ABSTRACT IMPACT: This translational study demonstrates a method for identifying possible mechanisms underlying a highly effective randomized control trial intervention so that a university-public health agency partnership might replicate intervention components in a scalable, feasible, community version of the program. OBJECTIVES/GOALS: The Barbershop Study was a cluster randomized control trial which demonstrated that clinical pharmacist directed care, provided to African American men in community barbershops, significantly improve hypertension control. We sought to understand which components of the intervention the participants and implementers considered most important. METHODS/STUDY POPULATION: Enrollment in the Barbershop Study included 319 men from 52 barbershops across Los Angeles. Two specialty trained clinical pharmacists led the intervention. We performed 32 structured interviews of 20 study participants, 10 barbers, and 2 clinical pharmacists approximately 1 year after the study’s completion. Interviews consisted of 27, 24 and 19 questions for barbers, participants, and pharmacists, respectively. Interviewees were asked about their experience in the study, barriers and facilitators to participation, effective aspects of the intervention, and less helpful components of the design. Interviews were recorded performed by a research assistant uninvolved in the study. Recordings were then transcribed for a qualitative thematic analysis. RESULTS/ANTICIPATED RESULTS: We anticipate facilitators of participant engagement to include the provision of care in the community and integration of services into a regular task (getting their hair cut). Based on prior conceptual models, we also anticipate the provision of care in a trusted setting to be an effective means to enhance participants’ willingness to follow clinical instructions. An anticipated barrier for participants includes the need to go to an offsite pharmacy to pick up their medications. For barbers, we anticipate themes including a desire to help their community, while barriers include potential decreased productivity due to time spent counseling participants. Pharmacists are expected to identify an enhanced sense of importance in their work, while identifying the need to travel as a barrier to the intervention. DISCUSSION/SIGNIFICANCE OF FINDINGS: Insights from this qualitative analysis may assist with adaptation of the highly effective Barbershop intervention, allowing it to be rolled out at scale. If done successfully, achieved reductions in blood pressure may result in reduced health disparities and prevent thousands of strokes, heart attacks and deaths.

ABSTRACT IMPACT: This study will showcase the importance if incorporating patient stakeholders in the development of an interview guide for a women of color with uterine fibroids, an understudied population. OBJECTIVES/GOALS: Black women and Hispanic/Latinas report having greater symptom burden from uterine fibroids (UF), non-cancerous neoplasms, compared to White women. These disparities may be linked to cultural factors resulting in treatment delays. The objective of this study is to provide insights to barriers and facilitators to timely treatment. METHODS/STUDY POPULATION: In partnership with the Fibroid Foundation, a UF advocacy organization, we plan to conduct a virtual community engagement (CE) studio to serve as a first step for a pilot study with a national cohort of Black women and Hispanic/Latinas who receive treatment in the United States for UF. The studios will include a presentation about UF treatment options and a facilitated discussion. The CE team will use past research and constructs from Model of Improvement and Health Belief Model to develop materials for the studio. A qualitative researcher will guide the discussion, a note-taker will take notes, and they will thematically code the notes. The results will be used to create and implement a cross-sectional in-depth qualitative study with a national sample. RESULTS/ANTICIPATED RESULTS: We hypothesize that timely treatment will be impacted by cultural factors, such as health literacy in uterine fibroids and menstruation. We expect that detailed feedback from this national cohort will contribute to greater insight to the experiences of women of color with UF and address barriers and facilitators to treatment. We anticipate the anecdotes will provide information about the influence of culture in seeking treatment for UF. We will utilize this experience to understand the impact of a virtual CE studio in elucidating open discussion among women of color on a challenging and personal topic. DISCUSSION/SIGNIFICANCE OF FINDINGS: Using CE process with advocates and research partners attains a deeper understanding in the development of an interview guide to examine the cultural impact on the treatment of UF for women of color. Understanding cultural barriers and facilitators can help overcome treatments delays in UF along with other gynecological diseases.

ABSTRACT IMPACT: By identifying clear gaps in our knowledge of racial and ethnic disparities in antibiotic-resistant infections, this research is informing the design of (a) community-based interventions and (b) patient-centered research studies that we are currently leading to address these disparities and improve human health. OBJECTIVES/GOALS: Antibiotic resistance (AR) is widely considered to be the next global pandemic. As with COVID-19, the potential for AR to disproportionately impact racial/ethnic minorities is a major concern. Our goal was to identify gaps in knowledge of AR disparities in order to inform the types of interventions that might be most appropriate to address this. METHODS/STUDY POPULATION: We reviewed the literature to examine evidence of racial/ethnic disparities in (a) infections with the most concerning drug-resistant bacteria in the United States, and (b) underlying social-economic or behavioral factors that could contribute to such infections. We searched PubMed and Google Scholar to identify studies published in English between August 1973 - August 2020. We used keywords that included: antibiotic resistance, antibiotic-resistant infections, antibiotic-seeking behavior, prescription/non-prescription antibiotic use, antibiotic education, or health literacy AND race, ethnicity, or socioeconomic status. We screened all abstracts to identify US-based studies that assessed (a) or (b) above. RESULTS/ANTICIPATED RESULTS: We identified 11 studies investigating racial/ethnic disparities for 5 of the 17 drug-resistant bacteria flagged in the CDC’s 2019 Antibiotic Resistance Threats Report. Black, Hispanic, and lower-income individuals were found to be at higher risk of some community-acquired antibiotic-resistant infections. We identified multiple factors that may contribute to disparities in AR-related morbidity and mortality, including reported differences in antibiotic use, higher likelihood of living in crowded/multigenerational homes, more frequent employment in potentially high exposure settings (e.g. slaughterhouses), lower health literacy, and more frequent underlying comorbidities, which increases risks for hospitalization and subsequent acquisition of drug-resistant infections. DISCUSSION/SIGNIFICANCE OF FINDINGS: Given the small number of studies on this topic, educational interventions that aim to raise awareness of this issue must target not only the public but also researchers. Community-based interventions that seek to address disparities in ‘antibiotic resistance literacy’ among minority and underserved groups could be particularly impactful.

ABSTRACT IMPACT: Triple negative breast cancer (TNBC) affects 10-20% of women with breast cancer and is biologically more aggressive than other subtypes. The novel compound we have developed, DL7076, would give clinicians a vital strategy to improve the commonly used cyclophosphamide (CPA) and doxorubicin (DOX) regimen in the treatment of TNBC. OBJECTIVES/GOALS: The objective of this research project is to develop a novel compound which can activate both 1) the constitutive androstane receptor (CAR) and subsequently enhance the CYP2B6-mediated activation of CPA, and 2) the nuclear factor erythroid- related factor-2 (Nrf2) leading to the cardiomyocyte protection from DOX-associated cardiotoxicity. METHODS/STUDY POPULATION: Following the identification of the compound candidate, DL7076 was evaluated for tissue specific induction of CAR and Nrf2 using qPCR, western blot analysis, and luciferase reporter assays.

Further, we have developed a multicellular coculture model incorporating human primary hepatocytes for metabolism, TNBC spheroids as the target, and cardiomyocytes as a side target of DOX. We have investigated the anticancer effects of CPA/DOX on TNBC cells and the toxic effects on cardiomyocytes with/without a CAR-Nrf2 activator, in a multicellular environment where hepatic metabolism is well-retained. RESULTS/ANTICIPATED RESULTS: We found that our dual activator of CAR and Nrf2, DL7076, exhibits tissue specific induction of CAR and Nrf2. Inclusion of DL7076 in combination with the CPA/DOX regimen improves anticancer efficacy, through the subsequent increase in the formation of the active CPA metabolite. With the addition of DL7076, DOX-mediated off-target cardiotoxicity was markedly reduced.

Lastly, utilizing the novel coculture system with human primary hepatocytes, TNBC spheroids, and cardiomyocytes, the inclusion of DL7076 to the CPA/DOX regimen shows decreased spheroid viability and improved cardiomyocyte viability and function. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that DL7076 can facilitate DOX/CPA containing regimens by increasing CAR-mediated metabolism and subsequent CPA bioactivation while selectively protecting cardiomyocytes from DOX-induced toxicity. This research is expected to translate our basic scientific findings into therapeutic interventions for women with TNBC.

ABSTRACT IMPACT: Indiana CTSI Team Science to maximize rapid collection, analyses and dissemination of biosamples collected from patients with Covid-19 to provide preliminary data for grant applications on the pathogenesis and outcomes of patients with Covid-19. OBJECTIVES/GOALS: When Covid-19 hit Indiana in April, there was an immediate need to respond rapidly to coordinate research across our healthcare systems. The CTSI became a point of contact for coordinating research endeavors including activation of clinical trials and use of precious samples from patients with Covid-19 to maximize preliminary data for grants. METHODS/STUDY POPULATION: The Indiana CTSI coordinated collection of biospecimens at multiple hospitals using in person and remote consenting via telephone or on a smartphone utilizing a QR code. We also retrieved existing samples from the Indiana Biobank previously collected for future research and from subject positive for Covid-19 by search of the linked electronic health record (EHR). A total of 224 subject samples (7 children, 36 previously collected, and 6 with both acute and recovered specimens) were obtained over a four month period. Our CTSI cores ran varied analyses collated to a single database, linked to the EMR for use as preliminary data for grant applications to avoid redundancy of measures on limited samples. RESULTS/ANTICIPATED RESULTS: The 224 subject samples were used for whole exome DNA sequencing, RNA seq, analyses of 48 plasma cytokine/chemokines by multiplex analyses, and PBMC isolated for culture and assessment of secreted cytokines. The clinical data were linked and included demographics, hospitalization length of stay and need for mechanical ventilation, max and min oxygen levels, liver function tests, IL-6, D-dimer, CRP, LDH, and ferritin, need for dialysis, and echocardiography. Additional clinical data were available upon request. A survey was sent to our CTSI email to query for potential interest in the data with 87 inquiries, and to date 46 investigators have requested data and/or additional samples. DISCUSSION/SIGNIFICANCE OF FINDINGS: During the first surge of Covid-19, the CTSI coordinated analyses for the dissemination of results for use by CTSI investigators to minimize duplication of assays and increase availability. The collaboration of research coordinators, biobank, research cores, and informatics demonstrates the power and agility of team science in the Indiana CTSI.

ABSTRACT IMPACT: This study highlights the importance of creatine in developmental myelination and remyelination to investigate whether creatine provides a therapeutic value during a central nervous system (CNS) demyelinating insult with a potential value in patients with Multiple Sclerosis. OBJECTIVES/GOALS: Creatine is vital for ATP buffering in the brain. Interestingly, the cells that generate myelin express the main enzyme for creatine synthesis, Gamt. Patients with Gamt mutations display intellectual delays and impaired myelination. Therefore, we hypothesize that creatine is essential for developmental myelination and improves remyelination. METHODS/STUDY POPULATION: To investigate these hypotheses, we developed a new transgenic mouse model with LoxP sites flanking exons 2-6 of the guanidinoacetate methyltransferase (Gamt) gene where excision leads to expression of a green fluorescent tag allowing us to track the cells normally expressing Gamt. We used immunohistochemistry techniques to look at the corpus callosum, the main white matter tract in the brain, and evaluate the number of oligodendrocytes (OL), glial cells responsible for generating myelin. We also used the cuprizone model of toxic demyelination to investigate whether dietary creatine and cyclocreatine, a planar analog of creatine that more efficiently crosses the blood-brain barrier, can enhance remyelination. RESULTS/ANTICIPATED RESULTS: In this mouse model, we show a 95% (+/-0.47%, n=3) co-localization of Gamt within mature OL during postnatal (P) day P14 with no co-localization in neurons or other glial cells. This suggests that mature OL are the main cells making creatine in the CNS. Next, we show that knocking out Gamt leads to a significant reduction in OL in the developing corpus callosum, at P14 and P21 (P14: 0.007, n=3; P21: 0.04, n=3). We also show that creatine supplementation causes a trending increase in mature OL density in the corpus callosum following cuprizone demyelination (2% creatine; p=0.052; n=4). Interestingly, cyclocreatine supplementation significantly increased mature OL density in the corpus callosum following cuprizone demyelination (0.1% cyclocreatine; p=0.034; n=4). DISCUSSION/SIGNIFICANCE OF FINDINGS: These studies highlight the important role creatine plays in developmental myelination and remyelination to investigate whether creatine and cyclocreatine provide a therapeutic value during a CNS demyelinating insult. This work investigates a potential therapeutic value of creatine to patients with Multiple Sclerosis.

ABSTRACT IMPACT: Lipidomics is emerging as a powerful strategy to identify biomarkers for Major Depressive Disorder, as well as therapeutic targets in lipid metabolic pathways. OBJECTIVES/GOALS: Lipidomics is increasingly recognized in precision psychiatry for global lipid perturbations in patients suffering from Major Depressive Disorder (MDD). We will test the hypothesis that lipid metabolism dysregulation is associated with familial risk of depression. METHODS/STUDY POPULATION: Patients with MDD (G1), children (G2), and grandchildren (G3) have been part of a longitudinal study since 1982. If a parent G2 and grandparent G1 have MDD, G3 is considered a high risk of depression. Biospecimens (saliva and serum) were collected for full exome sequencing and RNA analysis. Samples will also be extracted for lipid content and lipids will be identified by mass spectrometry. A panel of nearly 600 lipid species can reliably be identified and quantified using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). Dysregulated lipids will be correlated with familial risk of depression in samples of G3. RESULTS/ANTICIPATED RESULTS: We hypothesize that dysregulation of lipids and lipid metabolism will be apparent in biospecimens from the high risk compared to the low risk of depression. Also, alterations in RNA transcriptomics of genes involved in lipid metabolic networks are associated with familial risk of depression. Several differential lipid species were previously identified to be associated with MDD. Reduced phosphatidylcholine(PC), phosphatidylethanolamine(PE), phosphatidylinositol(PI), and increased LysoPC, LysoPE, ceramide, triacylglycerol, and diacylglycerol levels have been correlated to MDD. However, these results need to be replicated in independent studies using lipidomics analysis. DISCUSSION/SIGNIFICANCE OF FINDINGS: It is highly likely that completely novel cellular targets will emerge from these studies by uncovering the convergence of lipidomics and genetic variance of lipid metabolic enzymes as biomarkers for predisposition to MDD as well as potential targets for therapeutic development for MDD.

ABSTRACT IMPACT: This work will develop a novel drug delivery system that has improved biocompatibility and controlled release than current systems and allow for customizable loading and drug delivery to unique patient and treatment requirements. OBJECTIVES/GOALS: The goal of my project is a novel hybrid core/shell nanoparticle system for controlling in vivo chemotherapeutic concentration. The current goal is to confirm core and shell polymeric nanoparticle formation via emulsion technique and validate predictive model developed to optimize shell formation efficiency and control shell thickness. METHODS/STUDY POPULATION: Though early results are promising, they are not proof that the desired core/shell structure is being formed via my novel process. I constructed a theoretical model to use to optimize and control the process for precise shell thicknesses. Therefore, the current experimental plan focus is to not only visually confirm the predicted formation of my core/shell design but use these experiments to validate the model.

1. 1. Gel-Suspended SEM: nanoparticles suspended in gel matrix, bisected to reveal inner structure

2. 2. Fluorescent Conjugation Microscopy: visually-distinct dyes used to show polymer distribution and validated against the theoretical model predictions.

3. 3. Modified Hydrophobic Dye Release: different mixtures of polymers with release showing if previous promising results due to core/shell structure RESULTS/ANTICIPATED RESULTS: As stated, the experiments will confirm the core/shell nanoparticle structure, validate the developed theoretical model, or provide direct evidence against any formation. This core/shell structure is key to the current design for controlling payload release rate and thus in vivo drug concentration. For the gel-suspension experiment, the interior core will be labeled with ultrasmall SPIONs and thus any layers within the particles will be distinct. While this result is qualitative, high magnification fluorescent microscope images will be analyzed using image processing software to determine core/shell formation efficiency and compared to estimated efficiencies from the model. Finally, the mixed release will clarify previous experiments’ release mechanism and either support or disprove shell influence. DISCUSSION/SIGNIFICANCE OF FINDINGS: The significance of this work is twofold: core/shell particles have been proven to provide variable control of release on the micron scale but not yet at the nanoscale, allowing for a circulating, targeted system that can finely control release. The process is also novel for producing this type of structure, at highly consistent quality and size.

ABSTRACT IMPACT: This work will help to understand a novel therapeutic approach to a common type of acute myeloid leukemia. OBJECTIVES/GOALS: FMS-like tyrosine kinase 3 (or FLT3) mutations occur in ˜30% of acute myeloid leukemia (AML) cases. FLT3 tyrosine kinase domain (TKD) mutations are particularly important in relapsed/refractory FLT3 mutant AML, which portends poor prognosis. This study describes a therapeutic approach to overcoming resistance conferred by FLT3-TKD mutations. METHODS/STUDY POPULATION: To understand the efficacy of a novel type 1 FLT3 inhibitor (NCGC1481), as a monotherapy and combination therapy, several assays were utilized to interrogate functionality of these therapies. Cell lines and patient samples containing aspartate 835 to tyrosine mutations (D835Y, the most common TKD alteration) and phenylalanine 691 to leucine (F691L) were utilized to examine the effects of NCGC1481 with and without other targeted therapies like MEK inhibitors. Specifically, assays measuring viability, cell death using flow cytometry, in vitro clonogenicity, cellular signaling, and xenograft survival were examined in these FLT3-TKD AML models. Synergy was also measured using well described methods, which also allowed for appropriate dose finding for drug combination experiments. RESULTS/ANTICIPATED RESULTS: Our novel type 1 FLT3 inhibitor (NCGC1481) was particularly effective in the most common FLT3 TKD mutant, D835Y. NCGC1481 reduced viability and cell signaling, while also inducing cell death and prolonging xenograft survival in the FLT3-D835Y model system. In contrast, clinically approved FLT3 inhibitors were less effective at suppressing AML cells expressing FLT3-D835Y. In the case of FLT3-F691L, most of the FLT3 inhibitors tested, including NCGC1481, suppressed canonical FLT3 signaling, but did not significantly reduce viability or leukemic clonogenicity. However, when NCGC1481 was combined with other targeted agents like MEK inhibitors, at synergistic doses, eradication of the FLT3-F691L AML clone was substantially increased. DISCUSSION/SIGNIFICANCE OF FINDINGS: In AML, response to FLT3 inhibitor therapy is often short-lived, with resistance sometimes occurring via FLT3-TKD mutations. Given the dismal prognosis of relapsed FLT3 mutant AML, novel therapies are necessary. This study describes efficacy of a novel FLT3 inhibitor, along with its synergistic activity when combined with other targeted agents.

ABSTRACT IMPACT: This work sheds diagnostic insight on patients with idiopathic rare disease and has the potential to further their care and treatment as a result. OBJECTIVES/GOALS: Correct diagnosis is imperative to treating patients with idiopathic, suspected genetic conditions, yet sequencing approaches leave up to 70% of these patients undiagnosed. We sought to improve diagnosis rates for a cohort patients referred for sequencing by characterizing deleterious variants within the 5’UTR. METHODS/STUDY POPULATION: We retrospectively analyzed whole exome sequencing (WES) data from 472 unsolved rare disease patients within the Mayo Clinic Center for Individualized Medicine to identify variants within the 5’UTR that affect the presence of upstream open reading frames (uORFs). uORFs are short regions (typically 30bp - 600bp) that typically influence downstream gene translation by sequestering ribosomes. We specifically searched for variants with the potential to disrupt existing uORFs or introduce new uORFs within the 5’UTR, and developed a pipeline to annotate these variants with information including GnomAD allele frequency and gene loss of function intolerance (pLI) score. To aid in variant interpretation, we applied two deep learning tools to predict variant impacts on transcript ribosome load (TITER and FramePool). RESULTS/ANTICIPATED RESULTS: Our pipeline identified a median of 21 variants per patient that were predicted to have a deleterious impact on the translational efficiency of protein coding transcripts, primarily by introducing new start codons within the 5’UTR or by altering the Kozak consensus of existing start codons. A median of 10 of these variants occur upstream of haploinsufficient genes with an existing disease association. We also identified a subset of variants that are predicted to introduce translationally active N-terminal extensions to protein coding transcripts, with the potential to disrupt protein localization and processing. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work demonstrates that analysis of 5’UTR variants can be incorporated into existing WES pipelines, and identifies a group of variants with potential significance to patient disease. Further experimental evidence is necessary to ascertain the pathogenicity of these variants.

ABSTRACT IMPACT: Preliminary results will inform the formal evaluation of the reliability of point-of-care ultrasound (POCUS) done by the gastroenterologist compared to standard of care methods such as MR-Enterography. OBJECTIVES/GOALS: Evaluation of mucosal healing is standard for pediatric patients with inflammatory bowel disease (IBD). Point-of-care ultrasound is a non-invasive, cost-efficient tool for assessing intestinal inflammation. We aim to evaluate the agreement between POCUS and typical cross-sectional imaging, such as MR-Enterography (MRE). METHODS/STUDY POPULATION: In this cross-sectional study, we recruited consecutive patients newly diagnosed with IBD, presenting to the specialty outpatient clinic or hospitalized in a pediatric tertiary care center between August to November 2020. They underwent POCUS performed by a single gastroenterologist, in addition to MRE. The sonographer was blinded to MRE results. Bowel wall thickness (BWT) was measured across different bowel segments and recorded twice in longitudinal view and twice in axial view. An average segmental BWT of the four measurements of more than 3 mm was considered inflamed. Agreement between sections of the bowel measured as inflamed were compared to inflamed bowel segments seen by MRE, using Cohen’s kappa. RESULTS/ANTICIPATED RESULTS: Eight of 12 patients completed both MRE and POCUS.A total of 40 bowel segments were assessed, namely the terminal ileum, ascending, transverse, descending and sigmoid colon. There were 4 girls with a median age of 15 years (IQR 14.25-16 years), and 6 patients were diagnosed with Crohn’s disease. Median PCDAI was 32.5 (IQR 30.6-40), and median PUCAI was 75 (72.5-77.5). Agreement between MRE and point-of-care ultrasound was substantial to perfect for the terminal ileum *(κ= 0.75, 95%CI 0.31-1), transverse colon (κ= 1, 95%CI 1-1) and sigmoid colon (κ= 1, 95%CI 1-1). The agreement was poor for the ascending (κ= 0, 95%CI 0-0) and moderate for the descending colon. (κ= 0.6, 95%CI -0.07-1) DISCUSSION/SIGNIFICANCE OF FINDINGS: In pediatric patients with IBD, we found a high agreement between POCUS and MRE for imaging of the terminal ileum, transverse and sigmoid colon, areas commonly involved in IBD. This reinforces adult data, outlining the potential of POCUS as an evaluation tool of disease activity in clinical practice.

ABSTRACT IMPACT: The key to advancing precision medicine is to deepen our understanding of drug modes-of-action (MOA). This project aims to develop a novel method for predicting MOA of potential drug compounds, providing an experimental and computational platform for more efficient drug discovery. OBJECTIVES/GOALS: To develop (1) a targeted CRISPR-Cas9 chemical-genetic screen approach, and (2) a computational method to predict drug mode-of-action from chemical-genetic interaction profiles. METHODS/STUDY POPULATION: Screening drugs against a gene deletion library can identify knockouts that modulate drug sensitivity. These chemical-genetic interaction (CGI) screens can be performed in human cell lines using a pooled lentiviral CRISPR-Cas9 approach to assess drug sensitivity/resistance of single-gene knockouts across the human genome. A targeted, rather than genome-wide, library can enable scaling these screens across many drugs.

CGI profiles can be derived from phenotypic screen readouts. These profiles are analogous to genetic interaction (GI) profiles, which represent sensitivity/resistance of gene knockouts to a second gene knockout rather than a drug. To computationally predict a drug’s genetic target, we leverage the property that a drug’s CGI profile will be similar to its target’s GI profile. RESULTS/ANTICIPATED RESULTS: Five proof-of-principle screens will be conducted with compounds that have existing genome-wide profiles and well-characterized MOA. I will generate CGI profiles for these five compounds and identify genes that are drug-sensitizers or drug-suppressors. I will then evaluate whether targeted library screens can recapitulate the CGIs found in genome-wide screens. Finally, I will develop a computational tool to integrate these CGI profiles with GI profiles (derived from another project) to predict gene-level and bioprocess-level drug targets. These predictions (from both targeted and genome-wide profiles) will be benchmarked against a drug-target and drug-bioprocess standard. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work will develop a scalable, targeted chemical-genetic screen approach to discovering how putative therapeutics work. The targeted screen workflow provides a method for higher-throughput drug screening. The computational pipeline provides a powerful tool for exploring the MOA of uncharacterized drugs or repurposing FDA-approved drugs.

ABSTRACT IMPACT: This work identifies host immune perturbations in sepsis mortality that suggests targets for a precision medicine paradigm in the host response to infection. OBJECTIVES/GOALS: To compare the early whole blood transcriptome during sepsis between 30-day survivors and non-survivors in the Intensive Care Unit (ICU), and to evaluate for pathway enrichment that might explain sepsis lethality. METHODS/STUDY POPULATION: We enrolled 162 sepsis patients in the first 24 hours of ICU admission, particularly targeting individuals requiring vasopressors. Peripheral whole blood was collected in PAXgene vacutainers. Isolated RNA was analyzed with Affymetrix Human Genome ST 2.0 microarray. Differential gene expression was performed with Bioconductor/R/limma using log2 fold-change +/-0.6 as a threshold for differential expression, and a Benjamini-Hochberg adjusted p-value <0.05 to declare significance. Functional gene enrichment was performed using the Gene Ontology (GO) database with PANTHER overrepresentation test (Fisher’s Exact) on all transcripts with adjusted p-value <0.05. Pathways analysis was performed with the Reactome Project using the raw fold change and significance data to identify dysregulated pathways. RESULTS/ANTICIPATED RESULTS: There were 58 non-survivors (36% mortality). We identified 39 genes as differentially expressed between sepsis non-survivors and survivors; 31 were upregulated in non-survivors and 8 had reduced expression. Several of the most overexpressed transcripts are neutrophil-specific, including LCN, MPO, OLF4M4, DEFA3, and DEFA4. A functional gene overrepresentation test further supports this finding, as the most enriched gene ontologies were neutrophil-mediated killing, neutrophil cytotoxicity, neutrophil extravasation, and respiratory burst, all demonstrating higher than 10-fold enrichment and FDR < 0.02. Pathway analysis of the peripheral blood transcriptome was notable for immune response derangement, specifically downregulation of both innate and adaptive immune pathways (FDR < 0.00001). DISCUSSION/SIGNIFICANCE OF FINDINGS: We identified increased expression of neutrophil-related genes in sepsis non-survivors, replicating candidates previously identified in pediatric sepsis mortality and ARDS. These immune perturbations in sepsis mortality may represent key targets for eventually employing precision medicine strategies in sepsis.

ABSTRACT IMPACT: Genomic variation likely plays a role in differences in rates of adverse reactions and efficacy for African populations, and this research will add to the understudied field of pharmacogenomics in African populations. OBJECTIVES/GOALS: We aim to characterize the frequency of variants in clinically relevant genes in East and West African populations to assess the prevalence of potential drug-gene interactions. METHODS/STUDY POPULATION: Our pilot study will consist of 100 Somali patients enrolled at Mayo Clinic Rochester and 100 Ghanaian patients recruited at Teaching Hospitals in Ghana. Germline DNA will be extracted from pre-existing blood samples. Sequencing will be performed using Admera Health’s PGxOne Plus test, interrogating a panel of 62 genes. Variants will be reported along with the predicted response for a list of drugs. Differences between frequencies of variants in East and West African populations will be analyzed. We will look for correlations with reported adverse reaction rates. We will then compare our findings with allele frequency data from publicly available data bases. Additionally, we will analyze the flanking regions of the panel for novel variants, using machine learning to predict gene-drug interactions. RESULTS/ANTICIPATED RESULTS: African populations are known to have more genetic diversity than any other population. Additionally, only African-Americans, African-Caribbeans from Barbados, Esan and Yoruba Nigerians, Gambians, Kenyans, and Sierra Leoneans are accounted for within the publicly available data bases most often used for variant studies. Thus, it is anticipated that we will find differences in the variant allele frequencies of our populations compared to European allele frequencies, and differences in frequencies between the East and West African populations. In the 200 base pair flanking regions that are sequenced in the assay along with the known variant regions, we may find novel previously unreported variants. DISCUSSION/SIGNIFICANCE OF FINDINGS: The lack of knowledge of pharmacogenomic variation in African populations contributes to ethnic disparities in patient outcomes. This study addresses this gap by adding to our comprehension of variants in clinically relevant genes, giving insight into underlying mechanisms of ethnicity-based drug responses.

ABSTRACT IMPACT: Here, we describe extensive sex-specific differences in the transcriptomes of pancreatic neuroendocrine tumors (PNETs). Given that the clinical course of PNETs differs by sex (female sex is associated with better survival), achieving a greater understanding of the specific molecular sexual dimorphisms is invaluable for advancing personalized treatment. OBJECTIVES/GOALS: Epidemiologic studies demonstrate that pancreatic neuroendocrine tumors (PNETs) exhibit sexual dimorphisms with regards to prognosis, disease recurrence, and complication rates. We sought to compare the transcription and DNA methylation landscapes of PNETs by sex, to elucidate molecular differences that may underlie this sex disparity. METHODS/STUDY POPULATION: RNAseq data was generated from PNETs surgically resected at our institution (9 Female; 12 Male patients). RNA was extracted with the RNeasy Mini Kit, stranded sequencing libraries were prepared with TruSeq, and paired end sequencing was done on the HiSeq 2500/4000 systems. Transcript-level quantification was performed with salmon, and DESeq2 was used for differential expression analysis. To account for significant variation due to covariates other than sex, surrogate variables were computed with the SVA package and adjusted for. The goseq package was used for gene set over representation analysis. Matched DNA methylation (DNAm) and RNAseq data was downloaded from GEO (16 F; 16 M). Raw DNAm data was processed with minfi. Differential methylation analysis was done with limma and bumphunter. Analysis was done in R. RESULTS/ANTICIPATED RESULTS: We found that 826 autosomal genes were differentially expressed (DE) by sex in PNETs (at FDR ≤0.1). Gene set over representation analysis performed on the DE genes revealed significant enrichment for several processes, including ‘ascorbate & aldarate metabolism’ and ‘positive regulation of ERK1 & ERK2 cascade’ (all FDR ≤0.1). When we compared DNAm profiles between sexes, we found 8 CpGs which were differentially methylated by sex (at FDR ≤0.1), 7 of which were proximal to genes. Methylation of one of the sex-associated CpGs, overlapping the gene TIMM8B, was found to be negatively correlated with gene expression (rho=-0.41; p-value=0.02). Interestingly, TIMM8B deletion has been previously reported in other non-pancreatic neuroendocrine tumors. There were no differentially methylated regions between sexes. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings demonstrate that PNETs exhibit extensive sexual dimorphisms with regards to gene expression profiles but have largely congruent methylomes by sex. These molecular differences may contribute to the variability in clinical course between men and women, and their characterization is vital for the advancement of personalized medicine.

ABSTRACT IMPACT: This work assesses clinical implementation of a surface guided imaging system to improve the accuracy radiation delivery for treatment of brain lesions using a patient CT derived head phantom. OBJECTIVES/GOALS: Advancements in radiotherapy design have made clinical demand for efficient and accurate methods to deliver stereotactic radiosurgery (SRS) for treatment of intracranial lesions. This study assesses the potential of using surface guided imaging for setup using a 3D patient specific head phantom. METHODS/STUDY POPULATION: A single isocenter, multiple metastases SRS plan was generated on a CT derived RTsafe Prime phantom made of tissue equivalent materials and a polymer gel insert. Five targets of varying diameters were treated with 8Gy of radiation using two different positioning techniques. The first gel insert was irradiated within the phantom according to internal alignment with standard orthogonal x-ray imaging while the second setup used surface guided imaging, based on external anatomy. 42 hours after irradiation, the phantom was scanned in a head coil using a 1.5T MRI. MR images were fused with the patient CT data and structure set to further evaluate calculated and measured dose distributions. RESULTS/ANTICIPATED RESULTS: Discrepancies in phantom setup according to standard orthogonal x-ray imaging compared to surface guided imaging demonstrated to be <1mm in each translational (vertical, longitudinal, and lateral) and angular (rot, roll, pitch) directions. The 3D gel inserts permitted spatial analysis to compare dose distributions of measured values to those calculated in a treatment planning system (TPS). 3D GI (Gamma Index) analysis showed good alignment in high dose regions and resulted in passing rates >94% (5%/2mm) and >87% (3%/2mm). Finally, 3 of 5 targets showed better 3D GI passing rates and less geometric offset for positioning with the surface guided imaging. DISCUSSION/SIGNIFICANCE OF FINDINGS: 3D spatial analysis of human like phantoms demonstrated that patient positioning according to external anatomy performed comparable to standard methods aligning to the internal anatomy, for a multiple met SRS treatment.

ABSTRACT IMPACT: The implementation of DPYD and UGT1A1 pharmacogenetic testing, a promising tool of precision medicine, translates evidence-based research into clinical oncology practice with personalized dosing to better predict interpatient variability in chemotherapy tolerability. OBJECTIVES/GOALS: Patients with DPYD and UGT1A1 genetic variants are at risk for severe toxicity from fluoropyrimidines and irinotecan, respectively. We propose that providing clinicians with the option to order a pharmacogenetic (PGx) test with relevant dose recommendations will increase test uptake to guide pharmacotherapy decisions and improve safety outcomes. METHODS/STUDY POPULATION: We plan to conduct a non-randomized, pragmatic, open-label study in 600 adult patients with gastrointestinal (GI) cancers initiating a fluoropyrimidine- and/or irinotecan-based regimen at three cancer centers within a health system. Implementation metrics of a new, in-house laboratory developed PGx test will be measured, including feasibility of returning results within one week, fidelity of providers following dose recommendations, and penetrance via test ordering rates. Clinical aims will include assessing severe toxicity during the first six months of chemotherapy. Outcomes will be compared to a historical control of GI cancer patients enrolled in a biobank and treated with standard dose chemotherapy. RESULTS/ANTICIPATED RESULTS: We anticipate that there will be an increase in PGx test uptake given its shorter turnaround time to facilitate clinical decision-making prior to the first dose of chemotherapy. Through integration of test results in the electronic health record (EHR) and clinical decision support tools for patients with actionable genotypes, we also expect that providers will have a high level of agreement to the recommended dose adjustments. We anticipate a decreased incidence of severe (Grade >3) toxicity among prospectively genotyped patients in the first six months of chemotherapy compared to DPYD and UGT1A1 variant carriers in the historical control group. Exploratory clinical utility data on costs of hospitalizations, chemotherapy treatment, PGx test, and medical services will also be reported. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study aims to address barriers identified by key stakeholders to implementing PGx testing to better tailor chemotherapy dosing to the genetic profiles to patients. This may prevent adverse event-related hospitalizations, improve quality of life for patients, and reduce health system resource utilization costs.

ABSTRACT IMPACT: The implementation of nasal nitric oxide (nNO) as a diagnostic tool to understand the phenotypic/genotypic profiles of Primary Ciliary Dyskinesia (PCD) in Puerto Rico (PR) will be translated in early disease diagnosis, avoidance of comorbidities, and increase survival in our population. OBJECTIVES/GOALS: This study aims to evaluate the role of nNO levels in PCD diagnosis in the Puerto Rican population. Also, we aim to describe the clinical, genetic, and physiological characteristics of PCD in Puerto Ricans to develop a better understanding of the disease. METHODS/STUDY POPULATION: We plan to conduct a cross-sectional study on participants recruited from patients of the Pediatric Rare Lung and Asthma Institute in PR. We will compare nNO levels among genetically confirmed PCD patients, suspected PCD patients with variant of unknown significance (VUS) mutations, suspected PCD patients without genetic mutations, and age-matched healthy subjects. We plan to analyze clinical data and genetic variants to understand the natural history of the disease. The nNO measurements will be completed following previous published protocols. We will also assess the accuracy of the nNO measurements by repeating the measurements two weeks after the initial measurement. RESULTS/ANTICIPATED RESULTS: We hypothesize that many of the VUS present in our population may represent potential new founder mutations not previously reported in the literature. Our expectation is to identify new atypical PCD phenotypes contemplating the heterogenous genetic Puerto Rican pool. We anticipate that nNO levels will help to screen, identify, and confirm diagnosis of patients with clinical PCD in PR. Our findings will be translated in avoidance of further comorbidities and mortality due to earlier disease PCD diagnosis and will expand our genetic understanding about PCD in PR and other diverse populations with heterogenous genetic admixture. DISCUSSION/SIGNIFICANCE OF FINDINGS: We present a significant and novel research proposal that plan to impact the quality of life of patients living with PCD in PR. The implementation of state-of-the-art diagnostic tools like nNO measurement will positively impact and expand our current capabilities to diagnose rare lung diseases like PCD on the island.

ABSTRACT IMPACT: This study deepens knowledge with respect to the associations between depression, cardiometabolic conditions, and accelerated aging with a clinically accessible marker in a population with disproportionate risk for comorbidity. OBJECTIVES/GOALS: The aim of this secondary analysis is to examine associations between DNA methylation age acceleration (DNAm AA) and depressive symptoms in African American women (AAW) considering the presence of cardiometabolic conditions (CMCs) including hypertension, diabetes, obesity. METHODS/STUDY POPULATION: Genomic and longitudinal clinical data (collected 2015-2020) from the Intergenerational Impact of Genetic and Psychosocial Factors on Blood Pressure Study (InterGEN) cohort (n=227) were utilized for this analysis. DNA methylation age (estimated by the Horvath method) incorporates DNA methylation status at 353 CpG sites. DNAm AA is the residual of DNA methylation age regressed on chronological age in a linear model. Spearman’s correlations and linear regression examine the relationship between DNAm AA and depressive symptoms (Beck Depression Inventory) and cardiometabolic status. The potential association and impact of SES, trauma, substance use, and stress were also considered. RESULTS/ANTICIPATED RESULTS: Contrary to our hypothesis, DNAm AA did not associate with the severity of depressive symptoms. Correlation between DNAm AA and affective symptom subscore (BDI) approached significance (p = 0.06). We observed significant correlations between DNAm AA and specific depressive symptoms including participants’ reported disappointment, disgust, or hatred toward themselves (p < 0.05), difficulty with making decisions (p < 0.05), and worry about their physical health (p < 0.05). DNAm AA was also significantly correlated with BMI (p > 0.001). Significant relationships were not evident in the subsequent regression analysis examining potential relationships between DNAm AA and depression. To our knowledge, this is the first study to examine associations between DNAm AA and depressive symptoms in AAW. DISCUSSION/SIGNIFICANCE OF FINDINGS: Depression limits life quality and quantity and is highly comorbid in CMCs. AAW have a high risk of comorbidity. This study deepens knowledge with respect to the associations between depression, CMCs, and aging with a clinically accessible marker in a population with disproportionate risk.

ABSTRACT IMPACT: The pathophysiologic features of a metabolomic endotype that predicts patient outcomes due to sepsis have the potential to direct new therapies that target immune dysregulation and bioenergetic insufficiency. OBJECTIVES/GOALS: Acute respiratory failure (ARF) requiring mechanical ventilation is a frequent complication of sepsis and other disorders. It is associated with high morbidity and mortality. Despite its severity and prevalence, little is known about metabolic and bioenergetic changes that accompanying ARF. METHODS/STUDY POPULATION: In this study, semiquantitative and quantitative ultrahigh performance liquid chromatography mass spectrometry (UHPLC MS) analysis was performed on patient serum collected from the Trial with Acute Respiratory failure patients: evaluation of Global Exercise Therapies (TARGET). Serum from survivors (n=15) and nonsurvivors (n=15) was collected at day 1 and day 3 after admission to the medical intensive care unit as well as at discharge in survivors. Pathway analysis of the biochemical changes was performed to determine whether the disruption in specific metabolic pathways can identify the bioenergetic and metabolomic profile of these patients. RESULTS/ANTICIPATED RESULTS: Significant metabolomic differences were related to biosynthetic intermediates of redox cofactors nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), increased acyl-carnitines, and decreased acyl-glycerophosphocholines in nonsurvivors compared to survivors. The metabolites associated with poor outcomes are substrates of enzymatic processes dependent on NAD(P), while the abundance of NAD cofactors rely on the bioavailability of dietary vitamins B1, B2 and B6. Changes in the efficiency of the nicotinamide-derived cofactors’ biosynthetic pathways also associate with an alteration of the glutathione-dependent drug metabolism as characterized by the substantial differences observed in the acetaminophen metabolome. DISCUSSION/SIGNIFICANCE OF FINDINGS: This metabolomic endotype represents a previously unappreciated association between severity of outcomes and micronutrient deficiency, thus pointing to new pharmacologic targets and highlighting the need for nutritional remediation upon hospitalization to improve patient outcomes due to ARF.