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Dana-Farber/Harvard Cancer Center

CURE Continuing Umbrella of Research Experiences

Scientifi c Presentations Summer 2015

For more information about the IECD or CURE program, contact:

Karen Burns White, Deputy Associate DirectorInitiative to Eliminate Cancer DisparitiesP: (617) 632-3244, E: [email protected]

Ying Jiang, CURE Research Training Specialist P: (617) 632-3028, E: [email protected]

Dana-Farber/Harvard Cancer Center Continuing Umbrella of Research Experiences (CURE)

Launched in 2002, the Continuing Umbrella of Research Experiences (CURE) at Dana-Farber/Harvard Cancer Center (DF/HCC) is an important building block in research training initiatives. Under the direction of the DF/HCC Initiative to Eliminate Cancer Disparities(IECD), this program is designed to provide underrepresented minority high school and college students with a stimulating and rewarding hands-on research experience that encourages students to pursue education and training in the biomedical sciences and careers in basic, clinical, nursing, and population sciences, cancer research.

Dana-Farber/Harvard Cancer Center Initiative to Eliminate Cancer Disparities (IECD)

Th e IECD provides a centralized and coordinated structure for addressing cancer disparities. It was among the nation’s fi rst integrated, inter-institutional, multi-pronged approach for addressing cancer inequities. Its mission is to support and encourage disparities research in all disciplines and across all DF/HCC member institutions. Th e IECD focuses on four key areas: a) community engagement, b) training, recruitment and faculty development, c) increasing minority enrollment to cancer clinical trials, and d) enhancing cultural competency throughout DF/HCC institutions and membership.

CURE Scientifi c Presentations

Oral PresentationsTuesday, August 11, 2015 and Wednesday, August 12, 2015

2:30P.M.-5:00 P.M.

Digital Poster PresentationsThursday, August 13, 2015

3:30P.M.-5:00 P.M.

Dana-Farber Cancer Institute450 Brookline Avenue, Third Floor

Yawkey Conference Center

Boston, MA 02215

# Indicates students presenting a poster

Usman Ali Ahmed 1Detecting Binary Protein-Protein Interactions Using

the Yeast Two-Hybrid Assay

Kate Armstrong 2Examining Oncogenic Transformation of Transfected

FLT3 Gene Isoform in Hek293T Cell Line

Omar Badr 3Involvement of Signal Transduction Molecules in

Cardiomyocyte (CM) Proliferation

Katisha Bellegarde 4The Effect of Cyclin Dependent Kinases on the

Expression and Function of Enhancer of Zest

Homolog 2 (EZH2) and its Implications on Foxp3

expression

Roderick Brathwaite Jr. # 5Weight Loss in Overweight and Obese

Postmenopausal Women Reduces Breast Cancer

Risk

Kaelyn Brown # 6Investigating the Role of NRDE-2 in Non-Small Cell

Lung Cancer

Piseth Cheav 7Investigating the Reproducibility of Tumor

Measurements Between Different Image Analysts

Daniella Colombo 8BPA-Induced Stress Granules: Characterizing the

Cellular Stress Response

Eliezer Colon 9Eicosapentaenoic Acid Induces Survival of Human

Skin Fibroblasts Exposed to Ultraviolet Light

Kenesha Darlington # 10Analysis of Intestinal Pro- and Anti-infl ammatory

Transcription Factor Expression after Sleeve

Gastrectomy

Ilhan Esse # 11Library Construction Effi ciency in KAPA Library

Preparation and KAPA Hyper Prep Kits

Rachel C. Ezieme 12Understanding Barriers to Cancer Care Among

Black Bostonians

Armel Foade 13Towards Nanoparticle Drones: New Technology

to Kill Deadly Prostate Cancer Cells with Minimal

Collateral Damage

Nora Fortoul 14Patient Ancestry May be a Determinant

in Hematopoietic Stem Cell Transplantation

Outcomes

Ana Paola Garcia # 15The Interplay between Disrupted Peripheral

Circadian Rhythmicity and Cancer Cachexia

Heresa Guerrier # 16A Comparison of Two CRISPR-Cas9-based

Methods for Transcriptional Activation in Drosophila

Soham Gupta 17Reduction in Prostate Specifi c Antigen Screening

Following Contrary Recommendation

Adnil Gutarra # 18Cancer Genome Atlas Gene Mutation: Detection of

Co-occurring Variants and Network Community

Ted Hilaire # 19Angiogenic Properties of Breast Cancer Cell Lines

Lisa Hsiao 20Müllerian Inhibiting Substance Inhibits Growth of

Ovarian Cancer Cells In Vitro

Alexander R. Jeremiah # 21Epidermal Growth Factor Receptor Inhibition for

Targeted Neurofi bromatosis Therapies

Thamael Laurore # 22The Use of Functional Magnetic Resonance

Imaging (fMRI) to Prevent Post Operative Defi cits

Table of Contents

Nicholas F. Lawrence 23Id1 and p53 Expression and G691S RET

Polymorphism in Desmoplastic Melanoma: An

Analysis of Sixty Six Cases

Amy Luong 24Correlating Distances between Prostate

Biopsy Core and Cancer Lesion to Respective

Metabolomic Profi les

Khang Nguyen # 25Defective NHEJ Leads to Deregulated PI3-K

Pathway in Glioblastoma Multiforme Mice Model

Habiba Noamany 26Structural Study of Actin Nucleating Formins using

X-Ray Crystallography

Miracle Onyeoziri # 27Drugs (263,1781) Inhibit the Hypoxia Induced Cell

Death

Ambar Piña 28Optimization of Cell Migration Assay to Assess the

Role of CMG-2 in Endothelial Cell Migration

Brenda Romero # 29DREAM Complex Maintenance in Quiescence is

Regulated by CDK Phosphorylation

Naria Sealy 30Questionnaire Reliability in the Boston

Mammography Cohort Study

Adam Stevens 31Exploring Blood Platelets as a Liquid Biopsy

for Tumor-Derived Mutant RNA in Patients with

Glioblastoma

Mariano R. Suriel 32A Phase I/Ib Study of Ipilimumab in Patients

with Relapsed Hematologic Malignancies after

Allogeneic Hematopoietic Cell Transplantation

Judene Thomas # 33Numb Isoform Expression During MEL (Murine

Erythroleukemia) DMSO (Dimethyl sulfoxide)

-induced Differentiation

Emilio Vides-Curnen # 34Red Blood Cell Antigen Prediction Using Whole

Genome Sequencing (WGS) Data

Carolina Villalba 35Neuropilin 2 Infl uences Angiogenesis in Breast

Cancer

Katherina Yeager # 36BRG/BRM Assay Development Pipeline

1

Detecting Binary Protein-Protein Interactions Using the Yeast Two-Hybrid Assay

Usman Ali Ahmed

Principal Investigator: Marc Vidal, PhD

Mentors: Tiziana Cafarelli, PhD; Alice Desbuleux

Dana-Farber Cancer Institute

Macromolecules are involved in almost every function of a cell and tend to interact with each other in networks. Studying such macromolecular networks can help us understand genotype-phenotype relationships in living organisms. Perturbations occurring within these macromolecular networks can lead to complex disease phenotypes such as cancer. In order to improve our understanding of the relationships between macromolecules, specifi cally proteins, the Center for Cancer Systems Biology systematically maps complete and high-quality protein-protein interactions called “interactomes,” of various model organisms. Due to its evolutionarily well conserved pathways and easily manipulable genetics, we are currently mapping the interactome of the simple model eukaryote, Saccharomyces cerevisiae using the Yeast Two-Hybrid (Y2H) assay. In this assay, proteins of interest X and Y, are respectively fused to the DNA binding domain (DB) and activation domain (AD) of the transcription factor, Gal4. If an interaction occurs between these proteins, Gal4 is reconstituted, which then activates a reporter gene, allowing the yeast cells containing the interacting proteins to grow on selective media. Our pipeline screens ~6,000 AD prey proteins against ~6,000 DB bait proteins which covers a subset of the entire interactome. In our latest screen of a subset of bait and prey protein, we identifi ed a total of 1,263 possible interactions; these will later be verifi ed by a pairwise Y2H assay and subsequently validated with other orthogonal assays. Generating a high-quality, binary Saccharomyces cerevisiae interactome map will provide a deeper understanding of protein networks and through the study of their perturbations, insight into genetic variation, including disease.

2

Examining Oncogenic Transformation of Transfected FLT3 Gene Isoform in Hek293T Cell Line

Kate Armstrong

Principal Investigator: James D Griffi n, MD

Mentor: Sophia Adamia, PhD


Introduction: Acute myeloid leukemia (AML) is the most prevalent leukemia in adults. AML begins in blood stem cells in the bone marrow but is quick to spread, resulting in a low survival rate. Th ese outcomes have initiated the search for novel biomarkers and drug targets for selective therapy. Previously in our laboratory, genetic alterations were identifi ed in the FLT3 gene; this gene is known to be involved in hematopoesis and cell cycle regulation and we are currently testing the biological eff ects of these alterations in a cell line model system. We engineered the FLT3 gene transfected into HEK293T cells and are examining if the genetically altered FLT3 gene is transforming and has oncogenic characteristics.

Study Hypothesis: It was hypothesized that because of genetic alterations detected on the FLT3 gene, the novel form of this gene would encode a novel form of FLT3 protein with oncogeneic potential.

Methods: HEK293T cells were cultured and split prior to transfection. Previously generated plasmids containing the FLT3 gene isoform were transfected into the HEK293T cells using Lipofectamine 2000 Reagent. Polymerase chain reaction (PCR) and western blotting were conducted, and gel electrophoresis was performed to evaluate successful gene delivery.

Results: Our Real-Time PCR (RT-PCR) and western blotting analyses demonstrate that novel transcripts of FLT3 were successfully expressed in the HEK293T cells. We are currently monitoring biological eff ects of novel FLT3 and its transforming potential as demonstrated by rolling up and forming colonies.

Conclusion: Positive PCR results suggest that the FLT3 isoform is present in the nucleus. Th e presence of FLT3 protein suggests that the FLT3 transcripts encode protein and it is expressed in HEK293T cells. If these cells display oncogenic transformation, this isoform can be used as a novel biomarker and possible drug target for patients with AML.

3

Involvement of Signal Transduction Molecules in Cardiomyocyte (CM) Proliferation

Omar Badr

Principal Investigator: Sangita Choudhury, PhD

Boston Children’s Hospital

Background: Congenital anomalies of the cardiovascular system are the leading cause of morbidity and mortality in infancy and childhood. Congenital heart disease has an incidence rate between 0.4% and 1% among live-born babies. Many patients with congenital heart disease have associated heart failure at birth or develop heart failure as a complication, for which no eff ective therapies exist except for heart transplantation. It has been shown that CMs, the hearts muscle cells, proliferate in the fi rst weeks and in humans in the fi rst two decades of life. Understanding the molecular mechanism of CM proliferation will help us to identify specifi c factors to induce CM proliferation with the aim of enhancing cardiac regeneration. In this study we evaluate the involvement of signal transduction molecules in diff erent developmental time points.

Objective: Advance the mechanistic understanding of CM proliferation with the long-term goal of stimulating cardiac regeneration in pediatric patients.

Methods: Fetal, neonatal and adult cardiomyocytes were isolated with enzymatic dissociation. Proliferation capacity was evaluated with mitotic marker phospho histone H3. Expressions of diff erent signal transduction molecules were evaluated with western blot as well as immunostaining.

Findings and conclusions: ERK1/2 and AKT expression decreased in adult heart, where CM proliferation capacity is very low. Erk1/2 and AKT might play an important role in CM proliferation.

4

The Effect of Cyclin Dependent Kinases on the Expression and Function of Enhancer of Zest Homolog 2 (EZH2) and its Implications on Foxp3 expression

Katisha Bellegarde

Principal Investigator: Vassiliki A. Boussiotis MD, PhD

Mentor: Kankana Bardhan, PhD

Beth Israel Deaconess Medical Center

Allogeneic hematopoietic stem cell transplantation (HSCT) is a life-saving therapy for patients with leukemia. Graft versus host disease (GvHD) is a complication which compromises the curative outcome of HSCT. GvHD is caused by donor T cells, which recognize the recipient’s antigens, become eff ectors and damage host tissues. Th e cyclin dependent kinase 2 (Cdk2) enables the re-entry of T lymphocytes to the cell cycle but is not crucial for the survival of lymphocytes, hematopoiesis or thymocyte development. Based on this, the Cdk2 inhibitor (Roscovitine) was tested as a potential treatment of GvHD. Roscovitine decreased proliferation of T cells and induced expression of Foxp3 in a signifi cant T cell subset. Coherent with the expression of Foxp3 there was decrease in the expression and phosphorylation of the enhancer of zest homolog 2 (EZH2), a polycomb protein responsible for H3 lysine 27 trimethylation (H3K4me3), which induces gene silencing. Cdk2 phosphorylates EZH2 at Th r350 and without that phosphorylation the methyltransferase activity of EZH2 is halted. EZH2 is a critical regulator of FOX transcription factors, but its involvement in the expression of Foxp3 has not been determined. In this study we are evaluating the expression of EZH2 and Foxp3 and the interaction of EZH2 with the Foxp3 promoter by chromatin immunoprecipitation using primary human T cells. In the presence of a Cdk2 inhibitor we anticipate a decrease of EZH2 expression and phosphorylation, an increase of Foxp3 expression, and a diminished association of EZH2 with the Foxp3 gene promoter. We are also evaluating the eff ects of Cdk4 or Cdk6 inhibitors. Because Th r350 of EZH2 is uniquely phosphorylated by Cdk2, we anticipate that these inhibitors will not aff ect EZH2 activity and Foxp3 expression. We expect that this study will aid in a better understanding of the mechanisms necessary to prevent GvHD, allowing for the development of more eff ective treatments.

5

Weight Loss in Overweight and Obese Postmenopausal Women Reduces Breast Cancer Risk

Roderick Brathwaite Jr. #

Principal Investigator: Stephanie Smith-Warner, PhD

Mentors: Shiaw-Shyuan Yaun, MPH; Fred Tabung, PhD, MSPH; Tao Hou, MPH;

Emilie Zoltick, MS

Harvard T.H. Chan School of Public Health

Being overweight (body mass index [BMI] = 25-29 kg/m2) or obese (BMI ≥30 kg/m2) opens the door to a higher risk of contracting numerous health problems including diabetes, heart disease, and respiratory diseases. Being overweight or obese also increases breast cancer risk in postmenopausal women. However, few studies have evaluated if overweight and obese postmenopausal women could lower their breast cancer risk by losing weight. We analyzed the primary data in fi ve studies from the Pooling Project of Prospective Studies of Diet and Cancer (DCPP), an international consortium, to examine the association between sustained weight loss and breast cancer risk. Th ese analyses were conducted among women who: (1) were at least 40 years old or postmenopausal at study enrollment, (2) had a BMI ≥25 kg/m2 at enrollment, and (3) had body weight measures 5-6 and 9-11 years after enrollment. Th e analyses included 71,073 women among whom 3,048 developed breast cancer following the 9-11 year body weight assessment. Th e Cox Proportional Hazards Model was used to calculate study-specifi c relative risks with 95% confi dence intervals (CI) which were pooled using a mixed-eff ects model. Preliminary results show that 18,952 (27%) women lost weight between enrollment and 5-6 years later; of these, 9,982 maintained their weight loss at the 9-11 year assessment. Postmenopausal weight loss in overweight women reduced breast cancer risk. Women with sustained weight loss (n=348 cases) had a 16% (95% CI 2%-28%) lower risk of breast cancer compared to those who maintained their weight (n=357 cases; p-value, test for between studies heterogeneity = 0.85). Th ese fi ndings identify a modifi able lifestyle behavior that postmenopausal women could adopt to potentially reduce their breast cancer risk. Future analyses will incorporate the data from seven additional studies in DCPP and will examine whether these results vary by menopausal therapy, age, and smoking status.

6

Investigating the Role of NRDE-2 in Non-Small Cell Lung Cancer

Kaelyn Brown #

Principal Investigator: Frank Slack, PhD

Mentors: Jeff Haswell; Minlee Kim, PhD


Th e discovery of microRNAs (miRNAs) helps elucidate how cells control expression of diff erent genes. miRNAs are a class of small noncoding RNA, ranging from 20-25 nucleotides in size and are translation inhibitors, preventing messenger RNA (mRNA) from translating into protein. Cells use miRNAs as regulators, preventing the overexpression of certain genes. Over 60% of human protein-coding genes are thought to have miRNA-binding sites. Th erefore, fl aws in the miRNA silencing process are expected to hold a prominent role in the development of cancer. Cancer can develop if miRNAs fail to repress translation of oncogenes or if miRNAs extensively target tumor suppressor genes. Non-small cell lung cancer (NSCLC) composes 85-90% of lung cancer cases. In NSCLC, certain families of miRNAs target tumor suppressor genes. NRDE-2 is a gene believed to assist in miRNA-mediated silencing of gene transcription in the nucleus. Th us, we hypothesized that with the absence of NRDE-2, NSCLC cell lines would increase the expression of tumor suppressor genes and eventually lead to the death of the cancer cell. To test this, we grew six NSCLC cell lines along with two control lines (HeLa and 293-T), and transfected each with various amounts of small interfering RNA (siRNA) to inhibit NRDE-2 protein translation. We plan to perform reverse transcription quantitative polymerase chain reaction (RT-qPCR) to identify the amount of NRDE-2 present in the cells after treatment.

7

Investigating the Reproducibility of Tumor Measurements Between Different Image Analysts

Piseth Cheav

Principal Investigator: Annick Van den Abbeele, MD

Mentor: Keisha McCall, PhD


Functional imaging with ¬18FDG-PET/CT allows identifi cation of tumors and quantitative measurement of tumor metabolism due to the abnormally high glucose metabolism dependency of malignant cancer cells. Quantitative evaluation of 18FDG-PET/CT, using maximum and peak Standardized Uptake Values (SUV), is routinely used to assess tumor glucose metabolism in patients with a variety of cancers. Consistency in tumor measurements is important for radiologists and oncologists to determine the eff ectiveness of a cancer therapy on patients. A small previous study found variations in SUV when measurements were performed by diff erent image analysts (readers). Th e purpose of this project was to determine the reproducibility of the quantitative evaluation of tumor glucose metabolism on 18FDG-PET/CT between two diff erent readers.

Two readers made SUV measurements in 32 patients with gastrointestinal stromal tumors (GIST) at four time points: before (baseline), and after the fi rst, second, and fourth cycle of therapy. Th e percent change in SUV measurements during therapy relative to baseline was used to categorize metabolic response into three categories: partial metabolic response (PMR) if tumor SUV decreased below threshold, progressive metabolic disease (PMD) if SUV increased and/or if new lesions developed, or stable metabolic disease (SMD) if SUV neither increased nor decreased. Th e SUV, metabolic response, and survival curves were compared between the readers and variations tested for statistical signifi cance.

Linear regression analysis found signifi cant correlation between both readers (p<0.0001) for SUV and percent change in SUV. Bland Altman plots showed the reproducibility coeffi cient (95% CI) was 23% change in SUV. Response categories agreed between the readers (Fleiss’ Kappa = 0.86, p<0.05). Progression free survival and overall survival curves showed no signifi cant diff erence between the two readers (p > 0.05).

Tumor measurements obtained from diff erent image analysts did not yield signifi cant variations. 18FDG-PET/CT quantitative evaluations of GIST tumors glucose metabolism and metabolic response to therapy measurements were reproducible using SUV.

8

BPA-Induced Stress Granules: Characterizing the Cellular Stress Response

Daniella Colombo

Principal Investigators: Paul J. Anderson, MD, PhD; Pavel Ivanov, PhD

Mentor: Marta Fay, PhD

Brigham and Women’s Hospital

Use of water bottles and sales receipts are common avenues of human exposure to Bisphenol-A (BPA), a synthetic compound prevalently used to produce common materials such as polycarbonate plastics and thermal paper. BPA is an estrogen-mimic, endocrine disruptor, and neurotoxicant associated with various adverse health outcomes. Previous research has linked BPA exposure to hormonal, reproductive, neurodevelopmental, cardiovascular, and endocrine eff ects. Here, we investigated the link between BPA stress and translational arrest.

Stress granules are cytoplasmic aggregations assembled in response to stress-induced translational arrest often caused by phosphorylation of eukaryotic initiation factor alpha (eIF2α), an essential component of translation initiation. Th us, SGs act as transient signaling stations composed of translationally-stalled mRNA and associated proteins during translation initiation. We studied the eff ect BPA has on the cellular stress response, and whether the cytoplasmic aggregations observed to form upon exposure were SGs by characterizing their composition and kinetics.

We used immunofl uorescence microscopy to visualize granules composed of classical SG markers in the cytoplasm of U2OS cells upon BPA stress. We established that the SGs were canonical as eIF2α is required for SG formation. Stressing cells with BPA followed by cycloheximide treatment, an inhibitor of translation, prevented SGs from reforming. Th is indicated that inhibition at the translation initiation stage is required for SG assembly. In another experiment, we removed the BPA stress and found that the cells recovered a characteristic of SGs.

Th e results of these experiments allowed us to characterize how BPA inhibits translation by analyzing the composition and kinetics of the BPA-induced SGs. We were able to map out a piece of the BPA stress regulated pathway and concluded that BPA exposure induces classical SGs. Identifying the mechanism by which BPA has an eff ect on translational arrest provides a foundation for future studies which investigate implications on physiological processes.

9

Eicosapentaenoic Acid Induces Survival of Human Skin Fibroblasts Exposed to Ultraviolet Light

Eliezer Colon

Principal Investigator: Jose A. Halperin, MD

Mentors: Rupam Sahoo, PhD; Pamela Ghosh, PhD


Translation Initiation, the fi rst step in mRNA translation, plays a critical role in the regulation of cell growth and malignant transformation. Th e Halperin lab discovered several small molecules that induce phosphorylation of eukaryotic Initiation Factor 2α (eIF2α), inhibit translation initiation and exert anti-cancer activity both in vitro and in vivo. Th is is because phosphorylation of eIF2α preferentially aff ects the translation of mRNAs with complex 5’ un-translated regions (5’UTR), which mostly encode for oncogenic proteins such as Cyclin D1. Interestingly, phosphorylation of eIF2α paradoxically increases the translation of a subset of mRNAs such as activating transcription factor 4 (ATF4), a key regulator of genes essential for adaptive functions in response to cellular stressors. Ultraviolet light (UV) is an environmental stressor especially for skin cells. A tightly regulated balance between anti- and pro-apoptotic pathways determines the fate of UV-irradiated cells and causes diff erential cellular response. Preliminary evidence from our lab suggests that small molecules that induce phosphorylation of eIF2α may exert a protective eff ect against UV radiation. Eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid abundant in marine fi sh oil that induces eIF2α phosphorylation, as reported by the Halperin lab. Th e aim of the present study was to investigate whether EPA would protect human skin fi broblast from UV radiation stress. Human foreskin fi broblasts were grown and exposed to UV light in the presence or absence of EPA, and cell survival was quantifi ed with a colorimetric assay. My preliminary results suggest that EPA induces a dose response protection (increased cell survival) of skin fi broblast cells. Future work will aim at confi rming these fi ndings and, potentially, identifying the mechanism(s) by which EPA protects skin fi broblast from UV-stress. It will be interesting to investigate if this observation can be extended to other cell types and stressors including, for example, cardiac cells and hypoxia.

10

Analysis of Intestinal Pro- and Anti-infl ammatory Transcription Factor Expression after Sleeve Gastrectomy

Kenesha Darlington #

Principal Investigators: Ali Tavakkoli, MD; Eric Sheu, MD, PhD; Stanley Ashley, MD

Mentors: Eleanor Rudge, MD; Tara Deelman, MD


Weight loss surgery is the gold standard treatment for obesity and associated Type 2 Diabetes Mellitus (T2DM), with improved glycemic control often seen within days of operation. Sleeve gastrectomy is one such weight loss operation which has been shown to provide these benefi cial eff ects. Prior research has shown infl ammation in adipose tissue contributes to insulin resistance, and that the transcription factors T-bet and Foxp3, which are pro- and anti-infl ammatory, respectively, are critical mediators of this process. We hypothesized that changes in T-bet and Foxp3 levels are important in the anti-diabetic eff ects of sleeve gastrectomy. To test this hypothesis, we planned to compare intestinal T-bet and Foxp3 levels in SD rats that had undergone either sleeve gastrectomy or control laparotomy. Small intestinal tissue samples were harvested from each group of rats. mRNA of adequate quality was isolated and converted into cDNA. RT-PCR was used to analyze the expression of T-bet and FOXP3 accordingly. Th e study compared each sample of T-bet/FOXP3 to standard Beta-actin gene, which was used as a loading control for gel electrophoresis. Th is work will allow us to determine the impact of sleeve gastrectomy on mRNA expression of T-bet and its antithesis FOXP3. We anticipate that T-bet expression will be greater in the control sham-laparotomy rats and less in the sleeve gastrectomy rats. Th e data will serve as a reference for understanding the importance of intestinal T cell subsets in the remission of Type 2 Diabetes after sleeve gastrectomy.

11

Library Construction Effi ciency in KAPA Library Preparation and KAPA Hyper Prep Kits

Ilhan Esse #

Principal Investigator: Laura Macconaill, PhD

Mentors: Ling Lin, PhD; Andrea Clapp; Paul Van Hummelen, PhD; William Hahn, MD, PhD; Matthew Meyerson, MD, PhD


Th e recent application of Next-Generation Sequencing (NGS) in clinical and basic science research coupled with increasing sequencing capability and availability has resulted in a high demand for more effi cient and cost-eff ective NGS library construction. Currently, the KAPA Library Preparation protocol is a common, traditional kit used in various research settings that produces high library yields. However, this protocol requires large amounts of DNA input and is more sensitive to the quality of DNA. Recently, Kapa Biosystems developed a simpler and faster method, the KAPA Hyper Prep Kit, that is specially formulated for low-input, challenging samples. In order to compare the effi ciency of newly developed and traditional library construction, libraries were prepared from CEPH, cell-line samples of variable quality using the KAPA Library Preparation Kit and the newly designed KAPA Hyper Prep Kit. Typically, KAPA Library requires at least 100 ng of CEPH DNA to be successful, and our experiment aims to confi rm whether the KAPA Hyper method can be as effi cient with signifi cantly lower DNA input. Library triplicates of each sample size were created with both protocols using diff erent barcodes. Th e samples were captured with a subset of interested genes and sequenced using an Illumina MiSeq sequencer. Our fi ndings deepen our understanding of Next-Generation Sequencing and will contribute to the advancement of more eff ective sequencing methods.

12

Understanding Barriers to Cancer Care Among Black Bostonians

Rachel C. Ezieme

Principal Investigator: Karen M. Winkfi eld MD, PhD

Mentors: Elizabeth Powell, MPH; Elmer Freeman MSW; Linda Sprague Martinez, PhD

Massachusetts General Hospital

Objective: Black residents in Boston experience health disparities across many health conditions, including cancer. Community practices, educational levels and perception of health and health care have a large impact on patient decisions and may infl uence health outcomes. To assess how information about cancer and cancer care is obtained and perceived among Black Bostonians, a community needs assessments was performed with the goal of identifying strategies that could be implemented to help improve.

Methods: A mixed method approach was implemented. Data from key informant interviews with patients, health care providers, patient advocates, and community organizers was used to develop baseline questions for this community assessment. Nine focus groups were conducted in three Boston neighborhoods (Roxbury, Dorchester and Mattapan) with residents who self-identifi ed as Black. Th e focus groups were audio taped and transcribed. Transcriptions were uploaded to the Nvivo database to be coded by team members. Th emes were identifi ed and a communications strategy formulated.

Results: Transportation and cultural norms were described as major health care limitations for Black Bostonians. Other themes that arose as barriers to care include mistrust, institutionalized racism, and lack of adequate insurance. Information about cancer and cancer care was received primarily through school or workplace affi liations or social outlets. A paucity of education, communication, and/or advertisements about general cancer care and cancer clinical trials in the Black community was noted.

Conclusion: Interventions targeting improved education and communication within underserved communities may be a useful strategy to help improve access to cancer care and reduce associated barriers.

13

Towards Nanoparticle Drones: New Technology to Kill Deadly Prostate Cancer Cells with Minimal Collateral Damage

Armel Foade

Principal Investigator: Wilfred Ngwa, PhD

Mentors: Michele Moreau; Rajiv Kumar, PhD


Background: Radiotherapy is one of the major treatment modalities for prostate cancer, which is the second leading cause of cancer death in men in America. However, radiotherapy treatment is limited by the collateral damage caused by radiation to neighboring healthy organs. In this work, we are developing novel biomaterials loaded with 4th generation (4G) nanoparticles conferred with stealth, fl uorescence and precision targeting moieties that can be released in-situ to serve as smart biomaterials during radiotherapy. Th e released 4G nanoparticle drones can be activated by radiotherapy photons to emit micro-meter range, missile-like electrons to kill prostate cancer cells with minimal collateral damage.

Methods: As a fi rst step to developing our Nanoparticle Drones, radiotherapy biomaterials made of the polymer Poly(D,L-lactide-co-glycolide) were prepared and coated with chitosan loaded with the fl uorescein dye (used as surrogate of the fl uorescent nanoparticles). Th e release of the fl uorescein dye in vitro was investigated as a function of concentration and two polymer molecular weights (MW): medium and high.

Results: Th e preliminary results show that the release of the fl uorescent dye from the coated spacer was between 50% and 90% the fi rst three days, and then increased steadily over time. As the concentration of the chitosan increased from 0.5% to 4%, the loading capacity of the medium and high MW chitosan increased from 16 ± 3 μM to 24 ± 3 μM, and from 9 ± 3 μM to 28 ± 3 μM, respectively. As the polymer weighting increased, the rate of release decreased. Conclusions: Th e results indicate that the release profi le of nanoparticles or radiosensitizer from the biomaterial can be controlled as a function of diff erent parameters. Th is would enable synchronization of release to diff erent radiotherapy schedules. Th ese promising results provide a useful basis for further investigations to optimize the design of the nanoparticle drone technology to enhance therapeutic effi cacy during radiotherapy treatment of prostate cancer patients.

14

Patient Ancestry May be a Determinant in Hematopoietic Stem Cell Transplantation Outcomes

Nora Fortoul

Principal Investigator: Michelle Lee, MD, PhD

Dana-Farber/Boston Children’s Cancer and Blood Disorders Center

Allogeneic hematopoietic stem cell transplantation (HSCT) is a medically intensive procedure that can be used to cure cancerous and non-cancerous blood diseases. Th rough transplantation, the diseased blood stem cells of a recipient are replaced with healthy stem cells from a donor.

In HSCT, diff erences between recipient and donor initiate an immune response in the recipient. Degree of matching for human leukocyte antigens (HLA) impacts the intensity of that immune response and aff ects outcomes after transplant. Human leukocyte antigen (HLA) proteins must be compatible between each recipient-donor pair at alleles -A, -B, -C, -DRB1, DQB1. HLA-matched siblings are ideal donors because they share the same HLA alleles, but often they are not available. Alternative unrelated donor sources include adult volunteers or banked cord blood.

HLA composition relies on linkage disequilibrium, a population genetics principal that describes how diff erent populations accumulate distinct genes. Linkage disequilibrium dictates which HLA genes travel together genetically. As such, it is a determinant of the likelihood of having an HLA-compatible donor for transplantation and it also can illuminate one’s ancestral origins. Patient ancestry has been shown to aff ect transplant options and outcomes. For example, a large disparity is seen when trying to identify HLA-compatible donors for people of diff erent racial and ethnic backgrounds, where people of non-European descent have a signifi cantly worse chance of fi nding an optimal donor.

In order to investigate the impact of patient ancestry on HSCT, a clinical research protocol was written and IRB approval was obtained. A retrospective review of the medical records of 207 patients was carried out to investigate how race/ethnicity - as a proxy for patient ancestry - may aff ect transplant characteristics and transplant outcomes in a single institution, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center from 2007-2012.

Understanding the impact of patient ancestry on HSCT will have important implications for bridging the disparity in outcomes for patients of varying races and ethnicities. Information from this research could support community-targeted donor recruitment eff orts and may encourage applying modern genomic tools to maximize the likelihood of fi nding the best available donors for patients with limited options.

15

The Interplay between Disrupted Peripheral Circadian Rhythmicity and Cancer Cachexia

Ana Paola Garcia #

Principal Investigator: Bruce Spiegelman, PhD

Mentor: Serkan Kir, PhD


Cancer cachexia is a multifactorial metabolic syndrome leading to signifi cant weight loss and frailty in more than half of all cancer patients. Characterized primarily by atrophy of skeletal muscle and adipose tissue, this wasting disorder directly accounts for twenty percent of all cancer deaths. Cachectic patients often display elevated resting energy expenditure and are resistant to conventional nutritional supplementation. While muscle loss overtly impacts cancer patients’ quality of life and overall survival, the wasting of fat depots represents a sustained metabolic energy imbalance. Interestingly, a large body of evidence points to the intimate relationship between metabolism and core clock machinery. Specifi cally, it has been shown that the peripheral circadian clocks control the expression of essential genes within a wide array of metabolic pathways, and the disruption of these clocks causes metabolic disorders by altering rhythmic expression of important metabolic genes. Here, we used the Lewis Lung Carcinoma (LLC) murine model of cancer cachexia to identify cachexia-associated changes in circadian oscillations. We investigated diurnal gene expression of circadian rhythm regulators as well as other key mediators of energy metabolism. We analyzed rhythmic gene expression patterns in adipose tissue and skeletal muscle of LLC-tumor bearing mice using quantitative real-time PCR (qPCR). In both tissues, we observed a marked dampening of diurnal transcriptional rhythms in Cry2, Per2, and Rev-Erb-alpha genes. Additionally, the expression patterns of the clock genes Dbp, Per1, Per3, and Rorg were upregulated, with further distortions in circadian regulation of Npas2, Bmal1, and Clock. Ultimately, elucidation of the role of altered peripheral clock rhythmicity in cancer cachexia will shed light on the unknown contributions to the cachectic process, eventually paving the way for the development of focused treatment strategies for this and other metabolic diseases.

16

A Comparison of Two CRISPR-Cas9-based Methods for Transcriptional Activation in Drosophila

Heresa Guerrier #

Principal Investigator: Norbert Perrimon, PhD

Mentor: Ben Ewen-Campen, PhD

Harvard Medical School

In recent years, the CRISPR/Cas9 system has revolutionized the fi eld of genome engineering. Although CRISPR/Cas9 was initially used to create knock-out/knock-in mutations, this system can also be modifi ed to regulate gene expression in a number of ways. For example, a catalytically-inactive Cas9 (dCas9) can be fused to functional domains such as transcriptional activators, repressors, or chromatin modifi ers, which can then be targeted to specifi c genomic locations using guide RNAs. In this study, we compare two recently described dCas9-fusion proteins approaches for activating gene expression: one based on a chromatin modifying domain (from the acetyltransferase, p300), and one based on a transcriptional activator domain (“VPR”). We show that, in Drosophila cells, the transcriptional activator fusion robustly activates target gene expression, but that the acetyltransferase does not. We discuss possible explanations for this diff erence, and conclude that the dCas9-VPR system will be useful for future over-expression studies.

17

Reduction in Prostate Specifi c Antigen Screening Following Contrary Recommendation

Soham Gupta

Principal Investigator: Quoc-Dien Trinh, MD

Mentors: Christian Meyer, MD; Julian Hanske, MD; Michael Zavaski, MD


Purpose: Th e use of prostate specifi c antigen (PSA) testing for early detection of prostate cancer (PCa) is controversial and the subject of intense scrutiny. In 2008, the United States Preventative Services Task Force (USPSTF) recommended against PSA screening in men > 75 years of age. Following the publication of two large randomized trials originating from the United States and Europe, the USPSTF further downgraded the use of PSA screening to a grade ‘D’ recommendation against screening for all men. Despite suggestions of wide variation in provider practices, recent data shows that the use of PSA screening is broadly decreasing in the US. However, the mechanisms for these changes are currently unknown. To address this gap in knowledge, we examined the use of PSA screening among urologists vs. primary care providers before and after the latest USPSTF recommendations.

Materials and Methods: We examined the 2010 and 2012 National Ambulatory Medical Care Survey (NAMCS), an annual, nationally representative, survey of ambulatory care in the US. Our sample included men aged 50 and above, who visited an urologist or primary care (including general/family practice and internal medicine) physician for a preventive care visit. We excluded men already diagnosed with PCa and/or elevated PSA. To account for secular trends in PSA screening, a diff erence-in-diff erences analysis was employed to compare the decrease of PSA screening following the new recommendations among patients who were aged <75 and ≥75. Patients who were ≥75 years of age were used as controls, as this population was not subjected to a change in recommendations during the study period.

Results: In men aged 50-75, the use of PSA testing decreased from 35.6% to 16.3% for primary care visits, whereas it decreased from 43.9% to 37.5% for urology visits. When a diff erence-in-diff erences approach was used, the decrease in PSA testing was signifi cant among primary care physicians (p=0.015), but not among Urologists (p=0.095).

Discussion: In men aged 50-75, the use of PSA testing in the context of a preventive care visit has decreased signifi cantly among primary care physicians but not among urologists.

18

Cancer Genome Atlas Gene Mutation: Detection of Co-occurring Variants and Network Community

Adnil Gutarra #

Principal Investigator: John Quackenbush, PhD

Mentors: Cho-Yi Chen, PhD; Marieke Kuijjer, PhD


Cancer is the result of uncontrolled cell growth. When a certain gene is altered or mutated, it can disrupt the cell’s ability to produce the correct proteins needed for controlled growth and replication. Certain combinations of mutated genes may provide a selective advantage to cancer cells. To understand how cancer develops and how it is treated, we identifi ed shared patterns of gene mutations between cancers. We used the R programming language to analyze a large dataset containing mutation data for about 2,119 genes in over 6,000 tumors covering 22 cancer types from Th e Cancer Genome Atlas (TCGA). We eliminated the genes that were mutated in fewer than fi ve samples and at least 5% of all samples. We used the Jaccard Index to identify similarities between gene mutational patterns. We plotted our data using heat maps: a graphical representation of data where the individual values contained in a matrix are represented as colors. Finally, we used community detection analysis to determine what groups of genes are often mutated together and we ranked the genes based on modularity, a measure of how well defi ned the communities are. It was found that genes IDH1 and ATRX were frequently mutated together, which is known to occur in low grade gliomas and glioblastomas. Th ese similarities allow us to identify which mutations often co-occur or never occur together within a tumor which may help us to develop more eff ective treatment strategies for these cancers.

19

Angiogenic Properties of Breast Cancer Cell Lines

Ted Hilaire #

Principal Investigator: Randolph Watnick, PhD

Mentor: Anna Blois, PhD


Breast cancer is the second leading cause of death among women. Th ere are four breast cancer subtypes: Luminal A, Luminal B, Triple Negative/Basal like, and HER2 type. Although there are many treatments for breast cancer, there is no cure for the triple negative or basal subtype. Angiogenesis is the process in which new blood vessels form from pre-existing vessels. Angiogenesis is a staple point in the metastasis of breast cancer. Understanding the way angiogenesis is induced can help understand a form of treatment for basal like cancers.

We hypothesize that the triple negative basal like cancer cell lines Sum 159, MDA-MB 231 and HS578t will secrete growth factors that increase the proliferation of mammary endothelial cells compared to the conditioned media of the luminal cell lines BT549, MCF7, and T47D. With this experiment, we wanted to answer the question of whether angiogenesis and metastasis is dependent upon growth factors secreted by basal and luminal subtype cancers. Tumor angiogenesis is very important for endothelial cells to proliferate in order to support the growth of the tumor cells. Another important part of angiogenesis is migration: where cells go into the blood vessel and proliferate at other sites in the body. We tested our hypothesis with a proliferation assay using conditioned media with four parallels and two controls. We then used a CyQuant assay to quantify the proliferation of EC. We also used a scratch assay to evaluate the migration of the endothelial cells when treated with the conditioned media.

20

Müllerian Inhibiting Substance Inhibits Growth of Ovarian Cancer Cells In Vitro

Lisa Hsiao

Principal Investigator: Patricia K. Donahoe, MD

Mentor: David Pepin, PhD

Massachusetts General Hospital, Harvard Medical School

With current ovarian cancer treatment methods, there is a high rate of chemoresistant recurrence. Th us, new and more eff ective ovarian cancer treatments are needed to improve patient survival. Müllerian Inhibiting Substance (MIS) is an endogenous hormone that has been shown to inhibit the growth of stem-like cell populations in both murine and human ovarian cancer cell lines. We sought to investigate the potential therapeutic eff ects of MIS on ovarian cancer cells by using cell lines derived from serous malignant ascites of chemoresistant ovarian cancer patients. We plated each well of a round-bottomed 96-well plate with a single cancer cell spheroid and treated the wells with either 10 μg/ml recombinant MIS (LRF-MIS), 60nM doxorubicin (DOX), 10 μg/ml carboplatin (CBP), a phosphate buff ered saline (PBS) control, or an MIS vehicle control (mock MIS). We then monitored spheroid growth over a period of 21 days and found that LRF-MIS, DOX, and CBP inhibited the growth of spheroids in one patient (ptAV) compared to the PBS and mock MIS controls. However, in another patient (ptAS), LRF-MIS did not inhibit spheroid growth compared to PBS and mock MIS controls. Th is data suggests that MIS may be an eff ective treatment for some patients with chemoresistant ovarian cancers, though subsequent studies with more patient cell lines are needed. Furthermore, the in vitro spheroid assay could be used as a minimally invasive screening method to assess patient response to MIS before administering treatment.

21

Epidermal Growth Factor Receptor Inhibition for Targeted Neurofi bromatosis Therapies

Alexander R. Jeremiah #

Principal Investigator: Karen Cichowski, PhD

Mentors: Clare Malone, PhD; Becky Lock, PhD


Neurofi bromatosis is an inherited cancer predisposition syndrome caused by genetic loss of the NF1 gene. Th is loss of wildtype NF1 causes patients to have an 8-13% lifetime risk of developing malignant peripheral nerve sheath tumors (MPNSTs) and there are currently no eff ective therapies for NF1 patients. In MPNSTs, the receptor of epidermal growth factor (EGFR), a receptor tyrosine kinase, is often overexpressed and we postulated that this could serve as a potential therapeutic target for patients; we aimed to inhibit EGFR using various EGFR inhibitors. It was expected that such inhibitors might block the activation of EGFR and therefore the downstream RAS pathway by preventing autophosphorolation of the EGFR dimers. Human MPNST lines were employed to test the effi cacy of EGFR inhibitors against EGFR and other downstream targets.

We found that, EGFR-specifi c inhibitors had little eff ect on proliferation compared to drugs that inhibit both EGFR and HER2, another receptor tyrosine kinase. Afatinib, one of the dual inhibitors, had the greatest eff ect on proliferation. Interestingly, another dual inhibitor, Lapatinib, has also shown to have an inhibitory eff ect on cell proliferation. In conclusion, this suggests that both EGFR and HER2 contribute to proliferation of MPNST cells. Currently, we are examining this relationship further using genetic approaches via siRNA and EGFR/HER2 inhibition in combination with other known downstream inhibitors to explore new therapies for neurofi bromatosis patients.

22

The Use of Functional Magnetic Resonance Imaging (fMRI) to Prevent Post Operative Defi cits

Thamael Laurore #

Principal Investigator: Alexandra Golby, MD

Mentor: Laura Rigiolo, MA


Functional magnetic resonance imaging (fMRI) is an imaging technique that has become an important tool for neurosurgical planning. FMRI uses the Blood-Oxygenation-Level Dependent (BOLD) eff ect to estimate and localize neuronal activation in the grey matter of the brain. Th e BOLD technique utilizes T2* weighted MR images to measure the signal related to deoxygenated hemoglobin’s magnetic properties. Th e ratio of oxygenated to deoxygenated hemoglobin changes in the event of neural activation, therefore, an increase in MR signal can be detected. After statistical analysis, BOLD maps can be used to determine activation in areas of the brain related to stimulus compared to resting conditions. In neurosurgery, fMRI can be used to determine critical brain activations adjacent to abnormalities and malformations in the brain. Th is information is useful for pre-surgical planning as it allows the surgeon to avoid damage to critical areas causing post-operative neurological defi cits. In this case study, pre-surgical fMRI data is reviewed for a 27 year male with a lesion in the left temporal lobe. Clinical workup revealed an Arteriovenous Malformation (AVM) and epilepsy. Pre surgical fMRI indicated left lateralized language function adjacent to the lesion, however the initial surgery attempt was aborted due to a seizure event during surgical preparation. He was re-scanned about one year later yielding similar results, and an awake craniotomy was planned in order to monitor function during the procedure. MRI image guidance was used to aid the surgeons as they resected the AVM and seizure focus. Th e second operation was successful and subsequent anatomical imaging indicated that the AVM was removed. In conclusion the fMRI data aided in guiding the surgery and in ensuring that the patient would not suff er any post operative defi cits.

23

Id1 and p53 Expression and G691S RET Polymorphism in Desmoplastic Melanoma: An Analysis of Sixty Six Cases

Nicholas F. Lawrence

Principal investigator: Mai Hoang, MD

Mentors: Marc Hammond; Dennie Frederick, MS; Mai Hoang, MD


Introduction: Desmoplastic melanoma (DM) is a rare subtype of melanoma. Th e majority of DM lesions occur on the head and neck, implicating the role of ultraviolet radiation and TP53 mutations. A single nucleotide polymorphism at codon G691S of RET (rearranged during transfection) is present in up to 62% of cases. RET encodes a Receptor Tyrosine Kinase with functions in cell proliferation and survival. Id1 (inhibitor of DNA binding 1) protein has been shown to play a role in cell diff erentiation and tumor angiogenesis in neoplasms.

Methods: Sixty-six cases of pure DM were included. Immunohistochemical studies for Id1 and p53 expression were performed. DNA was isolated from archival tumor samples. Th e RET gene was amplifi ed via polymerase chain reaction and analyzed for G691S using Sanger Sequencing. Statistical analyses of the relationships between RETp, protein expression, histology and survival were performed.

Results: Patient ages ranged from 34 to 97 years (median, 71 years). Th e male to female ratio was 2:1. Th e locations of the tumors include: head and neck (49), trunk (11), and extremities (6). Follow-up was available for 65/66 patients (range, 2 months to 22 years 4 months). Recurrence was documented in 11/66 (17%) patients. Metastasis was developed in 26/66 (39%); 13/66 (20%) with nodal metastasis and 18/66 (27%) with distant metastasis. Death was noted in 25/55 (45%) patients. Nuclear Id1 expression was seen in 25/58 (43%) cases and signifi cantly correlated with perineural invasion (p= 0.0045), vascular invasion (p= 0.03), disease progression (p=0.0381), and worse overall survival (p=0.0501). High p53 expression (greater than 50%) was noted in 16/57 (28%) of cases. RETp was present in 8/40 (20%) of cases. Th ere was no correlation observed between p53 expression and RETp and clinical or histologic variables.

Conclusion: Id1 expression is associated with more aggressive histologic features, disease progression, and poor clinical outcome in desmoplastic melanoma.

24

Correlating Distances between Prostate Biopsy Core and Cancer Lesion to Respective Metabolomic Profi les

Amy Luong

Principal Investigator: Leo Cheng, PhD

Mentor: Taylor Fuss


Prostate Cancer (PCa) is the second leading cause of cancer death among men. Currently, any high concentrations of prostate specifi c antigen (PSA) suggest surgical biopsy to determine if PCa is present. Biopsies collect tissue from the right and left halves of the base, middle and apex zones to assign Gleason Scores (GS) through histopathology methods, if there is presence of cancerous glands. However, the random sampling of tissues raises the ambiguity in diagnosis as it can lead to false negative results. Our ongoing studies recognized potential applications to the diagnosis of PCa through the changes of metabolites found in the prostate. We scanned one tissue core from each biopsy set with high resolution magic angle spinning (HR MAS) proton magnetic resonance spectroscopy (1H MRS) to generate metabolomic profi les. Pathology reports from biopsies were then used to evaluate trends of metabolites from benign to malignant conditions. Here we propose a 3D spherical model to represent the eff ects of changing prostate volume on distance between biopsy core location and region of cancer lesion. Th rough a series of formulas inputted in excel, prostate volume was used to generate midpoints of each of the six zones from which distances were calculated. Quantifi ed data provided variation in distances to previous categorical groupings of proximity. Collected data indicated a quantifi able fi eld in which PCa is able to infl uence changes in the metabolomic profi le. Th e smaller the distance between the biopsy core and cancer lesion, the more distinctive the diff erent concentrations of metabolites are. Th is study could contribute to improving the accuracy of diagnosis and characterization of PCa.

25

Defective NHEJ Leads to Deregulated PI3-K Pathway in Glioblastoma Multiforme Mice Model

Khang Nguyen #

Principal Investigator: Catherine Yan, PhD

Mentor: Youn-Jung Kang, PhD


Glioblastoma multiforme (GBM) is the most common malignant brain tumor in humans. Despite recent intensive research on GBM, still very little is known about the disease. Mutations in DNA repair genes like XRCC4 are commonly found in human cancers due to accumulation of genomic instability and mutations associated with misrepair of DNA damage. In several cases, mutations that impair p53 combined with various mutations have been observed in human GBMs. In order to evaluate the interplay between the DNA repair gene in the non-homologous end-joining (NHEJ) DNA repair pathway, XRCC4 (X), and the tumor suppressor gene, p53 (P), in gliomagenesis, we have developed a novel glioma mouse model by conditionally deleting both genes in GFAP(G)-expressing cells in the brain. Th is reproducibly led to the development of high-grade gliomas, which we refer to as GXP GBMs, that exhibit common oncogenic signatures found in human-like proneural/classical GBMs. Mice expressing the XRCC4 and p53 conditional alleles were crossbred and genotyped to confi rm the ablation of both genes in the brain. Intriguingly, gene expression analysis identifi ed the murine GXP GBMs to routinely exhibit low PTEN functional activity levels, which cause the deregulation of PI3-K signaling. Because tumorigenesis is an indicator of DNA damage resistance, viability assays were conducted to identify how the conditional deletions of XRCC4 and p53 aff ected cell growth under induction of DNA double strand breaks through doxorubicin treatment. Th ese fi ndings may provide insight into the initiating molecular mechanisms driving GBM formation in response to accumulated DNA repair failure. We believe that understanding causal genetic alterations and the molecular mechanisms initiating and promoting these malignancies will be critical for the novel therapeutic stratifi cation against this deadly brain cancer.

26

Structural Study of Actin Nucleating Formins using X-Ray Crystallography

Habiba Noamany

Principal Investigator: Michael J. Eck, MD, PhD

Mentor: Hyesung Jeon, PhD


Formins are a family of proteins associated with actin nucleation of unbranched actin fi laments. While it is known that proper actin nucleation is crucial for cytokinesis, cell polarity, and cell migration, the mechanism by which these multidomain proteins nucleate actin has not been clearly defi ned. We are studying the structure of the Saccharomyces cerevisiae formin, Bni1, and its associated nucleation-promoting factor (NPF), Bud6. Previous work within the lab using X-ray Crystallography determined the structure of the Formin Homology-2 (FH2) domain of Bni1 to be of a tethered dimer architecture and the structure of the Bud6 core domain to be an elongated dimeric rod. It is hypothesized that the Bni1-FH2 dimer binds two Bud6 dimers, which then bind four actin subunits to form a nucleation seed with Bni1. Current eff orts are underway to solve the structure of a bigger part of Bni1, the FH2C domain, in a complex with the c-Bud6 domain, which includes an actin binding motif. Such a fi nding would illustrate the structural and functional relationship between the Bni1-FH2C tail and the Bud6 core and in turn elucidate the interactions between Bni1 and Bud6 which promote actin nucleation. Th e c-Bud6 and FH2C domains are unstable and therefore diffi cult to work with, however, we have crystallized the complex and are in the process of optimizing the conditions for X-ray experiments.

27

Drugs (263,1781) Inhibit the Hypoxia Induced Cell Death

Miracle Onyeoziri #

Principal Investigator: Bertal Huseyin Aktas, DVM, PhD

Mentor: Yan Lu, PhD


Tumor hypoxia is caused by abnormal blood vessels which lead to an insuffi cient supply of oxygen. Hypoxia is deadly to eukaryotic cells. However, tumor cells develop adaption to hypoxia, which is associated with the acquisition of an increased ability for invasive growth, distant tumor cell spreading, and a resistance to therapy. One mechanism tumors use to protect themselves from hypoxia is activating unfolded protein response (UPR). Once UPR is activated, it can degrade misfolded proteins, stop mRNA translation to restore the normal functions of the cell, and activate the signaling pathways to increase the production of molecules involved with protein folding. One important UPR response is eIF2α phosphorylation. Aktas lab developed small molecules that induce eIF2α phosphorylation. Upon eIF2α phosphorylation, protein synthesis is reduced which may either cause hypoxic tumor cells to die faster or survive longer. In order to determine the eff ect chemical induction of eIF2α phosphorylation has on hypoxic cells, we compared the consequences of culturing hypoxic or normoxic cells with two chemical inducers of eIF2α phosphorylation. One of two six-well plates was in hypoxia for one day while one plate was cultured in normoxic conditions; all wells were treated with an active compound or DMSO (solvent). After one day plates were washed, media was replaced with fresh medium without drugs, and was cultured under normoxic conditions. Once the experiment was performed we could see if chemical inducers of eIF2α phosphorylation accelerate or decelerate death of cancer cells under hypoxia. In conclusion, these studies may bring about a way to stop cancer cells from surviving and decrease therapy resistance.

28

Optimization of Cell Migration Assay to Assess the Role of CMG-2 in Endothelial Cell Migration

Ambar Piña

Principal Investigator: Michael Rogers, PhD

Mentors: Lorna Cryan, PhD; Jessica Stiles


Angiogenesis is the process by which new blood vessels are generated in the body. Angiogenesis is important for physiological processes such as wound healing and tissue development, but is also involved in tumor growth and metastasis. Endothelial cells line the inside walls of blood vessels. Previous studies have found that in endothelial cells there are two Anthrax toxin receptors known as Tumor Endothelial Marker-8 (TEM-8) and Capillary Morphogenesis Gene-2 (CMG-2) that are highly expressed during the process of angiogenesis. Diff erent cell growth factors are known to regulate the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF) and Epidermal Growth Factor (EGF). In this study, we proposed to analyze the role of CMG-2 and TEM-8 in VEGF and serum induced cell migration of Human Microvascular Endothelial Cells (HMVECs). HMVECs were used in several diff erent cell migration assays such as modifi ed Boyden chamber, scratch-wound healing and also combined with siRNA mediated CMG2 or TEM8 knockdown in order to assess the role of these receptors in cell migration. Th e migration of these cells was induced with both VEGF and full serum media. An antagonist ligand known as Protective Antigen (PA) was also used to assess the role of CMG-2 in endothelial cells. Cell migration in the scratch-wound healing assay was assessed using timelapse microscopy. We observed that the outer wells of the 48-well plates used in the scratch migration assay exhibited reduced cell migration compared to the inner wells independent of conditions previously established such as temperature and CO2. After several experiments, we quantifi ed a decrease of approximately 70% in migration in the outer wells compared to those wells that are located in the middle of the plate. We also assessed the eff ectiveness of siRNA mediated knockdown of CMG-2 and TEM-8 using western blotting. Optimization of these assays will allow for further detailed quantitative assessment of the role of these receptors in endothelial cell migration.

29

DREAM Complex Maintenance in Quiescence is Regulated by CDK Phosphorylation

Brenda Romero #

Principle Investigator: James DeCaprio, MD

Mentor: Amy Schade


Th e cell cycle progresses through G1, S, G2, and Mitosis for cells to replicate. Cells can also exit the cell cycle to the quiescent state commonly referred to as G0. Th e DREAM protein complex contains DP1, RB-like (p130), E2F, and Muv-B and maintains cellular quiescence. DREAM represses genes required for S and M phases of the cell cycle. Th e question of our project is how DREAM loses its ability to repress cell cycle genes when a cell leaves G0. When a mammalian cell enters the G1 phase, Muv-B and p130 disassociate from each other. We believe that cyclin-dependent-kinase (CDK) phosphorylation sites regulate Muv-B and p130 binding. To test if the phosphorylation of p130 is required for the disassembly of DREAM, we created mutations that changed Serine and Th reonine residues in the p130 protein sequence to Alanine. Th is removes phosphorylation sites since Alanine does not have the hydroxyl group that is required. We hypothesize that this prevents p130 phosphorylation and will keep a cell in G0. Using Wild Type (WT) p130 or a p130 with all CDK sites mutated to Alanine (PM19), we will test if the PM19 mutant can form a DREAM complex and maintain DREAM even when cells enter the cell cycle. We propose that PM19 will co-immoprecipitate more proteins associated with the DREAM complex versus WT because the phosphorylation site mutations on PM19 would allow for more total Muv-B proteins to remain. If we gain understanding in how DREAM is regulated, there may be a way to restore DREAM in rapidly proliferating cells, and provide a way to force cells into G0.

30

Questionnaire Reliability in the Boston Mammography Cohort Study

Naria Sealy

Principal Investigator: Erica Warner, ScD, MPH


Introduction: Questionnaires are essential data collection instruments in population health research. However, for questionnaires to yield meaningful data, they must produce reliable responses. Reliability is the degree to which one can expect relatively constant deviation scores of individuals across testing situations on the same testing instruments. Reliability has been often under-emphasized or misunderstood.

Objective: Th e objective of this research was to: 1) identify participants who completed multiple questionnaires; 2) determine how reliably participants responded to selected questions; 3) identify characteristics of questions and participants associated with reliable reporting.

Method: Th e Boston Mammography Cohort Study (BMCS) is a cohort of nearly 3,000 women who have had at least one mammogram at the Lee Bell Center for Breast Imaging at Brigham and Women’s Hospital in Boston, MA since 2007. At enrollment each woman completed a questionnaire that collected information on reproductive, lifestyle, and behavioral factors. A subset of women completed the questionnaire at multiple points. Duplicates were identifi ed and the fi rst and last completed questionnaires were compared to determine test-retest reliability.

Results: We anticipate that questions on milestone life events, like age at menarche or, age at fi rst birth, will be more reliably reported than questions regarding diet or lifestyle. Reliability for questions about early life events will be highest for subjects enrolled before age 40 and decrease with older age at enrollment.

Discussion: Th ere are many studies looking at individuals’ past activity and risk factors for developing cancer. Prospective cohort studies are ideal for this, but the most economical and time-effi cient way to collect data on participant exposures is often through retrospectively administered questionnaires. Assessing the reliability of the BMCS questionnaire will help determine what information reported by the participants can be used confi dently. Th e reliability of the data must be known to prevent misclassifi cation and incorrect conclusions.

31

Exploring Blood Platelets as a Liquid Biopsy for Tumor-Derived Mutant RNA in Patients with Glioblastoma

Adam Stevens

Principle Investigator: Bakhos Tannous, PhD

Mentor: Nik Sol, MD


Th e utility of liquid biopsies lies in their ability to capture trace amounts of genetic material shed into the blood by cancer cells, and to use this information to detect and monitor disease. In this way, liquid biopsies off er a non-invasive alternative to traditional cancer diagnostics typically plagued by problems of tumor accessibility and heterogeneity, on top of excessive cost and discomfort of the patient. Th e aim of the present study was to therefore expand upon the potential of this method by using blood platelets from clinical follow-up samples of glioblastoma (GBM) patients receiving standard of care. To do so, assay conditions were fi rst optimized for the detection of EGFRvIII, a GBM-specifi c mutation, by means of droplet digital PCR (ddPCR) on glioma cell lines overexpressing EGFRvIII and then applied to clinical samples. RNA, from cell lines and platelets from GBM patients were prepared using identical techniques, which included mirVana™ RNA isolation, Bioanalyzer quality control, and Easyscript™ cDNA synthesis. Assay components such as primer and probe concentrations and annealing temperature were identifi ed on cell lines to produce reliable ddPCR readouts, as indicated by a linearly quantitative dilution series assay ranging from as little as 0.005 ng total RNA input (r2=0.99). Th ese assay conditions were then applied to clinical samples to diff erentiate EGFRvIII positive from EGFRvIII negative tumors in GBM patients prior to surgery. Th is distinction became less clear, however, when using samples following treatment and consequent tumor resection. As a result, it appears that the indicated EGFRvIII mutation is not present—or not present in high enough quantities—in platelets to serve as a reliable marker for cancer monitoring. Future studies would therefore benefi t from further assay optimization, increased instrument sensitivity, or choosing to analyze additional blood components where EGFRvIII and other genetic variants may exist in higher concentrations.

32

A Phase I/Ib Study of Ipilimumab in Patients with Relapsed Hematologic Malignancies after Allogeneic Hematopoietic Cell Transplantation

Mariano R. Suriel

Principal Investigator: Jerome Ritz, MD

Mentors: Carol Reynolds, PhD; Marie Chammas


Allogeneic Stem Cell Transplantation (alloSCT) is commonly used for patients with chemotherapy resistant hematologic malignancies. Unfortunately relapse from graft rejection and infection is common in these patients due to the treatment, which compromises their immune system. Ipilimumab is a monoclonal antibody which is FDA approved for treating a variety of cancers. It has been shown to activate the immune system by binding to CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4) on the surface of T-cells. CTLA-4 acts as a check point that down-regulates immune response upon binding to the B7 receptor on antigen presenting cells (APC). Ipilimumab prevents this inhibitory binding of CTLA-4 to B7, which results in restoring functional activity to the cytotoxic T lymphocyte cell population. Th e study cohort consists of 16 patients. Ipilimumab is administered by intravenous infusion (IV) every 3 weeks for a total of 4 escalating doses. Peripheral blood (PB) samples are collected pre-alloSCT and post-alloSCT: day 1 and weeks 2, 4, 8, 10, and 14. Immunophenotypic analysis is performed looking at extracellular surface markers using a 4 tube antibody-cocktail-panel. Tube 1 focuses on T-cell subsets, tube 2 on B-cell subsets, tube 3 on natural killer-cell subsets, and tube 4 on dendritic cells. Th e PB samples are run on a BD LSRFortessa fl ow cytometry instrument and analyzed by BD FACSDIVA software. Overall the results showed total lymphocytes counts increases in the majority of the subjects. Helper T-cells, cytotoxic T-cells, B-cells, and NK cells demonstrated a similar increase in 3 out of the 4 subjects. T-cells counts (CD3) showed similar increase over a 10-week period. Th e data on lymphocyte subset progression throughout the established time points will aid clinicians as they continue to look at this drug for an immune enhancement agent in the allogeneic transplant setting.

33

Numb Isoform Expression During MEL (Murine Erythroleukemia) DMSO (Dimethyl sulfoxide) -induced Differentiation

Judene Thomas #

Principal Investigator: Shu-Ching Huang, PhD


Numb proteins play an essential role in the determination of cell fates during development. Using a Murine Erythroid Leukemia (MEL) cultured red blood cell diff erentiation model, we showed that Numb is distributed selectively to one daughter cell during progenitor asymmetric cell division. Th e inheritance of Numb guides this daughter cell towards red cell diff erentiation whilst the other daughter cell remains a progenitor cell. Numb consists of an amino-terminal phosphotyrosine binding domain (PTB), a C-terminal proline-rich (PRR) region, and an Eps15 homology region. Th e Numb gene expresses four isoforms generated from alternative splicing of exon 6 (11 amino acids in the PTB) and exon 12 (48 amino acids in the PRR). Th ese are Numb1 (+exon 6, +exon 12), Numb2 (+exon 6, -exon 12), Numb3 (-exon 6, +exon 12), and Numb4 (-exon 6, -exon 12). Th e diff erent forms of Numb have various progenitor-promoting and diff erentiation-promoting functions in the neuronal system. We hypothesized that regulated Numb isoform expression provides a tightly-controlled self-renewal and diff erentiation balance during red blood cell maturation. Using MEL system we set out to identify what specifi c isoform is expressed during diff erent stages of erythroid diff erentiation. To access this, MEL cells were cultured and chemically induced using DMSO from day 0 to day 6. Th e cells were collected and the RNA isolated, then the RNA samples were RT-PCRed using Numb-specifi c primers and the products were electrophoresed on a 5% polyacrylamide gel. Th e expression levels of the isoforms were then analyzed. Our analyses show that Numb3 and Numb4 are the predominant forms expressed during MEL erythroid diff erentiation. Furthermore, a signifi cant diff erentiation-induced switch of Numb3 to Numb4 occurs in early onset red cell diff erentiation. Th ese results suggest that the mammalian blood system produces Numb3 that maintain progenitor populations and Numb4 that support red blood cell diff erentiation, which we will verify through future experiments.

34

Red Blood Cell Antigen Prediction Using Whole Genome Sequencing (WGS) Data

Emilio Vides-Curnen #

Principal Investigator: William Lane, MD, PhD

Mentors: Peter Tonellato, PhD; Thiago Braga Rodrigues

Harvard Medical School

Whole Genome Sequencing (WGS) shows promise in regards to its possible applications in clinical medicine. Next Generation Sequencing (NGS) techniques, such as Illumina bridge amplifi cation, produce results in less time for less money, making WGS a more readily available tool for physicians. Th e importance of the genome cannot be overstated; it defi nes the genotype and the phenotype of organisms. Th e genome dictates the blood makeup of individuals: diff erent blood groups are the result of the presence or absence of antigens coded for in the genome. Currently, blood typing is achieved through serological testing. Serology is the gold standard for the evaluation of Red Blood Cell (RBC) antigens because it highlights the presence of specifi c antibodies that are created by the immune system in response to distinct antigens. We set out to compare the RBC and PLT antigen prediction capabilities of WGS results with those of serology.

In this study, we gathered the results from a clinical trial of 100 patients in order to judge predictive capabilities. We utilized an algorithm designed to scan the genome, highlight the alleles involved in deciding blood antigens, and use these alleles in comparison with a reference genome to predict the blood groups of each patient. We compared the WGS-generated predicted antigen frequencies in these 100 cases to known antigen frequencies and then evaluated the base coverage in the 100 genomes and how this impacted antigen prediction. Th e WGS-generated antigen predictions were compared to serologic RBC antigen predictions for the same individuals. Our team then developed a graphic that summarizes the antigen predictions, frequency, base coverage, and concordance with serology in one visual. Th is new means of predicting blood type may be more effi cient and accurate than serology, and thus may prove the better mechanism for use in blood typing and hematology.

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Neuropilin 2 Infl uences Angiogenesis in Breast Cancer

Carolina Villalba

Principal Investigator: Diane Bielenberg, PhD


Breast cancer is one of the leading causes of death in women. Angiogenesis, the sprouting of new blood vessels from preexisting ones, contributes to cancer progression by increasing tumor nutrients and oxygen and providing routes for dissemination. Neuropilins (NRP1 and NRP2) are non-tyrosine kinase cell surface receptors found on vascular and lymphatic endothelial cells that bind VEGF, a potent angiogenic protein. It is known that NRP1 is also expressed on epithelial cells (EC) and carcinoma cells. We sought to understand the role of NRP2 in breast cancer by fi rst evaluating its endogenous expression in human breast cancers and then by manipulating NRP2 expression in either the tumor cell or stromal cell compartment. We hypothesized that over-expression of NRP2 in the tumor cells would increase tumor growth and progression and that depletion of Nrp2 in the EC would decrease tumor growth and progression. Our results from western blotting demonstrate that most human breast cancers express NRP1 and a subset express NRP2. Immunohistochemistry staining shows that increasing NRP2 in lowly metastatic human breast cancer MCF7MFP1 cells results in greater microvessel density in xenografted tumors in immunodefi cient mice. In addition, tumor growth in syngeneic C57Bl6 wild-type and Nrp2 knockout mice injected with murine breast cancer cells E0771 does not dramatically diff er. Upon gross examination, however, tumors in Nrp2 knockout mice appear hemorrhagic and necrotic in contrast to tumors in wild-type mice. We predict that further investigation will reveal reduced microvessel density in the Nrp2 depleted mice tumors. Taken together, our results suggest that NRP2 may be a novel target for breast cancer therapy.

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BRG/BRM Assay Development Pipeline

Katherina Yeager #

Principal Investigator: Cigall Kadoch, PhD

Mentor: Roodolph St. Pierre


Rationale:Th e Switch/Sucrose Non-Fermentable (SWI/SNF) complex, found in both eukaryotes and prokaryotes, contains a core helicase/ATPase (BRG1 or BRM) domain that couples ATP catabolism to chromatin remodeling to facilitate or inhibit gene transcription. Roughly 20% of human cancers have been documented with various mutations in the subunits of the SWI/SNF complex. Specifi cally, BRG1 defi cient lung cancers occur in ~10-15% of lung adenocarcinomas. Th e loss of BRG1 leads to reliance on BRM, a highly homologous protein to BRG1. Furthermore down-regulation of BRM in BRG1-defi cient cells hampers cell growth suggesting that a small molecule inhibitor of BRM may help a subset of patients suff ering with lung adenocarcinomas.

Approach:We aim to develop an assay to monitor the enzymatic activity of both BRG1 and BRM. Both paralogs have identical ATPase domains, thus substituting BRG1 for BRM during the assay development stage is suitable. We will isolate BRG1 from embryonic kidney cells (293T) through cell cultures, nuclear extracts, and subsequent immunoprecipitations with appropriate antibodies. Isolated complexes will be subjected to an ATPase Time-Resolved Fluorescence Energy Transfer (TR-FRET) assay that monitors the concentration of ADP in solution. Assay set up will be amenable for high throughput screening to further launch a drug discovery eff ort to identify fi rst in class small molecule inhibitors of BRM.

Results: We have successfully purifi ed the ATPase viable complex. Previous purifi cation schemes maintained the SWI/SNF complex on the protein G agarose beads, which interferes with assay readout. We are currently optimizing various methods to compete off intact SWI/SNF complex of the beads. Once successful, the ATPase assay will be transferred to a high throughput format to initiate a drug screening eff ort to identify chemical probes for BRM.

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Notable Achievements 2015

In the past year a CURE student:

• Completed medical school and will begin an internship at Yale-New Haven Hospital

• Presented a cancer-related poster at the New England Science Symposium

• Gained acceptance into Michigan State University’s Doctor of Osteopathy PhD program

• Won a poster presentation award at the Annual Biomedical Conference for Minority Students in San Antonio, Texas

• Received a pre-doctoral Ford Foundation Fellowship Award

• Obtained acceptance into Princeton University Class of 2019

• Was a recipient of the Gates Millennium Scholarship and will be attending the University of Miami in Fall of 2015

• Gained acceptance into the Accelerated Nursing Program at the MGH Institute of Health Professions

• Secured an internship at Mondelez International - a food service company focused on ingredient research

• Received multi-year funding to support oral health research

• Gained acceptance into Ohio State University Medical School

• Was accepted into Yale School of Medicine’s Physician Associate Program

• Was a co-author on a CURE experience based manuscript accepted for publication in the Journal of Virology

• Received acceptance into the Howard Hughes Medical Institute - Exceptional Research Opportunities Program (EXROP) and is working as an intern in the research laboratory of Dr. George Daley at Boston Children’s Hospital

• Secured employment at Dana-Farber Cancer Institute as a Research Technician

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The CURE Program thanks:

A. David Mazzone Awards Program

Biogen Foundation

Cubist Pharmaceuticals/Merck

Friends of Dana-Farber Cancer Institute

Massachusetts General Hospital Cancer Center

National Cancer Institute Cancer Center Support Grant 3P30CA006516-50S1

Vertex Pharmaceuticals

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Dana-Farber/Harvard Cancer Center’s Continuing Umbrella of Research Experiences (CURE) wishes to acknowledge and thank the CURE Advisory Committees, principal investigators, mentors, scientifi c advisors, lecturers, and supporters for expanding the career horizons of our students.

Many thanks to the following organizations:

Biogen - Community Lab Harvard Integrated Life Sciences

Dana-Farber/Harvard Cancer Center and University of Massachusetts Boston U54 Comprehensive Cancer Partnership Program Offi ce of Diversity Inclusion and Community Partnerships, Harvard Medical School

A special thanks to: Fatih Aydogan, MDAlazar Ayele Shannon Bailey, PhDChristine BazeCarla BecerraKelly BietteTimothy BraniganTracy Callahan, PhD Juan Carmona, PhDLaura Cato, PhDAdán Colón-Carmona, PhDAngel CroninSusan DeCristofaro, RN, MS, OCNZainab DoctorRoberta Driscoll, EsqCatherine DuarteHilary Eaton, PhDGregory GalantiOmar Gandarilla, MDRaphael Gaudin, PhDCarla HawkinsJessica HawkinsElizabeth HayesJason Heustis, PhDAnthony Hill, PhDRachada HiranyaketTasmina Hydery, Pharm.D., MBA Elizabeth JaenschSonal Jhaveri-Schneider, PhDYing JiangNichole JonesHye-Jung Kim, PhD

Jonathan LeeMichaela Levin, PhDMelissa LisumDiego Martinez, PhDLuca Meoli, PhDTsega MesheshaBrenda MulliganNancy NguyenRichard OakleyKwadwo Owusu-BoaiteyJessica Pierre-FrancoisElizabeth PollinaYanny Qin, Pharm.D.Haley Ramesy, PhDBrianne RyanJerome SaundersJaclyn Sceneay, PhDAmy SchadeSandy SerizierMayuri Sharma, PhDJohnothan SmileyeRoodolph St. PierreLaura Stark, PhDVincent Streva, PhDAndy Tan, PhDVenee Tubman, MDJuan Villa, MDMichael WachalaColin WatersTodd WeissmanDiedra Wrighting, PhD

dana-farber/harvard cancer center cure · dana-farber/harvard cancer center cure continuing...

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