OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

OBJECTIVES/SPECIFIC AIMS: To learn the edit distance costs of a symbolic univariate time series representation through a stochastic finite-state transducer to predict patient outcomes in intensive care units. METHODS/STUDY POPULATION: High frequency data of patients in intensive care units were used as a data set. The nearest neighbor method with edit distance costs (learned by the FST) were used to classify the patient status within an hour after 10 hours of data. Several experiments were developed to estimate the parameters that better fit the model regarding the prediction metrics. RESULTS/ANTICIPATED RESULTS: Different metrics were obtained for the several parameters. These metrics were metrics (ie, accuracy, precision, and F-measure). DISCUSSION/SIGNIFICANCE OF IMPACT: Our best results are compared with published works, where most of the metrics (ie, accuracy, precision, and F-measure) were improved.

OBJECTIVES/GOALS: Social anhedonia is considered a key feature of schizophrenia that leads to social withdrawal. Research in general anhedonia suggests those with schizophrenia exhibit deficits in anticipated (predicted), but intact consummatory (in-the-moment), pleasure. This study will determine if these temporal differences apply to daily social anhedonia. METHODS/STUDY POPULATION: This project will use experience sampling methods (ESM) to measure real-world social pleasure in people with schizophrenia and healthy controls. Using electronic surveys, participants will predict social activities they plan to do throughout their day and report anticipated pleasure for each. Then, in subsequent surveys, participants will report their consummatory social pleasure shortly after engaging in each of these activities. We will use a time-lagged approach to match anticipated/consummatory pleasure ratings for specific social events and compute a difference score to determine discrepancy. Multi-level modeling will be used to determine if clinical status (schizophrenia/control) predicts anticipated pleasure, consummatory pleasure, and/or their discrepancy. RESULTS/ANTICIPATED RESULTS: Data collection for this project was recently completed. The schizophrenia and control groups (n = 30 per group) were demographically matched for age, sex, race, and ethnicity. Data processing and analyses are currently underway. We hypothesize that, in line with laboratory research in general anhedonia, those with schizophrenia will exhibit deficits in anticipated, but not consummatory, social pleasure throughout their daily lives. Moreover, when anticipated and consummatory pleasure ratings for the same social activities are matched, we expect those with schizophrenia to exhibit larger discrepancies between predicted and in-the-moment pleasure. DISCUSSION/SIGNIFICANCE: This project serves as a critical intermediate step to bridge the gap between laboratory research and patient treatment. ESM bypasses limitations of laboratory studies and increases ecological validity. Results will provide a nuanced understanding of social anhedonia and help identify precise targets to treat social withdrawal in schizophrenia.

OBJECTIVES/GOALS: To describe the impact of Trial-CARE (Coordination, Acceleration, and Recruitment Enhancement), a centralized service core at Washington University (WU), aimed at enhancing the capability of investigator-initiated multi-site clinical trials at WU and partner CTSA institutions. METHODS/STUDY POPULATION: Through review of our marketing materials and service tracking system we defined the following Trial-CARE offerings: Infrastructure: Trial-CARE is a centralized service core at WU. Whereby, a team member is sourced to an investigator-initiated multi-site clinical trial (II-MCT) to ensure the achievement of study milestones across any phase of the clinical trial life cycle. Team: Our team consists of research professionals with expertise in II-MCT project management, data management, and administrative/regulatory management. Services: * One free 60-minute case consultation * Tiers of Service * Academic Research Organization (ARO) support * Clinical Coordinating Center * Data Coordinating Center * II-MCT project management support * Time-limited targeted support RESULTS/ANTICIPATED RESULTS: Trial-CARE has completed 94 consultations in support of WU and partner CTSA institutions enabling the streamlining of study start-up; guidance for study recruitment, implementation, and operations; and offering resources to foster career development. Consultations can be completed at any phase in the clinical trial lifecycle, with Trial-CARE completing: * 12% in the idea phase * 45% in the grant development and submission stage * 28% after funding has been awarded Top reasons researchers connect with Trial-CARE about II-MCTs: * regulatory guidance (40%) * general information about Trial-CARE Services and II-MCTs (35%) * protocol development (21%) * data management (20%) * study budgeting (20%) DISCUSSION/SIGNIFICANCE: In response to a WU wide survey, Trial-CARE plans to generate informational webinars and is creating a clinical trial tracking dashboard, to pro-actively offer support services to researchers prior to grant funding. The goal is also to increase the percentage of consultations completed in the idea phase and the grant development and submission stage.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to assess how quality of life scores change in menopausal women before and after implementation of this aid. In addition, we are also interested in 2 process evaluation objectives: (1) determine if MyChart, the patient portal, is an effective way for this patient population to provide insight their quality of life to their providers and (2) to evaluate providers use of and reactions to the decision support tool. METHODS/STUDY POPULATION: This project is a collaboration between University of Rochester Medical Center and S.U.N.Y. Upstate Medical University. Participants were recruited through Upstate’s Family Medicine and OB/GYN practices via a MyChart invitation sent by the practices. Participating patients will be asked to complete a survey, through MyChart, every 3 months for 18 months. Participating health providers will be trained to use the decision support tool and participate in 3 interviews with the researchers to gain insight into the usefulness and effectiveness of the tool. RESULTS/ANTICIPATED RESULTS: Of the 465 eligible women, 117 women responded to our MyChart invitation to join our study. Of these, 105 agreed to participate and 98 met eligibility criteria. Only half of the women currently enrolled in our study had spoken to a provider about menopause related symptoms (56.1%) prior to study enrollment. DISCUSSION/SIGNIFICANCE OF IMPACT: The goal of this study is to improve menopause related symptoms in women, thus increasing their quality of life, but it will also provide important process evaluation for using EPIC and MyChart for future research studies.

OBJECTIVES/GOALS: Research in Non Alcoholic Fatty Liver Disease Genetics is ongoing. In this project, we aim to: 1.Identify and describe a histological NAFLD Cohort in the MVP Biobank by extracting liver biopsy proven NAFLD patients using natural language processing (NLP) 2.Confirm previously defined NALFD genetic loci (via look up-GWAS) in the histological NLP cohort METHODS/STUDY POPULATION: We will utilize the Million Veteran Program Biobank where a total of 10,959 subjects have been identified using liver biopsy reports in the EMR via CPT codes. Cases will be based on i) steatosis, steatohepatitis, inflammation or fibrosis in liver biopsy reports with ii) exclusion of other causes of liver disease. Controls will be comprised of the general VA population. In collaboration with the Applied NLP and Precision Medicine groups at the VA Informatics and Computing Infrastructure(VINCI); we will attempt to create and validate a histological cohort of NAFLD in the MVP database by: 1)Annotation of biopsy reports for the NLP algorithm 2)Automation/training of the NLP algorithm We will then perform a multi-ancestry genetic lookup of previously established genetic loci among the cases identified. RESULTS/ANTICIPATED RESULTS: Recently published data: the MVP NAFLD research led by Dr. Chang and Dr. Vujkovic had first validated a proxy NAFLD phenotype based on chronic alanine aminotransferase elevation (cALT). A GWAS was then performed using this phenotype which revealed 77 loci of genome-wide significance including 10 established NAFLD- and 52 ALT-associated SNPs were identified. Replication in external Liver Biopsy and Imaging Cohorts validated 17 SNPs of which 9 were novel. Preliminary data on Liver Biopsy reports: Using CPT codes for Liver Biopsy we estimated a total of 10,959 unique patients and 18,812 notes in the MVP biobank. We anticipate 2,000-3,000 NAFLD cases (based on the prevalence of NAFLD in the general population). Initial review reveals 90% concordance between analysts for the purposes of developing an NLP. DISCUSSION/SIGNIFICANCE: NAFLD is a major cause for morbidity and mortality in liver disease. Gold standard for diagnosis is based on biopsy. GWAS studies with biopsy proven phenotypes are limited. The study will aim to isolate a histologically defined NAFLD cohort in MVP to conduct further GWAS that can provide new clinically relevant knowledge for future research.

Contemporary Research Challenges

OBJECTIVES/GOALS: Genetic variation in L-type voltage-gated calcium channels, including CaV1.3, is associated with increased risk for psychiatric disorders including bipolar disorder and schizophrenia. Additionally, rare mutations in CaV1.3 have been linked to epilepsy, developmental delay, and autism. Deletion of CaV1.3 in mice is associated with impaired consolidation of contextual fear conditioning. Some studies have also observed affective behavior deficits in CaV1.3-deficient mice, but other studies have not found affective phenotypes, perhaps due to differences in genetic backgrounds, sex ratios, or task protocols. CaV1.3 is important for slow afterhyperpolarization in hippocampal and amygdala neurons, which prevents excessive firing in response to sustained excitatory input, and CaV1.3-deficient amygdala neurons exhibit hyperexcitability and impaired LTP. CaV1.3 is also expressed in the cerebellum, but its functional role there is not well understood. Given its importance in shaping neuronal activity in the hippocampus and amygdala, we hypothesized that loss of CaV1.3 would cause abnormalities in motor learning as well as affective and cognitive behaviors. METHODS/STUDY POPULATION: Wild-type (WT), haploinsufficient (Hap), and knockout (KO) mice were maintained on a congenic C57BL/6NTac genetic background and were subjected to behavioral tasks including open field, rotarod, ErasmusLadder, elevated zero maze, forced swim test, and tail suspension test. Data were analyzed with sexes combined and with sexes separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CaV1.3 KO was associated with impaired motor learning in the rotarod task (p < 0.05), as well as impaired associative learning in the ErasmusLadder task (p < 0.01), despite intact locomotor function on both tasks. When examined by sex, the rotarod phenotypes were driven by motor learning impairments in males (both Hap and KO, p < 0.05 and p < 0.01, respectively), whereas the ErasmusLadder associative learning phenotypes were present in both sexes only in the KO condition, consistent with previously reported impairments in CaV1.3-deficient mice in consolidation of contextual fear conditioning. Although KO mice learned the motor aspects of the ErasmusLadder task, they learned more slowly. They also failed to learn start cues, which requires intact associative learning. No differences were observed in overall exploration or locomotor activity in open field or elevated zero maze. Analyses from affective tasks are ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary studies provide new evidence that CaV1.3 is important for the function of neural circuits involved in motor learning, and concur with previous data showing its involvement in associative learning. Our data differ slightly from previous studies of CaV1.3 in motor learning, which could be attributable to differences in task protocols and/or genetic background. These results highlight the importance of CaV1.3 in a variety of behaviors, which may help explain why variation in CaV1.3 expression and function has pleiotropic effects in humans.

ABSTRACT IMPACT: By assessing function of mutant (patient-specific) tp53 in zebrafish embryonal rhabdomyosarcoma will inform clinicians of the severity of mutant tp53 alleles. OBJECTIVES/GOALS: This study aims to define loss- and gain-of-function TP53 mutations by comparing effects in tp53-null and wild-type tumors. In addition, it aims to generate a rapid in vivo analysis platform to assign function to patient specific TP53 mutations in the clinic METHODS/STUDY POPULATION: To define tp53 function in ERMS pathogenesis, we previously generated a new tp53-null mutant (tp53-/-) in zebrafish by deleting the entire tp53 genomic locus using TALEN mutagenesis. tp53-/- zebrafish spontaneously develop a spectrum of tumors including sarcomas, leukemia and germ cell tumors (Ignatius…Baxi et. al., eLife) reminiscent of tumors observed in Trp53-null mice. Using the tp53-/- mutants to generate kRASG12D-induced ERMS, we discovered that tp53 is a potent repressor of metastases but rather surprisingly had no effect on self-renewal (Ignatius…Baxi et. al., eLife). Here, using tp53-/- zebrafish, we assessed effects of wild-type and mutant (patient specific) tp53 on tumor initiation, proliferation and apoptosis. RESULTS/ANTICIPATED RESULTS: ERMS tumor initiation in the tp53-/- background is observed in > 97% of animals whereas only <40% of wild-type animals develop ERMS. Additionally, tp53 is a potent suppressor of ERMS proliferation and its effect on apoptosis is minor. Next, we expressed either WT zebrafish or human TP53 in tp53-/- animals along with kRASG12D and both genes suppressed tumor initiation and growth. We co-expressed TP53C176F (found in two ERMS patients) and TP53P153del (identified in a patient with osteosarcoma in our clinic) in zebrafish ERMS, and find that the TP53C176F allele significantly suppressed tumor initiation with effects predominantly on enhanced apoptosis. However, the TP53P153del allele initiated tumors at similar frequency compared to tp53-/- animals but increased the initiation of tumors in the head musculature. DISCUSSION/SIGNIFICANCE OF FINDINGS: Different TP53 alleles identified in patient tumors have very different effects on tumorigenesis in vivo and can respond differently to potentially therapeutic compounds. Thus, the type of precision modeling demonstrated here promises to help further define patient-specific TP53 biology and improve clinical strategies in the future.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if dopamine signaling is altered in a mouse model displaying excessive self-grooming and further elucidate the potential utility of compounds targeting the striatal DA system in modulating repetitive behaviors. METHODS/STUDY POPULATION: Here, we report studies using fast-scan cyclic voltammetry (FSCV) in mice lacking the postsynaptic protein SAP90/PSD95-associated protein (SAPAP3 KO mice) as well as control littermates. Rodent self-grooming provides a behavioral output with which one can monitor repetitive, self-directed, patterned behavior that has great translational value to OCD-like disorders. Total time spent grooming was monitored in SAPAP3KO mice and control littermates. To further examine the role of DA in regulating repetitive grooming behaviors the magnitude and kinetics of DA transients were assessed using FSCV in ex vivo slice preparations as well as in anesthetized mice in vivo. DA transients were elicited in the dorsolateral striatum (DLS), dorsomedial striatum (DMS); and nucelus accumbens core (NAcc). In some experiments mice were crossed with DAT-Cre animals and channelrhodopsin 2 (ChR2) was virally expressed in DA neurons to allow optical stimulation of DA transients. RESULTS/ANTICIPATED RESULTS: As previously reported, SAPAP3 KO mice showed excessive grooming compared to control littermates at the age assessed (4-5 months). DA transients evoked by a single electrical pulse in slices from SAPAP3 KO mice were not significantly different from those observed in slices from control littermates in any of the regions tested including the DLS, DMS and NAcc. However, when four electrical pulses were applied at a frequency of 10Hz to mimic DA neuron bursting, the magnitude of DA transients observed in the DMS and NAcc of SAPAP3 mice were greater than those evoked in control littermates.Interestingly, phasic stimulation produced similar DA transients in the DLS of both genotypes suggesting that phasic DA signaling was not globally altered. To confirm this finding we crossed SAPAP3 KO mice with DAT-Cre mice and injected ChR2 containing virus into the midbrain to selectively express ChR2 in DA neurons. Transients were then optically evoked resulting in selective activation of DA neurons. Optical stimulation produced a pronounced enhancement of DA release in SAPAP3 KO mice specifically in the DMS and only following phasic-like stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: These exciting findings suggest that DA signaling in SAPAP3KO mice is dysregulated in a very precise manner that is sub-region specific as well as dependent on the pattern of stimulation. These results suggest that targeted therapies that can modulate these specific modes of dopaminergic signaling in these distinct striatal subregions could provide improved efficacy in OCD patients that are resistant to SSRI treatment.

OBJECTIVES/SPECIFIC AIMS: Modulation of autophagy has the potential to treat inflammatory bowel disease (IBD). IBD is characterized by dysregulated inflammatory pathways and a defective intestinal epithelial barrier. We sought to better understand how autophagy can be utilized to regulate both inflammation and the intestinal barrier. METHODS/STUDY POPULATION: We examined mice with an autophagy defect in only macrophages in an animal model of IBD. To understand the phenotype, we utilized macrophages to investigate the mechanism behind autophagy and proinflammatory cytokine secretion. In addition, we analyzed the development of colonoids in a co-culture system with macrophages with or without a functional autophagy pathway. Lastly, pharmacological modulation of autophagy to control inflammation was assessed. RESULTS/ANTICIPATED RESULTS: Mice with autophagy-deficient macrophages were highly susceptible to intestinal barrier disruption. Susceptibility was due to enhanced proinflammatory cytokine secretion and intestinal permeability. Furthermore, proinflammatory macrophages (due to an autophagy defect) co-cultured with colonoids, significantly decreased the number of mucus producing goblet cells. Finally, pharmacologically modulation of autophagy reduced the secretion of proinflammatory cytokine by macrophages and reduced intestinal permeability. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results strongly suggest autophagy modulation can dampen inflammation and enhance the intestinal epithelial barrier.

OBJECTIVES/GOALS: A collaboration among Indiana CTSI community health partnerships (CHeP), bioethics, and regulatory programs identified and reviewed human research protection training programs targeting community engaged research, and pilot tested CIRTification with community partners working across a range of contexts. METHODS/STUDY POPULATION: We searched community human research protection training programs from across the county, identified three, examined each based upon criteria identified by community partners (time, relevance, online delivery) and our Human Research Protection Program (HRPP), and selected CIRTification (CIRT) to pilot. Ten community research partners volunteered to complete CIRT and a debriefing interview. Four completed CITI training previously. Participants included local and state-wide organizations, a resident, a state agency, and a hospital, and came from rural and urban communities. Interviews covered practical issues (ease of use, language, time), relevance, and comparison to CITI. Results were shared with HRPP for approval. RESULTS/ANTICIPATED RESULTS: Most felt CIRT was easy to navigate and engaging, and those who had done CITI felt CIRT was more relevant and engaging. The sections on historical background and recruitment were cited as most valuable. Suggestions were made to increase the diversity of examples beyond health care research. Community members mentioned several applications for CIRT including: (1) helping their own community work; (2) empowering them to be an advocate; (3) referring others to CIRT; (4) influencing approaches to recruitment and community engagement; and (5) applying ethics principles to their other community work. The Human Research Protection Program approved CIRT in place of CITI for community researchers. DISCUSSION/SIGNIFICANCE: Our process represents collaboration across the Indiana CTSI, HRPP and community partners, and use of best practices. Exemplifying “nothing about us without us”, actions were based on direct input from community partners, with the goal of decreasing barriers to engaging communities in research.

Academic discovery in biomedicine is a growing enterprise with tens of billions of dollars in research funding available to universities and hospitals. Protecting and optimizing the resultant intellectual property is required in order for the discoveries to have an impact on society. To achieve that, institutions must create a multidisciplinary, collaborative system of review and support, and utilize connections to industry partners. In this study, we outline the efforts of Case Western Reserve University, coordinated through its Clinical and Translational Science Collaborative (CTSC), to promote entrepreneurial culture, and achieve goals of product development and startup formation for biomedical and population health discoveries arising from the academic ecosystem in Cleveland. The CTSC Office of Translation and Innovation, with the university’s Technology Transfer Office (TTO), helps identify and derisk promising IP while building interdisciplinary project teams to optimize the assets through key preclinical derisking steps. The benefits of coordinating funding across multiple programs, assuring dedicated project management to oversee optimizing the IP, and ensuring training to help improve proposals and encourage an entrepreneurial culture, are discussed in the context of a case study of therapeutic assets, the Council to Advance Human Health. This case study highlights best practices in academic innovation.

Little has been published on the demographic composition of the clinical and translational science research workforce within the Clinical and Translational Science Awards (CTSA) Program despite the well-documented need for greater diversity in the biomedical research workforce. Analyses of workforce demographic reveal that women and members of underrepresented groups remain persistently underrepresented in the CTSA hub and training components principal investigators. In contrast, in the CTSA Program career development and training programs, females have greater representation as participants, and non-Whites were better represented in training programs.

OBJECTIVES/SPECIFIC AIMS: Issues with recruiting the targeted number of participants in a timely manner often results in underpowered studies, with more than 60% of clinical studies failing to complete or requiring extensions due to enrollment issues. The objective of this study is to develop and implement a scalable, organization wide platform to enhance accrual into clinical research studies. METHODS/STUDY POPULATION: We are developing and evaluating an informatics platform called Utah Utility for Research Recruitment (U2R2). U2R2 consists of 2 components: (i) Semantic Matcher: an automated trial criterion to patient matching component that also reports uncertainty associated with the match, and (ii) Match Delivery: mechanisms to deliver the list of matched patients for different research and clinical settings. As a first step, we limited the Semantic Matcher to utilize only structured data elements from the patient record and trial criteria. We are now including distributional semantic methods to match complete patient records and trial criteria as documents. We evaluated the first phase of U2R2 based on a randomized trial with a target enrollment of 220 participants that compares 2 treatment strategies for managing back pain (physical therapy and usual care) for individuals consulting a nonsurgical provider and symptomatic <90 days. RESULTS/ANTICIPATED RESULTS: U2R2 identified 9370 patients from the University of Utah Hospitals and Clinics as potential matches. Of these 9370, 1145 responded to the Back Pain study research team’s email or phone communications, and were further screened by phone. In total, 250 participants completed a screening visit, resulting in the current study enrollment of 130 participants. Forty-three of 1145 patients refused to participate, and 50 participants no-showed their screening visit. DISCUSSION/SIGNIFICANCE OF IMPACT: A recruitment platform can enhance potential participant identification, but requires attention to multiple issues involved with clinical research studies. Clinical eligibility criteria are usually unstructured and require human mediation and abstraction into discrete data elements for matching against patient records. In addition, key eligibility data are often embedded within text in the patient record. Distributional semantic approaches, by leveraging this content, can identify potential participants for screening with more specificity. The delivery of the list of matched patient results should consider characteristics of the research study, population, and targeted enrollment (eg, back pain being a common disorder and the possibility of the patient visiting different types of clinics), as well as organizational and socio-technical issues surrounding clinical practice and research. Embedding the delivery of match results into the clinical workflow by utilizing user-centered design approaches and involving the clinician, the clinic, and the patient in the recruitment process, could yield higher accrual indices.

ABSTRACT IMPACT: Insights from this project will provide clinical guidance in treatment of immunotherapy in triple negative breast cancer and identify early imaging biomarkers of treatment response. OBJECTIVES/GOALS: Significant research that addresses monitoring and predicting patient response of triple negative breast cancer (TNBC) to immunotherapy is needed. Using positron emission tomography (PET) imaging to probe the tumor microenvironment (hypoxia, T-cell activation), we aim to predict early response to immunotherapy for in mouse models of TNBC tumors. METHODS/STUDY POPULATION: Female Balb/c mice with 4T1-luciferase mammary carcinoma cell tumors were administered paclitaxel (PTX; 10 mg/kg), anti-PD1 (200 µg), both, or vehicle (saline) intraperitoneally. Treatment was given on days 0, 2, and 5 for cohort 1 (n=16) who underwent granzyme B specific (GZP) PET imaging (T-cell activation) and days 0, 2, 5, and 8 for cohort 2 (n=12) who underwent [18F]-fluoromisonidazole (FMISO)-PET imaging (hypoxia). Bioluminescence (BLI) imaging and caliper measurements were performed to track tumor size changes at multiple timepoints and tumors were collected for histological validation on day 20. Mean standard uptake value (SUVmean) was calculated as percent of day 0, and statistical analyses were performed with unpaired t-tests and Wilcoxon-rank sum tests. RESULTS/ANTICIPATED RESULTS: Non-responders to treatment had a significantly higher tumor volume compared to responders starting on day 6 (p<0.05). Although no significant differences in BLI between control and single-agent therapies were found, BLI data revealed that treatment with combination PTX and anti-PD1 significantly decreased viability signal between days 3 and 6 (p=0.04). SUVmean from GZP-PET was over 250% higher in responders compared to non-responders by day 6 (p=0.03). SUVmean from FMISO-PET was 80% less in responders compared to nonresponders, indicating less tumor hypoxia (p=0.04). DISCUSSION/SIGNIFICANCE OF FINDINGS: Non-invasive PET imaging of the tumor microenvironment can provide data on T cell activation and hypoxic response predicting response to combination immunotherapy and chemotherapy. Utilizing advanced imaging to understand biologically distinct features of the TNBC tumor microenvironment can aid in personalizing anti-cancer therapies.

OBJECTIVES/GOALS: Natural killer (NK) cells are a potential cancer therapeutic but expanding NK cells efficiently in vitro is difficult. Natural killer cell deficiency (NKD), a primary immune deficiency affecting only NK cells, is caused by defects in several DNA replication proteins. By studying NKD we will achieve better NK cell in vitro differentiation. METHODS/STUDY POPULATION: One patient with NKD has a compound heterozygous mutation in the essential DNA replication protein MCM10. We hypothesize that in individuals with NKD, dramatic telomere erosion from abnormal DNA replication leads to premature senescence and the loss of NK cells. To test our hypothesis, we will knockout one allele of MCM10 or over express MCM10 in NK cells isolated from blood. We will then monitor telomere length, expansion and cytotoxic activity of these NK cells. To understand the role of MCM10 in early stages of NK cell development we will deplete MCM10 in induced pluripotent stem cells and differentiate these cells into NK cells. During this differentiation we will monitor progression through NK cell developmental stages as well as telomere length and senescence markers. RESULTS/ANTICIPATED RESULTS: Telomeres insulate chromosomes and induce permanent growth arrest (senescence) when they are critically short. We have demonstrated that depletion of a DNA replication protein causes telomere erosion and increases senescence markers. NK cells have shorter telomeres and lower telomerase expression than other immune cells. We predict, this relatively poor telomere maintenance sensitizes NK cells to telomere loss upon depletion of replication proteins. During in vitro differentiation, we expect NK cell precursors to undergo premature senescence secondary to telomere shortening. Furthermore, we expect supplementation of DNA replication proteins will enhance NK cell expansion and maturation. DISCUSSION/SIGNIFICANCE OF IMPACT: NKD patients have provided the scientific community with clues as to what proteins NK cells rely on for their development. This project aims not only to understand why these proteins are critical, but to harness that information for cellular anti-cancer therapeutics.

OBJECTIVES/GOALS: Researchers include community health workers and promotoras (CHW/Ps) on research teams to increase community engagement; however, no formal training on research best practices exists for this group. Study objectives were to examine perceived relevance of a new culturally and linguistically appropriate CHW/P training and optimal delivery modes. METHODS/STUDY POPULATION: We conducted six focus groups (FGs), three each in English and Spanish, at three study sites, University of Florida, University of Michigan, and University of California Davis from February to August 2021. The CHWs/Ps were purposively selected to include diverse age, race/ethnicity, educational level, and work experience. Separate FGs were conducted for CHWs/Ps in English and Spanish as appropriate. All FGs were audio recorded, translated to English from Spanish, transcribed and analyzed using RADaR (Rigorous and Accelerated Data Reduction) technique. RESULTS/ANTICIPATED RESULTS: Forty CHWs/Ps (95% women, mean age 45) participated, with the majority (58%) identifying as Hispanic/Latino. Of the sample, most identified as White (50%) or Black (25%). The proposed training was mentioned as relevant and would help them to be confident, comfortable, knowledgeable and effective in the community. Online training, though advantageous due to its flexibility also reportedly had barriers such as internet access, computer availability and technological know-how of CHWs/Ps. A hybrid training approach, online plus peer-led, was recommended due to the importance of personal guidance by an experienced CHW/P’ especially for a newly recruited CHW/P. DISCUSSION/SIGNIFICANCE: Findings indicated that a culturally and linguistically appropriate CHW/P training that is flexible and easily accessible in its mode of delivery is relevant and useful. In-person guidance to a new CHW/P was reported as an important training component. Poster will include the detailed quotes on relevance, usefulness, and mode of delivery of training.

OBJECTIVES/SPECIFIC AIMS: CFTR is the mutant protein that causes cystic fibrosis (CF), a fatal respiratory diseases affecting 1 in 3500 children. CFTR modulators are small molecules that directly address mutant CFTR function. Improving correction of the F508del CFTR mutation (affecting 90% of CF patients) is one of the most pressing unmet needs in CF. Currently available F508del therapeutics only marginally improve CF, In vitro, we have identified a miRNA that impairs utility of CFTR directed therapies. miR-145 is upregulated by TGF-β (a genetic modifier of CF lung disease) with a direct binding site on the 3’-untranslated region of CFTR mRNA. Binding of miR-145 to CFTR destabilizes mRNA transcript and impedes protein translation. Overexpression of miR-145 abolishes benefit of F508del CFTR correction. Antagonists to miR-145 block TGF-β suppression of CFTR function and augment response to CFTR correction. This project evaluate in vivo impact of TGF-beta and miRNA manipulation on CFTR functional readouts including nasal potential difference (NPD) and short circuit current (Isc) across tracheal explants in addition to standard biochemical measures. METHODS/STUDY POPULATION: Wild-type Sprague-Dawley rats were inoculated with an adenoviral vector containing bioactive TGF-beta or sham at 1×109 pfu/animal placed in the left nares. Seven days post-inoculation, functional, and biochemical measures were conducted. NPD was measured with a microelectrode placed in the left nare and grounded the tail. The nare was sequentially perfused with standard Ringer’s solution, amiloride (to block the ENaC sodium channel), low chloride Ringer’s (to stimulate chloride efflux), forskolin (to open the CFTR channel) and CFTRinh-172 (to block the CFTR channel. Tracheal explants were harvested, microdissected, and placed on modified Ussing chambers. RESULTS/ANTICIPATED RESULTS: We have inoculated WT rats with bioactive TGF-β Versus sham delivered by intranasal inoculation of an adenoviral vector. Functional readout of CFTR function is by Isc across tracheal epithelia and NPD. Lung homogenates are analyzed for TGF-β signaling, miRNA expression, and CFTR transcripts. Both tracheal explants and NPD indicate TGF-β stimulation diminishes CFTR function in vivo. In tracheal explants, TGF-β exposure diminishes CFTR response to forskolin-stimulation by 75%. Loss of current after CFTR inhibition (CFTRinh-172) is halved. By nasal PD, TGF-β inoculation similarly halves the bioelectric response to low chloride and forskolin stimulation. Evaluation by qPCR reveals a strong increase in TGF-β signaling demarcated by PAI-1, prompting a reduction in CFTR mRNA. miR-145 is expressed highly in rat pulmonary tissue, but no change in overall miR-145 levels was detected between TGF-β and sham exposed rats. This finding reflects what we have observed in human lungs, with a localized increased miR-145 expression in CF epithelia, but similarly high levels of miR-145 in both CF and non-CF whole lung homogenates. Although expressed at lower levels than miR-145, we did find increased expression in TGF-β relevant miR-101, miR-494, and miR-144 that have a predicted binding site on rat 3’-UTR in TGF-β exposed Versus sham lungs. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data indicate the relevance of TGF-β stimulation to suppress CFTR synthesis and function in vivo. Future work will evaluate whether these additional miRNA with CFTR binding sites may mediate TGF-β suppression of CFTR in the rat model, and the utility of miRNA manipulation to augment F508del CFTR correction.