxidized by
redoxosomes have remained unclear. Here we report that platelet-derived
growth factor (PDGF), as well as epidermal growth factor (EGF), transiently
generates RAB5-positive, EEA1-negative redoxosomes that contain NADPH
oxidase (NOX) 1/4. Using a newly developed method to visualize oxidized
proteins, we find that Src homology 2 domain-containing protein-tyrosine
phosphatase 2 (SHP2) is oxidized at these growth factor-evoked
redoxosomes. Preventing redoxosomal ROS production alters the kinetics of
PDGFR-evoked signaling events. Our results expand the “redoxosome”
concept to receptor tyrosine kinase (RTK) signaling and show that SHP2 is
regulated in space and time by redoxosomal ROS.
Abstract Number 22
Rebeca Vasconcelos
Radiation Oncology
Microbial Biomarkers of Oral Mucositis Onset
Rebeca Vasconcelos; Bruce J. Paster; Nicholas Sanfilippo; Alexander Ross
Kerr; Yihong Li; Lina Faller; Beverly Smith; Kenneth Hu; Catherine Concert;
Julie Moya; Erica Queiroz; Cynthia Howard; Kira Nightingale; Marta Gabinskiy;
Luciana Ramalho and Patricia Corby*
Introduction: Oral mucositis (OM) is the most common, painful and
debilitating toxicities of cancer regimen-related treatment. The aim of this
study is to explore the changes in the microbiome associated with OM onset
in head and neck cancer patients undergoing radiotherapy alone (RT) or
chemoradiotherapy (chemoRT). Methods: We recruited patients with
oropharynx squamous cell carcinoma scheduled for receiving RT alone or
chemoRT. Site-specific oral biofilms samples were collected at two time
points: before RT/ChemoRT (pre-OM), and at the onset of OM. Changes in
microbial abundance were detected by Human Oral Microbe Identification
using Next Generation Sequencing and metagenomic analyses. Results:
Relative changes in abundance of microbial biomarkers in 11 subjects were
discriminative between pre and post OM onset. Species such as
Streptococcus spp., Gemella haemolysans, Granulicatella elegans and
Haemophillus spp. were found to be significantly overabundant in post-OM
onset samples as compared to pre-OM, in the tongue body site. Gemella
haemolysans and Granulicatella elegans were also significantly overabundant
in samples from the buccal mucosa at the OM onset. Conclusion: Our results
suggest a dynamic shift in the oral microbiome during the onset of OM and
further investigation is warranted to explore if they facilitate further tissue
damage and subsequent pain.
Abstract Number 23
Deepika Vasudevan
Cell Biology
Translational Regulation by Thor Drives the Innate Immune Response
Deepika Vasudevan, Jessica Sam, Hyung Don Ryoo*
Although 4E-BP (Thor in Drosophila) has been extensively studied in the
context of lifespan extension, the first described observations about thor
mutants showing immune-compromisation remains unexplained. Here, we
identify a role for 4E-BP in the innate immune response pathway in
Drosophila. The immune response begins with pathogen recognition followed
by transcriptional induction of anti-microbial peptides (AMPs), their
subsequent synthesis and secretion. Transcriptional induction of AMPs was
unaltered in Δ4E-BP, suggesting that 4E-BP regulates AMP translation or
secretion. 4E-BP is a known inhibitor of cap-dependent translation. In-vitro
experiments show that the 5’UTRs of AMPs are capable of cap-independent
translation. Additionally, AMP levels in hemolymph of Δ4E-BP were decreased
in comparison to controls. Together, these data suggest that translation
inhibition by 4E-BP drives preferential translation of AMPs during infection.
We find that 4E-BP is transcriptionally induced during infection by the
integrated stress response transcription factor, ATF4. A reporter driven by an
ATF4-responsive element in the 4E-BP locus is elevated in the gut of infected
larvae. Additionally, ATF4 mutants are immune-compromised similar to Δ4EBP. ATF4 can be activated by two upstream kinases, GCN2 or PERK. RNAi
experiments show that GCN2 and not PERK is the kinase responsible ATF4
activation in this context.
Abstract Number 24
Narendra Kumar Verma
Microbiology
MZF1 and GABP Novel Transcriptional Regulators in Osteoprogenitor Lineage
Narendra Kumar Verma, Upal Basu-Roy, Abhilash Gadi,Alka Mansukhani** and
Claudio Basilico**
The Hippo pathway was discovered in Drosophila about ten years ago as a
regulator of organ size. It consists of a phosphorylation cascade that restrains
the activity of the transcriptional coactivator, YAP a growth and proliferation
promoting factor. Hippo signaling in mammals controls cell proliferation and
stemness. This pathway also has a significant tumor suppressive role, it
restrains cancer development by promoting the phosphorylation and nuclear
exclusion of YAP which is emerging as a potent oncogene. We have shown
that the stem cell transcription factor SOX2 antagonizes the Hippo pathway
and affects the lineage differentiation fates of mesenchymal stem cells (MSCs)
through direct induction of YAP (Seo et al, Cell Reports, 2013). SOX2 and YAP
also maintain cancer stem cells in osteosarcomas, a cancer that arises from
the MSC lineage. We recently showed that like SOX2, YAP maintains these
cancer stem cells and that Hippo upstream activators, are repressed by SOX2
(BasuRoy et al, Nature Comm, 2014). Thus SOX2 antagonizes the Hippo
pathway by directly inducing YAP and repressing Hippo upstream activators.
We are now determining how SOX2 activates YAP by examining the
importance of putative SOX2 binding sites in the YAP genomic regions. In
particular we are using nucleotide substitutions to delete putative functional
elements in the 243 bp enhancer region of YAP. Together with proteomic
analysis we aim to determine the coactivators of YAP that are cooperating
with SOX2 to regulate YAP1 by mutagenesis apparoach. We have generated
luciferase reporter constructs driven by Sox2 bound genomic regions of YAP1,
and identified the enhancers for YAP1 expression in mesenchymal stem cells
(MSCs) and osteoprogenitor cells. We have identified additional transcription
factor (TFs) Myeloid Zinc Finger 1 (MZF1) and GA Binding Protein (GABP) that
play a role in regulation of YAP1 in cancer stem cells. Our reporter data
revealed that Myeloid Zinc Finger domain (MZF1) and GA binding protein
(GABP) are required to maintain basal activity of YAP1 in osteoproginator
cells. Knocking down of MZF1/GABP decreases the transcriptional activity as
well as the biological functions of YAP while, overexpression of MZF1
activates YAP1 transcriptional activity. Elevated YAP1 activity due to
mutations in Hippo pathway components or YAP1 amplification is observed in
several types of human cancers such as osteosarcoma and glioblastomas an
aggressive type of brain cancer. Therefore disruption of YAP transcriptional
activity by these TFs could be a therapeutic strategy for YAP1 dependent
tumours.
Abstract Number 25
Elizabeth Vink
Microbiology
Sustained mTORC1 Activation in HSV1-Infected Cells Determines Resistance to
Physiological Stress
Elizabeth Vink, Sora Lee, James R. Smiley, Ian Mohr*
By activating the host kinase mTORC1, HSV1 disables the cellular translational
repressor 4E-BP1 to stimulate viral protein production and replication.
However, whether physiological cues that naturally regulate mTORC1,
including energy and nutrient availability, control productive HSV1 replication
has not been explored. This is critical as the complex interplay of signals
controlling mTORC1 can be integrated in numerous ways to either activate or
inhibit the kinase, potentially restricting mRNA translation and HSV-1
productive growth. Here, we establish that mTORC1 function in HSV1-infected
cells, unlike uninfected cells, is preserved despite energy or amino acid (aa)
insufficiency. Overcoming energy insufficiency signals transmitted through
AMPK is dependent upon the Us3 protein kinase, which prevents the tuberous
sclerosis complex (TSC) from inhibiting mTORC1. Indeed, Us3-deficient
viruses are unable to stimulate mTORC1 activation and are hypersensitive to
energy insufficiency. HSV1 also preserves mTORC1 activity during aa
insufficiency, which controls mTORC1 in a TSC-independent manner.
Surprisingly, maintaining mTORC1 activation during aa insufficiency is
dependent upon Us3 and an additional viral function mediated by VP11/12, a
protein that activates the host kinase Akt. While sustained mTORC1 signaling
and replication of mutants deficient for either Us3 or VP11/12 was modestly
reduced during aa insufficiency, mTORC1 activity and productive growth of
viruses doubly-deficient for both Us3 and VP11/12 was substantially impaired.
Thus, HSV1 not only enforces mTORC1 activation, but also addition-ally
subverts host nutrient and energy sensing programs that allow mTORC1 to
monitor fundamental, physiological indicators of homeostasis. This identifies a
complex viral program to sustain mTORC1 signaling, supporting high level
HSV1 protein synthesis and viral productive growth irrespective of
physiological stress. Moreover, as inhibiting mTORC1 triggers reactivation
from latency, it suggests mechanisms whereby HSV1 enables viral protein
synthesis and productive growth by overriding physiological stress cues and
cell intrinsic responses that restrict mTORC1 activation.
Abstract Number 26
Haizhen Wang
Medcine
Apolipoprotein B Regulates Lipid Droplets Metabolism via KLHL12 and FIT2
Haizhen Wang, Edward A Fisher*
Apolipoprotein B (apoB) is the main structural molecular of VLDL and highly
related to obesity-related diseases. Lipid droplets (LDs) are important lipid
storage and metabolism organelle. Aggregating evidences suggest that apoB
could bind to the surface of some LDs in hepatocytes. Meanwhile, knock down
of apoB by siRNA lead to expansion of LDs. Hence, we speculate that the
apoB-associated LDs are required for apoB metabolism in hepatocytes. We
found genes involved into apoB metabolism also affect LDs metabolism, such
as apoC3, UBXD8, Tm6sf2 and KLHL12. KLHL12 is a key factor for transfer
lipidated apoB from ER to golgi. Knock down KLHL12 result in expansion of
LDs and more LDs with apoB crescent. On the other hand, LDs regulator also
regulate apoB metabolism. Such as oleic acid and FIT2, a key factor for LDs
synthesis in ER. Exogenous oleic acid stimulates expansion of LDs and apoB
level. Knock down FIT2 inhibit expansion of LDs. Interestingly, FIT2 knock
down also inhibit the oleic acid induced increase of apoB level. Next steps, we
will study the molecular mechanism of how apoB regulate LDs metabolism via
KLHL12 and FIT2 and separate apoB-related and unrelated LDs by IP and
perform proteomics and lipidomics.
Abstract Number 27
Ada Weinstock
Medicine (Cardiology)
Novel Regulation of Macrophage Polarization and Atherosclerosis Regression
by Wnt/β-Catenin
Ada Weinstock, Hitoo Nishi, Or Yaacov, Prashanthi Menon, Edward A. Fisher*
The leading cause of mortality worldwide is coronary artery disease, driven by
atherosclerosis. The disease usually remains undiagnosed until atherosclerotic
plaques progress to an advanced stage. At this point, interventions are
pursued to halt further progression. While the ideal is to promote disease
resolution, this has thus far proven elusive. Our laboratory has developed
mouse models in which atherosclerotic plaques are shown to regress, with
dramatic changes in plaque size as well as in the number and characteristics
of immune cells plaques, namely macrophages. Our previous studies suggest
that macrophages acquire anti-inflammatory properties and drive
atherosclerosis regression and that this process may be regulated by the
canonical Wnt/β-catenin pathway. Importantly, impaired Wnt/β-catenin has
been reported to associate with increased risk of atherosclerosis in humans.
Herein we investigated how inhibition of Wnt affects atherosclerosis
regression. Thus, we utilized a regression model and compared plaque
regression in mice treated with a Wnt inhibitor or vehicle. Results show that
mice treated with the Wnt inhibitor have impaired regression, manifested by
larger plaques and greater inflammatory macrophage content/phenotype and
composition. In conclusion, our exciting data strongly suggest that the Wnt/βcatenin are novel factors promoting the polarization of pro-resolving/tissue
repair macrophages that enhance atherosclerosis regression.
Abstract Number 28
Donna Whelan
Biochemistry
Visualization of in Vivo Homologous Recombination Kinetics Using Super
Resolution Microscopy
Donna R Whelan, Yandong Yin, Keria Bermudez-Hernandez, Sarah Keegan,
David Fenyo & Eli Rothenberg*
Homologous recombination (HR) is an important repair pathway for double
strand breaks (DSBs) during S phase, and affords exceptionally high fidelity.
Collapsed replication forks resulting in one-sided DSBs are the predominant
endogenous target of HR along with exogenous damage from ionizing
radiation and chemotherapeutic agents. Despite the clear importance of the
HR pathway in maintaining genomic integrity, elucidation of the physical
organization and progression of HR machinery has previously been limited by
the techniques available. Now, using single molecule super resolution (SR)
microscopy we can directly visualize the structures and interactions of HR
proteins at collapsed replication fork DSBs in vivo at spatial resolutions as
good as 20 nm. A dozen key proteins involved in HR were investigated by
immunolabeling fixed cells for three-color imaging and the resulting SR
images analyzed using in-house correlation and colocalization approaches.
We have observed loading of Ku and MRE11 onto the same DSB, colocalization
of RPA, RAD52 and RAD51 on resected DNA, and RAD51 nucleofilament
formation in the absence of BRCA2. This has allowed new insights into the
spatiotemporal kinetics of these proteins at a DSB and has also established a
novel experimental approach for probing the interactions and kinetics of DNA
damage repair.
Abstract Number 29
Yuliya Zilberman
Developmental Genetics
Regulation of Cell Shape Change by Local Inhibition of CDC-42 at Epithelial
Junctions
Yuliya Zilberman, Dorian C. Anderson, and Jeremy Nance*
During epithelial tissue morphogenesis, adherens junction (AJ) remodeling
must be regulated to allow cell shape change without compromising cell
adhesion. The Rho GTPase signaling protein CDC-42 can influence AJ protein
levels by directing the trafficking of AJ components to and from junctions.
CDC-42 oversees numerous cellular events, and it remains unclear how its
activity is locally controlled at junctions to guide AJ protein trafficking in vivo.
Here, we examine the function and regulation of CDC-42 in C. elegans embryo
elongation. Analysis of maternal-zygotic cdc-42 mutants and cdc-42
overexpression reveal that CDC-42 activity must be controlled for the cell
shape changes of elongation to occur normally. We identify the RhoGAP
PAC-1/ARHGAP21 as a local inhibitor of CDC-42 activity at AJs. Loss of PAC-1,
the interacting coiled-coil protein PICC-1/CCDC85A-C, or an increase in CDC42 activity blocks elongation in embryos with compromised AJ function and
results in an overall increase in AJ protein levels. Increasing AJ protein levels
alone is sufficient to arrest elongation in mutants with compromised AJ
function, indicating that precise control of AJ protein levels by PAC-1, PICC-1,
and CDC-42 is essential for proper cell shape changes during elongation. Our
findings identify a previously unrecognized molecular mechanism for
regulating junctional CDC-42 activity and AJ protein levels during epithelial
morphogenesis.
2016