Pathology Retreat 2022 - Research Retreat Wednesday, May 25, 2022 The 21st Annual Department of Pathology
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2022
Pathology
Retreat
The 21st Annual Department of Pathology
Research Retreat
featuring the
Pathology 32nd Annual Research Day
Wednesday, May 25, 2022
Program Contents
Research Day Agenda ............................................................................................................................... page 3
Poster Session & Abstracts ........................................................................................................................ page 5
Trainee Awards ........................................................................................................................................ page 78
Poster Winners, 2021 Pathology Retreat ................................................................................................. page 79
Retreat Planning Committee
Faculty Andrew Duncan, PhD, Chair
Sameer Agnihotri, PhD
Aaron Bell, PhD
Charleen Chu, MD PhD
Marie DeFrances, MD PhD
Roy Frye, MD
Wendy Mars, PhD
Octavia Palmer, PhD
Alex Soto, MD PhD
Jian Yu, PhD
Graduate Students Rithika Behera
Joud Mulla
Coordinators Shanning Wan
Amanda Bytzura, MBA
2022 Pathology Retreat • May 25, 2022
Keynote Presentation
Keynote Presentation Location: S120 BST & https://pitt.zoom.us/j/96992431085
12:00 – 1:00 pm Pilar Alcaide, PhD T Cell Immune Responses in Heart Failure: An Interplay
Associate Professor of Immunology Between Cardiac Inflammation and Fibrosis
Tufts University School of Medicine
Faculty & Trainee Presentations Location: S120 BST & https://pitt.zoom.us/j/96992431085
1:30 pm Opening Remarks
Andrew Duncan, PhD George Michalopoulos, MD, PhD
Associate Professor of Pathology Maud L. Menten Professor and Chair of Pathology
Session 1: Graduate Student Research
Moderator, Joud Mulla, BS
1:35 – 1:55 pm Jeremy Rich, MD, MHS, MBA Killing Brain Tumor Stem Cells
Professor of Neurology
1:55 – 2:05 pm Raphael Crum, BS Matrix Bound Nanovesicles as a Novel Extracellular Matrix
PhD Candidate, CMP, MSTP Therapy for the Treatment of Rheumatoid Arthritis
2:05 – 2:15 pm Daniel Zuppo, BS foxm1 is Required for Cardiomyocyte Proliferation After Adult
PhD Candidate, MGDB, CATER Zebrafish Cardiac Injury
2:15 – 2:25 pm Taylor Gatesman, BS Inhibiting Insulin Signaling Reverses Resistance to PI3K-Mtor
PhD Candidate, CMP Inhibitors in Aggressive Pediatric High-Grade Gliomas
Session 2: Basic Research
Moderator, Justin Sui, BA
2:30 – 2:50 pm Mo Ebrahimhkani, MD Stem Cell-based Multicellular Systems: New Platforms for
Associate Professor of Pathology Regenerative Technologies and to Study Human Development
2:50 – 3:00 pm Katherine Killian, DO The Receptor for Advanced Glycation Endproducts Promotes
Pathology Resident Allergen Driven Neutrophil Dominant Airway Inflammation Via
NLRP3 Inflammasome Activation
3:00 – 3:10 pm Michelle Drewry, BS Scaffold-Free Conduits Formed from Dental Pulp Stem Cell
PhD Candidate, BioE, CATER Sheets Provide Neurotrophic and Directional Support for
Regenerating Axons
3:10 – 3:20 pm Sowmya Mekala, PhD Antagonizing Activin A/P15ink4b-Signaling as a Therapeutic
Postdoctoral Fellow Strategy for Liver Disease
Session 3: Clinical Research
Moderator, Simmi Patel, MD
3:30 – 3:50 pm Sarah Wheeler, PhD, FACB, CC Data Driven Improvement for Diagnostic Testing in Special
Assistant Professor of Pathology Populations
3:50 – 4:00 pm Azfar Neyaz, MD Loss of PTEN and Expression of GLUT1 Predict the
Pathology Resident Metastatic Progression of Pancreatic Neuroendocrine Tumors
4:00 – 4:10 pm Shikha Malhotra, MD Persistent NUP98 Rearrangement may Portend Poor
Pathology Resident Prognosis
4:10 – 4:20 pm Akram Shalaby, MD Gastric Intestinal Metaplasia as a Risk Factor for Progression
GI Pathology Clinical Instructor to Gastric Dysplasia and Carcinoma in Patients Who Undergo
Gastric Biopsy: a Population-Based Study
Poster Session & Reception University Club, Ballroom B
5:00 – 7:00 pm Back in person! Join your colleagues at the Pitt University Club for an interactive poster session,
hors d'oeuvres, and drinks. Say hello to old friends and make some new ones.
Notes
Participation by all individuals is encouraged. Advance notification of any special needs will help us provide better service. Please notify
us of your needs at least two weeks in advance of the program by emailing Shanning Wan at shw126@pitt.edu.
All individuals in a position to control the content of this education activity are required to disclose all relevant financial relationships with
any entity producing, marketing, re-selling, or distributing health care goods or services, used on, or consumed by, patients.
Keynote Speaker Presentation: This activity is approved for AMA PRA Category 1 Credit™. Sponsored by The Department of
Pathology in conjunction with the Cellular and Molecular Pathology Graduate Program and University of Pittsburgh School of Medicine
Center for Continuing Education in the Health Sciences. The University of Pittsburgh School of Medicine is accredited by the
Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The University of
Pittsburgh School of Medicine designates this live activity for a maximum of 1.0 AMA PRA Category 1 Credits™. Physicians should
claim only the credit commensurate with the extent of their participation in the activity. Other health care professionals are awarded 0.1
continuing education units (CEU’s) which are equal to 1.0 contact hours.
The University of Pittsburgh is an affirmative action, equal opportunity institution.
2022
Poster Session
& Abstracts
May 25, 2022, 5:00 – 7:00 pm Presentation Schedule
Ballroom B, University Club 5:00 – 6:00 pm
University of Pittsburgh Odd-numbered posters
6:00 – 7:00 pm
Even-numbered postersGraduate, Basic Research
1. Anu Balogun, Beta-catenin inhibition as a novel 21. Jie Bin Liu, Characterizing and Targeting ERBB2
therapeutic strategy for porphyria Mutations in Invasive Lobular Carcinoma
2. Maria Beecher, MALT1 as a mediator of treatment 22. Joseph Maggiore, Human kidney organoids
resistance in triple-negative breast cancer demonstrate HDAC8 as a therapeutic target to attenuate
3. Rithika Behera, Transcriptional regulation of SSc EMT in kidney disease
dermal myofibroblasts by FOSL2 and FOXP1 23. Meagan Makarczyk, Joint Pain on a Chip: Mechanistic
4. Hannah Butterfield, Evaluating small extracellular Analysis, Therapeutic Targets, and an Empirical
vesicles as immunomodulatory components of the Strategy for Personalized Pain Management
microenvironment in p95HER2+ breast cancer 24. Philip Mannes, In vivo molecular imaging of chemokine-
5. Julianna Castle, The Impact of Profilin-1 Mutations on like receptor 1 (CMKLR1) in experimental lung injury
Protein Homeostasis in Amyotrophic Lateral Sclerosis 25. Nicole Martucci, Inhibition of Phosphoinositide 3-kinase
6. Margaret Champion, Investigating the role of alternative delta (PIK3CD) Suppresses Hepatocyte Proliferation by
splicing in reproductive aging and maternal-fetal health More than 50% in the Regenerating Liver after Partial
Hepatectomy
7. Mona Chatrizeh, Microbiome targeted nutrition to
support lymphopoiesis during critical illness 26. Meghan Mooring, CYR61 orchestrates NASH fibrosis
through IRAK4-SYK-NFκB-PDGF signaling in monocyte-
8. Grace Conway, Development of an in vitro System to derived macrophages
Understand the Mechanisms of Ultrasound-Targeted
Microbubble Cavitation-Mediated Blood Brain Barrier 27. Joud Mulla, The Role of Caspase-11 in Trauma-
Opening Induced Coagulopathy (TIC) in a Murine Polytrauma
Model
9. Raphael Crum, Matrix Bound Nanovesicles as A Novel
Extracellular Matrix Therapy For The Treatment Of 28. Alexis Nolfi, Therapeutic Use of an Interleukin-4 Eye
Rheumatoid Arthritis Drop in a Rabbit Model of Dry Eye Disease: A Pilot
Study
10. Andrea Cruz, The Role of Tumor Microenvironment
Derived Growth Factors in Pediatric Brain Tumors 29. Matthew Poskus, Overcoming Stroma-Mediated Drug
Resistance in HER2+ Breast Cancer
11. Michelle Drewry, Scaffold-free conduits formed from
dental pulp stem cell sheets provide neurotrophic and 30. Tamandeep Saggi, Hindlimb Muscle Representations in
directional support for regenerating axons Mouse Motor Cortex Defined by Viral Tracing
12. Taylor Gatesman, Inhibiting insulin signaling reverses 31. Justin Sui, Temporal Assessment of Single-Cell RNA
resistance to PI3K-mTOR inhibitors in aggressive Sequencing in a Mouse COPD Model Identifies Aberrant
pediatric high-grade gliomas Macrophage Inflammatory and Metabolic Functions
Dependent on Adenine Nucleotide Translocase 1 (ant1)
13. Shohini Ghosh-Choudhary, Whole Genome CRISPR
Screen Reveals Vulnerabilities of Senescent Cells 32. William Tennant, Phosphorylation state of TDP-43
alters both liquid-liquid phase separation and
14. Matthew Halbert, Loss of MAT2A Compromises nuclear/cytoplasmic localization
Methionine Metabolism and Represents a Vulnerability
in H3K27M Mutant Glioma by Modulating the 33. Rick van der Geest, BATF2 mediates pro-inflammatory
Epigenome cytokine production in alveolar macrophages and
enhances the early host defense against pulmonary
15. Shea Heilman, Uncovering Differentiation-Inhibitory Klebsiella pneumoniae infection
Mechanisms in the Developing tet2-/-;tet3-/- Zebrafish
Retina 34. Jeremy Velazquez, Directing morphogenesis of human
liver organoids in vitro via engineering inter- and intra-
16. Richard Herron, Identification of a direct role of CPSF6 cellular regulatory networks
and a key cis-acting motif in promoting TRIM9 proximal
polyA site usage 35. Susannah Waxman, Multicolor DiOlistic Labeling
Reveals Astrocyte Morphology in the Collagenous
17. Joshua Hislop, Synthetic Morphogenesis of Human Lamina Cribrosa
Blastocyst-to-Gastrula Bilaminar Disc
36. Sierra Wilson, Diploid Hepatocytes Resist
18. Yekaterina Krutsenko, Investigating the therapeutic Acetaminophen-induced Acute Liver Injury and Drive
efficacy of a novel mTORC1 inhibitor, RMC-6272, on Compensatory Regeneration
liver tumors with b-catenin activation
37. Jiazhen Xu, The study of Circular RNAs and their
19. Ryan LeGraw, Understanding and Engineering Human regulation of TAR DNA binding protein 43 (TDP-43)
Hematopoiesis using a Genetically Engineered Fetal pathological aggregation
Liver Niche
38. Daniel Zuppo, foxm1 is required for cardiomyocyte
20. Mingjun Liu, H3K4me2 regulates perivascular cell proliferation after adult zebrafish cardiac injury
participation in microvascular remodeling in mouse
hindlimb ischemia modelPost-Graduate, Basic Research
39. Chhavi Goel, β-catenin Overexpressing Hepatocytes 42. Sowmya Mekala, Antagonizing activin A/p15INK4b-
Reduce Bile Stasis by Contributing to Cholangiocyte- signaling as therapeutic strategy for liver disease
Like Phenotype in Murine Model of Intrahepatic 43. Vineet Mahajan, Multicellular Human Liver-on-Chip
Cholestasis Microphysiological Platform Predicts Immune Response
40. Katherine Killian, The receptor for advanced glycation to CRISPR-based Gene Editing
endproducts promotes allergen driven neutrophil 44. Ravi Rai, Hepatic stellate cells promote fibrosis
dominant airway inflammation via NLRP3 inflammasome progression by regulating immune cell recruitment to the
activation injured liver
41. Quitterie Larrouture, Osteoblast form an epithelial-like 45. Yuehua Zhu, Role of RUNX1 intragenic rearrangements
layer which play an important role in ion transport in TNBC immune evasion
Post-Graduate, Clinical Research
46. Robert Bubar, Four-year donor center experience with 58. Davsheen Bedi, Genetic and Genomic Landscape of
GCSF and dexamethasone-stimulated granulocyte Myxoid Liposarcoma and their Association with Round
collections Cell Phenotypes
47. Adam Davis, Clinicopathologic Features of IDH2 R172 59. Nathan Cook, Loss of SMARCA2 is an Adverse
Mutated Myeloid Neoplasms Prognostic Finding for Patients with Hepatocellular
48. Mitchell Ellison, Discrepancy Rates Between Histology Carcinoma and Correlates with Early Disease
(HIST) and Molecular Diagnosis (MDX) of T-cell Recurrence After Hepatectomy
Mediated Rejection (TCMR) Depend on Choice of 60. Daniel Geisler, A Prospective Multi-Institutional Study
Bioinformatics Pipeline Reveals the Combination of Next-Generation
49. Mitchell Ellison, Immunologic risk stratification of Sequencing and Cytology Improved the Evaluation of
pediatric heart transplant patients by combining HLA- Pancreatic Cyst Patients
EMMA and PIRCHE-II 61. Daniel Geisler, The Prognostic Significance of the
50. Pooria Khoshnoodi, Clinically Unsuspected Histologic Station 8a Lymph Regional Node in Resectable
Findings in Submitted Total Joint Arthroplasties: An Adenocarcinoma of the Pancreatic Head
Institutional Experience in Quality Assurance and Patient 62. Hae-Sun La, Ductal or Lobular: A Correlation Study of
Care Histomorphology and E-cadherin/p120 Immunoprofile in
51. Jeffrey Kleinberger, Targeted Next-Generation Mixed Ductal and Lobular Carcinoma of Breast
Sequencing of Biliary Tract Specimens from Patients 63. Katherine Killian, A 22-Year Study on Lung Asbestos
with Primary Sclerosing Cholangitis Improves the Early Fiber Burden: Amphibole Asbestos Levels on the
Detection of Cholangiocarcinoma Decline, a Trend Easy to Digest
52. Azfar Neyaz, Loss of PTEN and Expression of GLUT1 64. Aofei Li, Methylomic Profiling of Pediatric Primary
Predict the Metastatic Progression of Pancreatic Malignant Neurocutaneous Melanocytic Neoplasm
Neuroendocrine Tumors Involving Central Nervous System
53. Akram Shalaby, Gastric intestinal metaplasia is a risk 65. Shikha Malhotra, Persistent NUP98 Rearrangement
factor for progression to gastric dysplasia and carcinoma may Portend Poor Prognosis
in patients who undergo gastric biopsy: a population- 66. Simmi Patel, Molecular Characterization of Secretory
based study Myoepithelial Carcinoma (SMCA)
54. Pooja Srivastava, Metastatic Neuroendocrine Tumors 67. Pranav Patwardhan, Iso-chromosome 12p Analysis by
Involving Serous Fluid: A Cytopathologic Review Fluorescent In-Situ Hybridization: An Academic
55. Clayton Wiley, Non-Inflammatory Neuroinflammation Institutional Experience
Associated with Neurodegeneration 68. Pranav Patwardhan, Multi-Institutional Surveys of
56. Michelle Wood-Trageser, Adoption of NanoString Breast Pathologists and Clinicians Reveal Variability on
GeoMx® with nCounter readout to the study of liver: a Pathologic Reporting Practices and Perspectives for
pilot study of differential expression in cirrhotic and Breast Carcinoma Specimens after Neoadjuvant
inflamed liver specimens Chemotherapy (NACT)
57. Fawaz Almutairi, Malignant Germ Cell Tumors: A Large 69. Rayan Rammal, SOX10 can help distinguish triple
Academic Center Experience Of 47 Cases in Men Aged negative breast cancers from gynecologic carcinomas
50 Years and Over 70. Rayan Rammal, Utility of SOX10 in distinguishing
atypical ductal hyperplasia and DCIS from usual ductal
hyperplasiaGraduate, Basic Research
Author: Anu Balogun, BS Poster Number: 1
Contact: olb25@pitt.edu
Mentor: Kari Nejak-Bowen
Co-Authors: Pamela Cornuet, Kari Nejak-Bowen
Beta-catenin inhibition as a novel therapeutic strategy for porphyria
The porphyrias are metabolic disorders caused by enzymatic defects in heme biosynthesis, leading to
excessive accumulation of porphyrins and their precursors. Heme precursors in the various porphyrias
initially accumulate in the liver or bone marrow, the most active tissues in heme biosynthesis. The specific
patterns of accumulation of the heme precursors δ-aminolevulinic acid (ALA) and other intermediate
compounds involved in creating heme define clinical features of these diseases such as acute neurovisceral
attacks, skin lesions, and mental changes. The porphyrias are managed through prevention and palliative
care which focus on alleviating the acute symptoms. These debilitating diseases remain incurable, and
there is an unmet need to develop effective therapies to treat them. Because the liver is either a source or
sink for porphyrins, we investigated the role of Wnt/beta-catenin pathway in this disease. Wnt/beta-catenin
signaling pathway regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural
patterning, and organogenesis during embryonic development through TCF/LEF transcription factors that
mediate induction of target genes. Dysregulation of Wnt/beta-catenin signaling contributes to various human
diseases.
3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) induces hepatic porphyria in mice. DDC diet-induced
porphyria is a model utilized to identify signaling pathways involved in injury progression and testing
potential therapies to treat hepatic porphyrias. The metabolism of DDC creates a potent inhibitor of
ferrochelatase (FC), the terminal enzyme in the heme pathway, which causes the buildup of porphyrin
precursors. These precursors can cause many cellular abnormalities, including oxidative stress,
mitochondrial dysfunction, protein aggregation and inhibition of autophagy. We studied pharmacological
inhibition of Wnt/beta-catenin signaling by utilizing a beta-catenin DsiRNA formulated into a lipid
nanoparticle in mice fed DDC diet.
Our data demonstrates that mice with pharmacologically inhibited beta-catenin have decreased liver injury
due to prominently fewer porphyrin deposits. This is due to suppression of ALA-S and ALA-D enzymes
which catalyze the first and rate limiting steps of heme biosynthesis respectively. Significantly, our data
identified a strong TCF4 binding site in the intron region of the Alad gene, implicating ALA-D as a direct
Wnt/beta-catenin target. Our data, therefore, establishes a novel role of the Wnt/beta-catenin pathway in
regulating heme biosynthesis by inhibiting early steps in heme biosynthesis pathway and reducing toxic
porphyrin accumulation. Furthermore, our data reveal that mice lacking beta-catenin have increased
induction of autophagy over baseline that contributes to the protection from injury by clearing accumulated
toxic porphyrins.
These observations collectively offer a novel opportunity to remedy porphyria by targeting the Wnt/beta-
catenin signaling.Graduate, Basic Research
Author: Maria Beecher, BS Poster Number: 2
Contact: mlb207@pitt.edu
Mentor: Peter Lucas
Co-Authors: Dong Hu, Prasanna Ekambaram, Linda M. McAllister-Lucas, Peter C. Lucas
MALT1 as a mediator of treatment resistance in triple-negative breast cancer
Background: Breast cancer is the most commonly-diagnosed malignancy in American women. The triple-
negative breast cancer (TNBC) subtype has among the worst prognosis due to high rates of recurrence and
metastasis. Unlike estrogen receptor-positive or HER2-positive subtypes, TNBC lacks expression of
targetable receptor proteins. Therefore, treatment relies upon non-specific toxic chemotherapy, which can
become ineffective upon onset of resistance. There is urgent need to identify drivers of resistance to inform
development of new combination therapy regimens that increase response and survival in patients with
TNBC. One potential driver of treatment resistance in TNBC is MALT1, the effector component of the
CARMA-BCL10-MALT1 (CBM) protein signaling complex. This complex mediates activation of NF-kB in
response to stimulation of specific cell-surface receptors. MALT1 performs two functions, serving as a
scaffold to recruit downstream NF-kB signaling machinery and as a protease to cleave and inactivate
specific substrates, several of which inhibit NF-kB. Emerging evidence suggests that unrestrained MALT1
activity underlies the pathogenesis of multiple cancers. As such, many pharmaceutical companies have
developed MALT1 protease inhibitor pipelines, with one compound already in Phase I clinical trial. Notably,
breast cancer cells demonstrate increased sensitivity to chemotherapies such as doxorubicin and cisplatin
when MALT1 is depleted. We hypothesize that MALT1 protease is a key, pharmaceutically-targetable
determinant of TNBC treatment resistance. Mechanistically, we hypothesize that MALT1 drives NF-kB
induced DNA repair and upregulation of pro-survival genes.
Methods and Results: We utilized public databases to identify TNBC cell lines that demonstrate greatest
resistance to doxorubicin. In highly-resistant MDA-MB-231 cells, we find that MALT1 protease is activated in
response to doxorubicin. Using CellTiter Glo and Incucyte Caspase 3/7 assays, we find that MALT1
blockade, either via siRNA-knockdown or pharmaceutical MALT1 protease inhibitor treatment (JNJ-
67856633;Johnson& Johnson or MLT-748;Novartis) results in decreased cell viability and increased
apoptosis in response to doxorubicin.
Conclusions and Future Directions: Initial studies support our hypothesis that targeting MALT1 protease
enhances TNBC sensitivity to chemotherapy. We will also assess the impact of MALT1 blockade on pro-
survival gene expression and DNA repair in doxorubicin-treated TNBC cells. To expand our observations,
we will test additional treatment-resistant TNBC lines and will evaluate response to other treatments,
including cisplatin and radiation. Future studies will examine the impact of MALT1 inhibition on treatment
resistance in murine models of TNBC and in primary patient TNBC organoids. Through these studies, we
hope to elucidate new approaches for improving treatment response and survival in patients with TNBC.Graduate, Basic Research
Author: Rithika Behera, BS Poster Number: 3
Contact: rib35@pitt.edu
Mentor: Robert Lafyatis
Co-Authors: Mengqi Huang, Tracy Tabib, Nina Morse, Robert Lafyatis
Transcriptional regulation of SSc dermal myofibroblasts by FOSL2 and FOXP1
Background: Systemic Sclerosis (SSc) is characterized by fibrosis, vasculopathy, and immune
dysregulation. Skin fibrosis is the hallmark of SSc and is driven by the contractile action of myofibroblasts.
The number of myofibroblasts in the skin correlates with the modified Rodnan skin score, the most widely
used clinical measure of skin severity. Using single cell RNA sequencing, we have identified different
dermal fibroblast populations and shown that SSc dermal myofibroblasts arise in two steps from
SFRP2hi/DPP4 expressing progenitor population. Bioinformatic analyses of the SSc dermal fibroblast
transcriptome implicated the role of transcription factors FOSL2 and FOXP1 in the first and second step of
SSc myofibroblast differentiation respectively. Our aims are to understand the transcriptional regulation of
FOSL2 and FOXP1 in dermal myofibroblast activity and SSc pathogenesis.
Methods: We used si-RNA to knockdown the RNA expression of FOSL2 and FOXP1 in primary dermal
fibroblasts from SSc patients. The perturbed transcriptome, signaling pathways, and epigenetic changes
were characterized using bulk RNA sequencing, Western blotting, and ATAC sequencing.
Results: We found that knocking down FOSL2 RNA using si-RNA led to a reduction in fibrotic genes and
biomarkers for SSc disease progression such as: COL1A1, alpha-SMA, THBS1, PRSS23, THY1, and FN1.
On generating activity modules of the perturbed transcriptome, we found that the genes downregulated by
si-RNA activity had a high expression in the SFRP2hi/DPP4 expressing progenitor population.
Conclusion: Our study provides a novel understanding of the transcriptional and epigenetic regulation of
SSc dermal myofibroblasts by FOSL2 and FOXP1 and provides evidence of their role in the pathogenesis
of SSc. These findings can be beneficial in finding new therapies for targeting myofibroblast activity.Graduate, Basic Research
Author: Hannah Butterfield, BS Poster Number: 4
Contact: HEB62@pitt.edu
Mentor: Peter Lucas, Linda McAllister-Lucas
Co-Authors: Juliana Azambuja, Dong Hu, Saigopalakrishna Yerneni, Linda M. McAllister-Lucas,
Peter C. Lucas
Evaluating small extracellular vesicles as immunomodulatory components of the
microenvironment in p95HER2+ breast cancer
Background: Amplification of the gene encoding the HER2 growth factor receptor occurs in approximately
20% of breast cancers. HER2-targeted therapies, such as monoclonal antibodies trastuzumab and
pertuzumab, have dramatically improved outcomes for this patient population. However, resistance to these
therapies is common, and it is important to develop more nuanced approaches to diagnosis and treatment.
A subset of HER2+ breast cancers express a truncated form of HER2 called “p95HER2.” p95HER2 lacks
the HER2 extracellular domain region targeted by antibody-based therapies, and clinical studies reveal that
a high p95HER2/HER2 ratio predicts poor response to trastuzumab. In contrast, both p95HER2 and FL-
HER2 are susceptible to inhibition by tyrosine kinase inhibitors, which target their shared intracellular
domains. Therefore, evaluating p95HER2 as a potential mediator of treatment resistance in HER2+ breast
cancer is clinically important and actionable.
p95HER2 exerts cellular effects that are distinct from those of full-length (FL) HER2. These effects may
provide additional levels of pathogenicity that go beyond therapy resistance. Expression of p95HER2, but
not FL-HER2, leads to induction of the immune checkpoint ligand PDL1. In an immunocompetent syngeneic
mouse model, tumor cell expression of p95HER2 results in enhanced tumor growth relative to FL-HER2
expression. In contrast, p95HER2 expression does not lead to a growth advantage in an immunodeficient
mouse model. These results suggest a role for p95HER2 in promoting an immunosuppressive tumor
microenvironment (TME). Small extracellular vesicles (sEVs) are known to play a crucial role in tumor-
induced immunosuppression. Here, we evaluate the immunomodulatory effects of sEVs produced from
p95HER2-expressing breast cancer cells. We hypothesize that p95HER2 expression leads to production of
sEVs that create an immunosuppressive TME in HER2+ breast cancer.
Methods and Results: We utilized a mini-size exclusion chromatography (SEC) sEV isolation system and
validated the quality of breast cancer cell sEVs using transmission electron microscopy (TEM) and
nanoparticle tracking analysis (NTA). Using this system, we have identified PDL1 in the sEV-containing
isolates of p95HER2-expressing breast cancer cells.
Future directions: We will analyze the effects of sEVs from p95HER2-expressing breast cancer cells on
tumor growth and tumor immune infiltrate in an immunocompetent mouse model. We will also characterize
the proteomic contents of sEVs from p95HER2-expressing cells using liquid-chromatography/mass-
spectroscopy (LC/MS). Proteomic signatures unique to p95HER2-expressing breast cancer cells may be
evaluated for diagnostic application of sEVs in liquid biopsy.
Conclusions: Our overall goal is to evaluate the immunomodulatory effects and diagnostic utility of sEVs
produced by p95HER2-expressing breast cancer.Graduate, Basic Research
Author: Julianna Castle Poster Number: 5
Contact: jic142@pitt.edu
Mentor: Christi Kolarcik
Co-Authors: Jay Ferrara, David Gau, Amanda Gleixner, Partha Roy, Christi Kolarcik
The Impact of Profilin-1 Mutations on Protein Homeostasis in Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal disease characterized by the dysfunction and
degeneration of motor neurons in the central nervous system. ALS has been linked to a number of genetic
mutations, including point mutations in the actin-binding protein profilin-1 (Pfn1). While the prevalence of
Pfn1 mutations is low, around 1% of inherited forms of the disease, an understanding of their impact on
motor neuron cell death can provide insight into the mechanisms underlying ALS onset and progression. As
Pfn1 is an important regulator of the actin cytoskeleton, we hypothesize that ALS-linked mutations of Pfn1
promote defects in protein homeostasis and synaptic communication. To test this hypothesis, neuronal cells
were transfected with Pfn1 constructs (including wild-type, C71G, and M118V) and subjected to cellular
stressors (including heat shock and sodium arsenite treatments). Immunostaining was then used to
visualize and quantify differences in protein aggregation and stress granule formation. Our results have
established the baseline response with the wild-type construct and indicate that there is an increase in
protein aggregation as well as in the number and size of stress granules with the mutant constructs. Our
ongoing studies are aimed at validating these preliminary results with the goal of offering mechanistic
insight pertaining to Pfn1’s role in ALS disease pathogenesis.Graduate, Basic Research
Author: Margaret Champion, BS Poster Number: 6
Contact: mac575@pitt.edu
Mentor: Arjumand Ghazi
Co-Authors: Francis Amrit, Arjumand Ghazi
Investigating the role of alternative splicing in reproductive aging and maternal-fetal health
Precursor messenger RNA (pre-mRNA) splicing is a fundamental cellular process that generates mature
mRNA for subsequent translation. Alternative splicing, in which non-canonical exons are used to generate
the mature mRNA, allows the generation of diverse functional proteins from a single gene. This process is
essential to maintaining protein diversity. Dysregulation of alternative mRNA splicing is a broad signature of
age, but different organ systems show different patterns of dysregulated splicing. The female reproductive
system is one of the first organ systems to exhibit age-related decline, yet much is not known about the
molecular and genetic mechanisms underlying reproductive ageing. Here we examine the role that the
protein TCER-1, a transcriptional regulator involved in maintaining alternative splicing patterns, plays in
maintaining reproductive health during ageing in the model organism C. elegans.Graduate, Basic Research
Author: Mona Chatrizeh, BS Poster Number: 7
Contact: moc28@pitt.edu
Mentor: Michael J. Morowitz
Co-Authors: Jianmin Tian, Lauren Cass, Brian Firek, Mathew Rogers
Microbiome targeted nutrition to support lymphopoiesis during critical illness
Immune suppression and bone marrow dysfunction are ubiquitous among critically ill patients. Short term,
this places an already vulnerable population at additional risk of life-threatening infections. Long term,
immune suppression can persist in the form of chronic critical illness which significantly worsens functional
outcomes. There is a need to rescue immune function early in the care of critically ill patients to avoid
detrimental short and long term consequences of critical illness. Many studies have attempted to do this but
have generally failed, and targetable therapies are still lacking. This may be in part due to lack of
consideration about the integral role of the gut microbiome in regulating hematopoiesis and immune
function.
Emerging research has identified fundamental links between immunity and the gut microbiome. An
onslaught of publications have shown the microbiome shapes the immune system at various stages,
including during hematopoiesis. Recent murine studies have illustrated antibiotic induced dysbiosis impairs
hematopoiesis and suppresses bone marrow function. Clinically, our group completed some of the first
genomic studies illustrating microbiota derangements in critically ill patients, likely as a result of liberal use
of antibiotics. In addition to antibiotics, most critically ill patients rely on enteral nutrition which shapes their
microbiome. Previously we have shown artificial enteral nutrition (AEN), the default and most commonly
used formula for patients requiring enteral nutrition promotes dysbiosis. In contrast, high fiber plant based
enteral nutrition is well tolerated, promotes the growth of healthy commensal gut anaerobes, and improves
outcomes in murine models. Our unpublished data demonstrates PBEN randomized mice exhibit improved
immune recovery with higher lymphocyte and white blood cell counts following antibiotic induced bone
marrow suppression. We also provide evidence that critically ill patients randomized to PBEN have higher
lymphocyte counts than those that received CEN. These findings highlight nutrition as a previously
underappreciated therapeutic target for improving immune recovery of critically ill patients.Graduate, Basic Research
Author: Grace Conway, BS Poster Number: 8
Contact: gec36@pitt.edu
Mentor: Flordeliza Villanueva
Co-Authors: Anurag N. Paranjape, Xucai Chen, Flordeliza S. Villanueva
Development of an in vitro System to Understand the Mechanisms of Ultrasound-Targeted
Microbubble Cavitation-Mediated Blood Brain Barrier Opening
Background: Ultrasound-targeted microbubble cavitation (UTMC) is being explored as a technique to
transiently open the blood brain barrier (BBB) for the treatment of many neurologic diseases. While UTMC-
mediated BBB opening is an exciting drug delivery strategy, its underlying mechanisms are incompletely
understood. Here, we sought to develop an in vitro model of the BBB that simulates increased endothelial
barrier permeability after UTMC.
Methods: We developed a transwell model with murine brain microvasculature endothelial cells (EC;
bEnd.3) on the abluminal and murine C8-D1A astrocytes on luminal side of a 1.0 um transwell support
membrane. Lipid microbubbles (MB) were added at a 3:1 MB to EC ratio before applying pulsed ultrasound
(1 MHz, 10 µs pulse duration, 10 ms pulse interval) at 250 kPa for 20 s. To determine optimal parameters
for permeability studies, cells were stained with Hoechst 33342 (nucleus), propidium iodide (sonoporation),
CellMask Deep Red (cell coverage), and calcein-AM (viability). Two methods of endothelial barrier function
were assessed: transendothelial electrical resistance (TEER) and permeability using 10 kDa Texas Red
dextran. Live cell imaging was performed using Fluo-4 AM (calcium indicator). Live cell imaging and TEER
studies were performed in media with and without calcium. Differences between 0 and 250 kPa were
compared using unpaired 2-tailed t-test. Significance was defined as pGraduate, Basic Research
Author: Raphael Crum, BS Poster Number: 9
Contact: rjc105@pitt.edu
Mentor: Stephen F. Badylak
Co-Authors: K. Hall, C. P. Molina, G. S. Hussey, E. Graham, H. Li, S. F. Badylak
Matrix Bound Nanovesicles as a Novel Extracellular Matrix Therapy
for the Treatment of Rheumatoid Arthritis
Purpose/Objectives: Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation and
destruction of synovial joints that affects approximately 7.5 million people worldwide. Disease pathology, while
multifactorial in etiology, is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells,
especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory
transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based
immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles
(MBV) embedded within ECM. While it is known that an M1:M2 disequilibrium contributes to RA disease progression,
there are no therapies available that specifically modulate macrophage phenotype to promote disease remission
through an M2, anti-inflammatory phenotype. There is thus a clear unmet need for developing approaches to modulate
rather than suppress the pro-inflammatory immune response for the treatment of autoimmune diseases such as RA.
The evidence supporting ECM- and MBV-mediated immunomodulation of macrophage phenotype, combined with the
clinical evidence of pro-inflammatory M1 macrophages as a key mediator of RA, provides the premise of the present
research. Using the pristane-induced, pre-clinical rat model of RA, it was hypothesized that MBV would reduce
inflammatory arthritis disease development, decrease synovial inflammatory cell infiltration, prevent adverse cartilage
remodeling, modulate synovial and systemic macrophage populations from a pro-inflammatory M1 phenotype towards
an anti-inflammatory M2 phenotype, and thus promote disease resolution.
Methodology: Isolated MBV were delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-
induced arthritis (PIA). The results of MBV administration were compared to those following intraperitoneal (i.p.)
administration of methotrexate (MTX), the clinical standard of care, using disease scoring, microCT imaging,
histopathology, multiplex cytokine analysis, and multi-parameter flow cytometry.
Results: Relative to the vehicle treated animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both
acute and chronic phases of pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint
remodeling, and reduced the ratio of synovial and splenic pro-inflammatory M1 macrophages to anti-inflammatory M2
macrophages (pGraduate, Basic Research
Author: Andrea Cruz, BS Poster Number: 10
Contact: anc232@pitt.edu
Mentor: Sameer Agnihotri
Co-Authors: Abigail Locke, Katherine Halligan, Lauren Sanders, Allison Cheney, Ann-Catherine
Jean Stanton, Robert F. Koncar, Alberto Broniscer, Olena Morozova, Thomas Pearce,
Daniel Marker, Clayton Wiley, Stephen C. Mack, Mariella Filbin, Ian F. Pollack, Sameer
Agnihotri
The Role of Tumor Microenvironment Derived Growth Factors in Pediatric Brain Tumors
Introduction: High-grade gliomas (HGGs) are the most common fatal intrinsic brain tumors in pediatric
patients. H3K27-altered diffuse midline gliomas (H3K27-DMGs), a subgroup of HGGs defined by a histone
3 position 27 alteration, are especially aggressive and result in the poorest patient outcomes. Despite in-
depth genomic characterization, the 5-year survival rate has yet to improve beyond 2% following diagnosis.
A common feature of H3K27-DMGs is infiltration of microglia, macrophages, other myeloid cells, collectively
referred to as GAMs, and a small population of T-cells. The contribution of non-tumor cells in the tumor
microenvironment (TME) can both promote and or inhibit tumor growth, thus representing an opportunity in
the pursuit of novel therapeutics. We recently determined that H3K27-DMG cells stimulate microglial cell
secretion of heparin-binding EGF-like growth factor (HBEGF). HBEGF expression is elevated in many
human cancers and is an important ligand for the epidermal growth factor receptor (EGFR), which is
involved in cell signaling pathways that control cell proliferation and survival.
Hypothesis: Microglial-derived HBEGF binds to and activates EGFR via paracrine signaling, thus promoting
H3K27-DMG tumor cell proliferation and growth.
Methods/Preliminary Results: In preliminary studies, we show that H3K27-DMG cell proliferation is
increased under co-culture conditions with microglia. Moreover, microglial HBEGF blockade attenuates
glioma cell proliferation in vitro.
Relevance: No significant progress has been made in the development of new therapies for PHGGs. This
research uncovers an HBEGF-EGFR axis between GAMs and H3K27-DMGs, respectively, and highlights
an underappreciated role of EGFR signaling in pediatric HGGs.Graduate, Basic Research
Author: Michelle Drewry, BS Poster Number: 11
Contact: mdd64@pitt.edu
Mentor: Fatima Syed-Picard
Co-Authors: Matthew T. Dailey, Kristi Rothermund, Fatima N. Syed-Picard
Scaffold-free conduits formed from dental pulp stem cell sheets provide
neurotrophic and directional support for regenerating axons
Current treatments for peripheral nerve injuries (PNIs) result in slow and inefficient nerve regeneration,
yielding poor clinical outcomes. The delivery of neurotrophic factors (NTFs), growth factors specialized in
supporting axon growth and repair, to sites of PNIs can enhance this regeneration. Moreover, extracellular
matrix (ECM)-scale nanofibers have been shown to orient axon extension towards the end organ, thus
improving functional recovery. The dental pulp is a soft, innervated within the tooth that contains a
population of stem/progenitor cells embryonically derived from the neural crest. These dental pulp cells
(DPCs) express higher levels of NTFs than other mesenchymal stem cells, likely because of their
developmental origin. We have recently shown that DPCs can form scaffold-free cell sheets that act as NTF
delivery vehicles and promote regeneration when wrapped around PNIs in rats. The goal of this study is to
now engineer a bioactive, scaffold-free nerve conduit using DPC sheets with an endogenous, aligned ECM
capable of providing both neurotrophic and guidance cues to regenerating axons. To accomplish this, we
formed DPC sheets on a substrate comprised of linear microgrooves to induce the cells to align and deposit
a linearly aligned ECM. Histological analysis and scanning electron microscopy showed that the resulting
DPC sheets were solid and cellular, and immunofluorescent analysis indicated that these sheets were
composed of an aligned, collagenous ECM. Additionally, the aligned DPC sheets expressed high levels of
NTFs (brain-derived NTF, glial cell-derived NTF, and neurotrophin-3), similar to that of the un-aligned DPC
sheets previously shown capable of inducing axon regeneration. Furthermore, we found that the aligned cell
sheets were able to functionally induce and orient neurite extension in co-cultured neuronal cells in vitro.
The neurites cultured on the aligned cell sheets also contained fewer branches than those on the un-aligned
sheets, further highlighting the potential of the aligned DPC sheets to reduce aberrant reinnervation. We
assembled these bioactive cell sheets into robust, scaffold-free cylindrical constructs approximately 7mm in
length and 500 µm in diameter. Multiphoton and scanning electron microscopies showed that the DPC
sheets maintained their aligned ECM, and thus their ability to orient extending axons, when formed into
conduits. Overall, scaffold-free nerve conduits formed from aligned DPC sheets are capable of enhancing
nerve regeneration by providing both a continuous supply of NTFs, to promote axon regeneration, and
guidance cues from an aligned ECM, to direct axon extension, thus improving the clinical outcomes of PNI
treatment.Graduate, Basic Research
Author: Taylor Gatesman, BS Poster Number: 12
Contact: tag85@pitt.edu
Mentor: Sameer Agnihotri
Co-Authors: Katharine E. Halligan, Matthew E. Halbert, Ann-Catherine J. Stanton, Andrea F. Cruz,
Brian J. Golbourn, Ian F. Pollack, Sameer Agnihotri
Inhibiting insulin signaling reverses resistance to PI3K-mTOR inhibitors
in aggressive pediatric high-grade gliomas
Primary central nervous system (CNS) tumors are now the most common cause of childhood cancer–
related deaths. In particular, pediatric high-grade gliomas (pHGGs) are among the most lethal brain tumors
with patients having a 5-year survival rate of only 20%. In the molecular era, pHGGs, like many cancers,
can be classified into clinically and biologically relevant subgroups. MYCN pHGGs represents one such
fatal subgroup with an unmet need for therapeutics. MYCN belongs to the family of MYC transcription
factors that regulate numerous cancer hallmarks such as proliferation, apoptosis, and metabolism. While no
direct inhibitors of MYCN are in clinical trial, current strategies focus on targeting the MYCN mediated
transcriptional machinery or cell cycle regulators of MYCN. A lack of relevant pHGG models for pre-clinical
testing and understanding the etiology of the disease contribute to limited therapeutic efficacy. To address
these knowledge gaps, our lab developed a novel mouse model of MYCN pHGG using the FLEx-Cre (flip-
excision Cre-recombinase) switch system, whereby neural stem cells are selectively delivered with MYCN
cDNA and shRNA targeting the tumor suppressor genes p53 and Pten and form tumors in vivo. We have
identified that this model harbors hyper-activation of the phosphatidylinositol 3-kinase
(PI3K)/AKT/mammalian target of the rapamycin (mTOR) signalling pathway, a pathway that is of universal
interest in cancer biology. Using this model, we demonstrate that dual PI3K-mTOR blood brain barrier
(BBB) penetrant inhibitors are effective in reducing pHGG growth and MYCN protein levels. However, drug-
resistance is a fundamental feature of pHGGs. This prompted us to develop a novel drug-resistance model
of MYCN pHGG as a mechanistic tool to identify relevant resistance mechanisms to re-acquisition of the
PI3K-AKT-mTOR pathway. Using transcriptome analysis, we identified the insulin growth factor signaling
pathway as our top mechanism of resistance. We hypothesized that MYCN is a critical driver of pHGG and
can be effectively targeted via dual inhibition of the PI3K-mTOR and IGF/Insulin signalling pathways. To test
this hypothesis, we tested next generation inhibitors of the IGF and PI3K-mTOR pathways and performed
genetic and pharmacological assays in our MYCN pHGG gliomas. We also investigated this mechanism in
human MYCN pHGG cells. Inhibition of both pathways in our MYCN pHGG model and human MYCN cells
were synergistic, leading to significant decreases in cell growth and MYCN signaling. In summary, the
generation of novel mouse models of pHGG allow for development of novel treatments and testing of
promising therapies.Graduate, Basic Research
Author: Shohini Ghosh-Choudhary, BS Poster Number: 13
Contact: skg46@pitt.edu
Mentor: Toren Finkel
Co-Authors: Jie Liu, Mhedi Pirooznia, Jon Alder
Whole Genome CRISPR Screen Reveals Vulnerabilities of Senescent Cells
The accumulation of senescent cells with age promotes a pro-inflammatory environment ultimately leading
to a host of age-associated pathologies including neurodegenerative diseases, atherosclerosis,
osteoarthritis and diabetes. The pro-inflammatory components of the senescence associated secretory
phenotype (SASP) also promote a pro-tumor microenvironment. Therefore, there is an established rationale
to develop pharmacologic therapies that specifically kill senescent cells (senolytic therapies). Current
senolytic drugs suffer from a lack of potency and a narrow therapeutic index limiting their clinical utility.
Cellular senescence can be triggered by DNA damage, reactive oxygen species and replicative stress. All
of these triggers of senescence converge on telomeric stress. We propose to identify essential components
of the senescence response to telomeric stress and to evaluate them as candidate targets for senolytic
therapy.
Here, we intend to use a cell-based model of telomere stress-induced senescence (TSIS) that employs an
immortalized cell with a floxed allele of the telomere binding protein, TRF2, along with an estrogen-
responsive Cre-recombinase. These cells undergo rapid and uniform senescence with the addition of 4-
hydroxy-tamoxifen (4-OHT). We have deployed an unbiased whole genome CRISPR-based screen to
reveal the unique genetic vulnerabilities of senescent cells using the concept of synthetic lethality. Following
the completion of the screen and subsequent deep sequencing of the cell populations, we have, through
bioinformatic pathway analysis, determined that senescent cells are particularly vulnerable to perturbations
in ER stress pathways. Specifically, our screen determined that senescent cells, but not their normal cell
isogenic counterparts, uniquely rely on the ER resident proteins PARP16, Sec63 and BiP for survival.
Therefore, we hypothesize that senescent cells are particularly vulnerable to the modulation of ER stress
pathways likely because these cells persist in a metabolically demanding, high secretory state.Graduate, Basic Research
Author: Matthew Halbert, BS Poster Number: 14
Contact: meh237@pitt.edu
Mentor: Sameer Agnihotri
Co-Authors: Brian J. Golbourn, Katharine Halligan, Ann-Catherine Jean Stanton, Abigail L. Locke,
Stephanie M. Casillo, Michelle Wassell, Taylor A. Gatesman, Andrea F. Cruz, Ian F.
Pollack, Sameer Agnihotri
Loss of MAT2A Compromises Methionine Metabolism and Represents a Vulnerability
in H3K27M Mutant Glioma by Modulating the Epigenome
H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health
Organization. DMGs are inoperable, chemo and radio-resistant. Median survival ranges from 8-11 months,
with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and
transcriptional reprogramming driven by global loss of H3K27 trimethylation (H3K27me3). H3K27me3 is a
potent negative regulator of transcription. Loss of H3K27me3 is an initiating event in glioma-oncogenesis.
This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we
developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach
identified a reliance of H3K27M but not isogenic controls to the amino acid methionine and the enzyme
Methionine Adenosyltransferase 2A (MAT2A), a key enzyme in one-carbon metabolism. Methionine-
dependent cancers rely on methionine to sustain polyamine synthesis, purine/pyrimidine metabolism,
transsulfuration signaling, redox balance, and folate metabolism. MAT2A is a central regulator of one-
carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl donor
for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these
findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation,
genetic/pharmacological, would alter DMG viability by disrupting the methylation landscape. The current
MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a
synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M-mutant cells are sensitive to
MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1)
overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein
levels, suggesting MAT2A could be exploited by further inhibition of residual MAT2A. Genetic and
pharmacological aberrations to MAT2A resulted in reduced proliferation and cell cycle. We observed that
loss of MAT2A or methionine restriction (MR) resulted in global depletion of H3K36me3, a transcriptional
super-enhancer. Parallel H3K36me3 ChIP and RNA-seq identified loss of oncogenic and developmental
transcriptional programs associated with MAT2A loss. Moreover, multiple MAT2A and MR phenotypes
could be rescued with SAM. In vivo syngeneic and patient-derived xenograft models (PDXs) with both
inducible MAT2A knockdown or MR diets showed extended survival. These results suggest novel
interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence
that MAT2A, a central regulator of one-carbon metabolism, presents exploitable therapeutic vulnerabilities
in histone mutant gliomas. Moreover, our model can be further utilized as a discovery tool or validation of
promising pre-clinical agents.Graduate, Basic Research
Author: Shea Heilman, BS Poster Number: 15
Contact: shh136@pitt.edu
Mentor: Jeff Gross
Co-Authors: Gross JM
Uncovering Differentiation-Inhibitory Mechanisms in the Developing
tet2-/-;tet3-/- Zebrafish Retina
TET-mediated DNA hydroxymethylation (5hmC) is an epigenetic modification that broadly impacts gene
expression, and is known to regulate terminal differentiation in many developmental contexts. Our lab
recently showed that tet2-/-;tet3-/- zebrafish show global 5hmC loss and impaired differentiation of retinal
neurons and glia. While this and other studies demonstrate the need for 5hmC in development,
differentiation-inhibitory mechanisms are still unknown. To answer this question, we perform scRNA-seq on
sibling control and tet2-/-;tet3-/- zebrafish retinas at several timepoints spanning retinal development in
order to (1) reveal cell type-specific, TET-responsive changes in differentiation-associated gene expression
events, and (2) develop a high-resolution map of putative differentiation-inhibitory signals and their cellular
sources. Here, we highlight the work completed thus far to generate this comprehensive multi-timepoint
dataset. This includes generation and preliminary analysis of several late-timepoint datasets, and
optimization of F0 CRISPR gRNA injections as a tool for tet2-/-;tet3-/- embryo selection and analysis at
early timepoints. When completed, these studies will reveal 5hmC-responsive, cell population-specific
targets that are required in retinal development.Graduate, Basic Research
Author: Richard Herron, BS Poster Number: 16
Contact: rsh35@pitt.edu
Mentor: Hun-Way Hwang
Co-Authors: Alexander K. Kunisky, Jessica R. Madden, Hun-Way Hwang
Identification of a direct role of CPSF6 and a key cis-acting motif in
promoting TRIM9 proximal polyA site usage
Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3’
termini, is a rapidly expanding area of research that in recent studies has been linked to the mechanistic
target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism.
Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling
and APA remains poorly understood. We previously leveraged our cTag-PAPERCLIP technique to generate
a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin
ligase with a role in neurodevelopment, as a gene of interest. In this study, we characterized the regulation
of the mTOR-induced TRIM9 APA shift to establish human relevance. Additional study of the regulation of
TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed
CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms,
with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Study into the 3’UTR sequence
elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the
CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence
elements essential for CPSF6-mediated regulation of the proximal TRIM9 PAS, we developed a RT-qPCR
PAS competition assay to quantify sequence-mediated usage of PASs. Utilizing this assay, we assessed
usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we
generated constructs containing mutated UGUA sequences in order to ascertain the importance of the
UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the
TRIM9 proximal PAS (TRIM9S). Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a
reduction in TRIM9S PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis-
acting motif in promoting TRIM9S PAS usage. Furthermore, our results also suggest a possible link
between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and
neurodevelopment through TRIM9.Graduate, Basic Research
Author: Joshua Hislop, BS Poster Number: 17
Contact: joshua.hislop@pitt.edu
Mentor: Mo Ebrahimkhani
Co-Authors: Amir Alavi, Jeremy Velazquez, Ryan LeGraw, Tahere Mokhtari, Matthew Rytel, Donna
Stolz, Susana M Chuva de Sousa Lopes, Samira Kiani, Berna Sozen, Ziv Bar-Joseph,
Mo R. Ebrahimkhani
Synthetic Morphogenesis of Human Blastocyst-to-Gastrula Bilaminar Disc
Implantation of human embryo commences a critical developmental stage that comprises profound
morphogenetic alteration of embryonic and extraembryonic tissues, axis formation and gastrulation events.
Our mechanistic knowledge of this window of human life remains limited due to restricted access to natural
healthy samples for both technical and ethical reasons. Here we describe a system that employs human
induced pluripotent stem cells engineered with an inducible GATA6 gene circuit to demonstrate that GATA6
alone is sufficient to drive genetic and spatial symmetry breaking into tissues with single-cell transcriptional
profiles similar to post-implantation embryonic and extraembryonic lineages. We describe the self-
organization of these populations into epiblast-like wild-type compartments surrounded by a GATA6+
endoderm-like cellular assemblies. They exhibit self-organization and tissue boundary formation that
recapitulates yolk sac-like tissue specification, the formation of bilaminar disc-like embryonic morphology,
and the development of the amniotic-like cavity. This approach provides a simple platform for studying peri-
implantation embryonic fate decisions and for the exploration of synthetic developmental engineering using
a small number of genetically encoded core control factors.You can also read
