OBJECTIVES/SPECIFIC AIMS: Deficits in reward-based learning have been shown in youth at risk for developing substance use disorders (SUD). Here, we investigated whether computational models can be used to more precisely delineate the additive effects of such risk loading (i.e., the comparison between youth with ADHD, and those with ADHD and familial SUD) on reward-based learning in youth. METHODS/STUDY POPULATION: In total, 41 drug-naïve youth, stratified into 3 groups based on ADHD diagnosis and parental SUD: healthy controls (HC, n=13; neither ADHD nor parental SUD), low risk (LR, n=13; ADHD only), and high risk (HR, n=15; both ADHD and parental SUD), performed a reward task. Learning rates, prediction and congruence t-scores were computed using a reinforcement learning model and analyzed via a multivariate ANOVA. RESULTS/ANTICIPATED RESULTS: The analyses showed a significant linear effect in task accuracy, which decreased with increasing risk profiles. Analyses of the model-derived variables also showed similar significant linear effects in learning rates and the congruence t-score, but not in the prediction t-score. These effects were primarily driven by significantly higher learning rate and congruence t-score in HC compared with HR youth. DISCUSSION/SIGNIFICANCE OF IMPACT: These results show most profound deficits in reward-learning in HR youth. These findings also show that computational analyses can offer added value over conventional behavioral analyses by more precisely evaluating group differences in relation to SUD risk.

OBJECTIVES/SPECIFIC AIMS: Despite aggressive chemotherapy, surgical resection, and radiation therapy, glioblastoma remains almost universally fatal. In a pilot, randomized, and blinded clinical trial, we recently demonstrated that administration of RNA-loaded DC vaccines was associated with significantly improved progression-free and overall survival in patients with glioblastoma (Mitchell et al., Nature, 2015). Furthermore, clinical outcomes correlated with DC migration to vaccine-site draining lymph nodes measured by Indium-111 labeling of RNA-loaded DCs and SPECT/CT imaging. Although these studies demonstrated that tracking DC migration may be an important clinical biomarker for response to DC vaccination, the complexity and regulatory requirements associated with nuclear labelling to track DC migration limits widespread application of this technique. We have therefore developed RNA-loaded magnetic nanoparticles (RNA-NPs) to enhance DC migration to LNs and track that migration with a widely available imaging modality (i.e., MRI). METHODS/STUDY POPULATION: Cationic liposomes were loaded with iron oxide nanoparticles with or without cholesterol. The resulting nanoparticles were complexed with RNA and used to transfect DCs ex vivo. RNA-NP-loaded DsRed+ DCs were then injected intradermally into mice and tracked noninvasively with T2-weighted 11T MRI before excision and quantification with flow cytometry. RESULTS/ANTICIPATED RESULTS: In vitro experiments demonstrate that iron oxide loading does not reduce RNA-NP-mediated transfection of DCs. Additionally, replacement of cationic lipids with cholesterol increased RNA-NP transfection of the DC2.4 cell line and enhanced the T cell stimulatory capacity of treated bone marrow-derived dendritic cells (BMDCs). Compared to electroporation, RNA-NPs enhanced DC migration to lymph nodes and reduced T2 MRI intensity in DC-bearing lymph nodes. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that iron oxide-loaded RNA-NPs enable noninvasive cell tracking with MRI and enhance DC migration to lymph nodes. We have further shown that inclusion of cholesterol in RNA-NPs augments the stimulatory capacity of transfected DCs. Future work will consider effects of RNA-NPs on antitumor immune responses and the utility of MRI-detected DC migration as a biomarker of vaccine efficacy.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to further evaluate underlying disease parameters in respiratory syncytial virus (RSV) infection, that is reduction in antioxidant potential, and determining if supplementation of the antioxidant enzyme catalase could be employed as a potential therapeutic. METHODS/STUDY POPULATION: Nasopharyngeal secretions were obtained from patients (<2 years old) verified for RSV infection, and assessed for catalase activity and correlated with disease parameters. In addition, the BALB/c animal model of RSV infection was utilized to directly study the effect of supplemental catalase on RSV-related disease parameters in vivo. The catalase formulation used in these studies is pegylated, and has been tested to provide long-term increased catalase activity in vivo. We are also currently working on designing an in vitro model of catalase supplementation in A549 bronchial epithelial cells. RESULTS/ANTICIPATED RESULTS: Our preliminary data shows that patients with more severe disease (based on hospitalization, oxygen supplementation) have significantly lower levels of catalase activity (p<0.02). Additionally, when pegylated-Catalase (PG-CAT) treatment is utilized in RSV infection of mice, there is significant improvement in several disease parameters. PG-CAT-treated mice show an attenuated body weight loss (p<0.001) and clinical disease (p<0.02), and also have lower levels of key pro-inflammatory cytokines including CXCL1 and TNF-α. PG-CAT treatment also resulted in a minor decrease in viral titer, which is being further evaluated. In addition, PG-CAT treatment resulted in an improvement in airway hyperresponsiveness observed at baseline, we are further characterizing this improvement and also conducting methacholine challenges. Currently, we are working to determine the underlying mechanism through which PG-CAT results in these improvements, and whether it is through changes in immune cell populations, cellular signaling or apoptosis signaling pathways (i.e., caspases). DISCUSSION/SIGNIFICANCE OF IMPACT: RSV is the leading cause of viral pneumonia and bronchiolitis in infants, with no vaccines or effective therapeutics available currently. Our study indicates that catalase activity could be used as a potential correlate for disease severity and be used as an indicator of disease during patient treatment. Additionally, and more importantly supplementation of catalase could be used as a potential therapeutic for treatment of RSV.

OBJECTIVES/SPECIFIC AIMS: Treatment of acute myeloid leukemia (AML) is challenging, as apoptosis-resistant AML cells often persist within the bone marrow microenvironment despite chemotherapy. The overall goal of our laboratory is to identify and ultimately target the bone marrow factors that protect AML cells. METHODS/STUDY POPULATION: Using cell cultures, we previously reported that SDF-1 (CXCL12), an abundant bone marrow chemokine, induces apoptosis of isolated CXCR4+ AML cells, including freshly isolated bone marrow-derived AML cells from approximately one-third of AML patients. However, co-culture of AML cells with differentiating osteoblasts protected AML cells from apoptosis. RESULTS/ANTICIPATED RESULTS: Histone deacetylase inhibitors (HDACi) abrogated the ability of osteoblasts to protect AML cells and altered expression of matrix mineralization genes including tissue nonspecific alkaline phosphatase (TNAP). A different drug, cyclosporine A (CSA), similarly inhibited osteoblast-mediated protection of AML cells and reduced TNAP expression. Specifically targeting osteoblast TNAP via siRNA was sufficient to prevent osteoblasts from protecting AML cells in co-cultures. In addition, we are targeting TNAP enzymatically. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that targeting TNAP may be useful in AML treatment to render the bone marrow microenvironment more hostile to leukemic cell survival.

OBJECTIVES/SPECIFIC AIMS: The goals of our study are: (1) To test the hypothesis that the presence of any autoimmune cytopenia (ITP, AIHA, or ES) at time of cSLE diagnosis is associated with decreased risk of developing LN. (1b) To test the hypothesis that there is a lower risk of LN in patients with cSLE and any co-existing autoimmune cytopenia (ITP, AIHA, or ES) who had treatment with immunomodulatory or immunosuppressive therapy (intravenous immunoglobulin, corticosteroids, rituximab, or cyclophosphamide) before diagnosis of cSLE. (2) To test the hypothesis that in patients with cSLE who develop LN, the presence of any co-existing autoimmune cytopenia (ITP, AIHA, or ES) at time of cSLE diagnosis is associated with less severe LN. (3) To test the hypothesis that at the time of cSLE diagnosis, there is a lower incidence of double-stranded DNA (dsDNA) and a higher incidence of ribonucleoprotein autoantibodies in those with co-existing autoimmune cytopenias (ITP, AIHA, or ES). METHODS/STUDY POPULATION: This is a retrospective study of a large cohort of patients from the Emory Children’s Center, Children’s Healthcare of Atlanta (CHOA) satellite clinics and pediatric rheumatology inpatient services at any of the 3 CHOA hospitals (Egleston, Scottish Rite, and Hughes Spalding) with ICD 9 or ICD 10 codes corresponding to a diagnosis of SLE between January 1, 2000 and January 31, 2015. We will include patients diagnosed at age 2–16 years who meet at least 4 of the 11 American College of Rheumatology (ACR) classification criteria for SLE. We will consider these patients as having cSLE. We will exclude patients with less than 2 years of follow-up data and patients with a pre-existing diagnosis of cSLE who transferred care to our Emory/CHOA center. We will define time of diagnosis as time from initial evaluation for cSLE by a pediatric rheumatologist up to 28 days post cSLE diagnosis. We will define co-existing autoimmune cytopenia as preceding diagnosis of a primary autoimmune cytopenia or the presence of an autoimmune cytopenia at the time of initial evaluation for cSLE and up to 28 days post cSLE diagnosis. We will define AIHA as hemoglobin ≤10 g/dL with positive direct Coombs and/or reticulocytosis. We will define ITP as thrombocytopenia <100,000/mm3 and Evans syndrome as concurrent or sequential AIHA and ITP. We will define lupus nephritis (LN) as the presence of urine protein to creatinine ratio>0.5 in a patient with cSLE and/or biopsy demonstrating LN. IRB approval of the study protocol with waiver of informed consent has been obtained from the CHOA IRB. RESULTS/ANTICIPATED RESULTS: We have approximately 40 newly diagnosed cSLE patients annually; therefore, a study population of 400 patients with cSLE is possible. Therefore, assuming 50% of cSLE patients without autoimmune cytopenias have LN and 22% of cSLE patients with autoimmune cytopenias have LN, at an alpha of 0.05, we will have > 80% power to detect significant differences. We expect to show phenotypic differences in patients with co-existing autoimmune cytopenia and cSLE from other newly diagnosed cSLE patients. We expect that the presence of a co-existing autoimmune cytopenia and cSLE is associated with decreased risk of developing LN. We expect that there will be a decreased prevalence of LN in cSLE patients pretreated with immunosuppression further highlighting that earlier indicators of LN risk and early interventions are necessary. We expect to find decreased severity of LN in patients with a co-existing autoimmune cytopenia at time of cSLE diagnosis. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study will be conducted on one of the largest single-center cohorts of cSLE patients. We will determine whether pediatric patients with SLE and autoimmune cytopenias have a distinct clinical or serological phenotype and less severe disease. Our results will be significant in developing hypothesis for further retrospective or prospective multi-center or large database and immunological studies to understand the relationship of each individual autoimmune cytopenia to cSLE. It will provide the necessary background for further clinical and immunological studies to identify predictive biomarkers of cSLE severity.

OBJECTIVES/SPECIFIC AIMS: Pre-clinical and clinical observations have noted that increased aortic dilation is associated with male sex. Using an experimental model of severe, syndromic thoracic aortic aneurysms, we quantify aortic dilation and elastin stability in male Versus female mice. METHODS/STUDY POPULATION: Ascending aortas from male and female FBN1mgR/mgR mice and their wild type littermates were assessed every 4 weeks from 6 to 18 weeks of age by ultrasound. Measurements were taken luminal edge to luminal edge in diastole. At termination, aortas were harvested for RT-PCR analysis of extracellular matrix genes. Aortas were serially sectioned and elastin fragmentation was imaged by auto-fluorescence. RESULTS/ANTICIPATED RESULTS: At 12 weeks of age, differences of aortic diameters between male and female FBN1mgR/mgR mice were significantly different (2.24±0.43 vs. 1.57±0.22 mm; p=0.002), while there were no significant differences between sexes of wild type littermates (1.29±0.13 vs. 1.23±0.08 mm; p=0.71). Male sex was associated with increased elastin but not fibrillin-1 mRNA expression. Ascending aortas from male and female FBN1mgR/mgR mice significantly differed in the degree of elastin fragmentation (2.76 vs. 1.85 breaks/ 100 µm aorta; p=0.03). DISCUSSION/SIGNIFICANCE OF IMPACT: Sexual dimorphism of thoracic aortic dilation observed in human TAA patients was recapitulated in the fibirllin-1 hypomorphic mouse model of syndromic thoracic aortic aneurysms. Differences in this mouse model could be explained by the differential expression of extracellular matrix genes.

OBJECTIVES/SPECIFIC AIMS: Lung cancer claims 160,000 lives in the United States every year, and lung adenocarcinoma (LADC) is the most frequent type. Early diagnosis is crucial. Computed tomography (CT) is very sensitive in identifying early-stage lung nodules, but has low specificity. Increased glucose uptake is a hallmark of cancer measurable in vivo by fluorodeoxyglucose (FDG) positron-emission tomography (PET). FDG PET is widely used for cancer staging but has low sensitivity in the diagnosis of solitary lung nodules. We have previously identified an alternative glucose transporter, SGLT2, expressed in different types of cancer but not detected by FDG PET. SGLT2 activity can be measured in vivo with the PET tracer methyl-4-fluorodeoxyglucose (Me4FDG). The objective of this study was to test the hypothesis that SGLT2 is a novel diagnostic and therapeutic target in FDG-negative, early stage LADC. METHODS/STUDY POPULATION: To study glucose transporter expression in LADC, we performed immunohistochemistry with SGLT2- and GLUT1-specific antibodies in human lung pre-malignant lesions and LADC samples. To verify the possibility of detecting SGLT2 activity in vivo, we performed microPET imaging with the SGLT-specific tracer Me4FDG in a Kras-driven, p53-null genetically engineered mouse model and in patient-derived xenografts of LADC. Finally, we performed therapeutic trials in genetically engineered and patient-derived mouse models of LADC with the FDA-approved SGLT2 inhibitor canagliflozin. RESULTS/ANTICIPATED RESULTS: We observed a switch in the modality of glucose transport during lung carcinogenesis: SGLT2 was highly expressed in pre-malignant lesions and well-differentiated LADC, whereas GLUT1 was upregulated in advanced, poorly differentiated lesions. This pattern was observed both in human samples and in murine models. This observation led us to hypothesize that early-stage LADCs are often negative on FDG PET because this imaging modality does not detect the activity of SGLT2, which is expressed in early lesions. Therefore, we performed PET imaging with the tracer Me4FDG, that measures SGLT2 activity, in our mouse model, and observed that Me4FDG accumulated in small nodules that were negative with FDG. We confirmed the functionality of SGLT2 in human LADC by Me4FDG PET in patient-derived xenografts. To investigate the role of SGLT2-mediated glucose uptake in the early stages of LADC development, we treated both genetically engineered mice and patient-derived xenografts with FDA-approved SGLT2 inhibitors, showing that SGLT2 inhibition effectively reduced LADC growth and prolonged survival in mouse models. In addition, Me4FDG uptake predicted response to SGLT2 inhibition. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results show that sodium-dependent glucose transport is a critical metabolic supply strategy in the early stages of lung adenocarcinoma development, and that Me4FDG is a novel biomarker of early LADC and of SGLT-dependent tumor growth. The discovery of SGLT2 in LADC highlighted the need for a re-interpretation of FDG-negative lung nodules, which might rely on SGLT2 for glucose uptake, and therefore may be detected by the new tracer Me4FDG. We anticipate our findings will lead to clinical studies evaluating Me4FDG as a diagnostic tracer for solitary lung nodules and early LADC, and as a biomarker for the selection of patients eligible for treatment with SGLT2 inhibitors.

OBJECTIVES/SPECIFIC AIMS: Computed tomography (CT) enables 3-dimensional (3D) visualization of cortical bone structures with high spatial resolution, and thus has been the gold-standard method for evaluation and diagnosis of craniofacial skeletal pathologies. However, ionizing radiation and, in particular, repeated scanning for presurgery and postsurgery assessments, is of concern when applied to infants and young children. Recent advances in solid-state MRI allow the capture of the short-T2 signals in cortical bone while suppressing the signal from soft-tissue protons having T2 relaxation time 1–2 orders of magnitude longer (50–100 ms). One approach, a dual-radiofrequency (RF) pulse and ultrashort echo time (UTE) imaging based method, exploits different sensitivities of bone and soft tissue to different RF pulse widths and TEs. This study aims to demonstrate the feasibility of producing 3D renderings of the human skull and visualization of cranial sutures using the bone-selective MRI technique in comparison to CT. METHODS/STUDY POPULATION: Imaging technique: Two RF pulses differing in duration and amplitude are alternately applied in successive repetition time (TR) along the pulse train. Within each TR, 2 echoes are acquired. Acquisition of the first echo starts at the ramp-up of the encoding gradient (TE1), allowing for capture of signals with very short lifetimes (bone), while that of the second starts after a longer delay (TE2). In total, 4 echoes are obtained: ECHO11 (RF1TE1), ECHO12 (RF1TE2), ECHO21 (RF2TE1), and ECHO22 (RF2TE2). During reconstruction, ECHO11 is combined with ECHO21 and ECHO12 is combined with ECHO22, resulting in 2 images. The subtraction of these 2 images yields an enhanced bone contrast. Data acquisition/processing: The pulse sequence described above was applied for MR imaging of a human cadaveric skull and 2 adult human subjects in vivo, at 3T field strength (Siemens Prisma, Erlangen, Germany). Imaging parameters: TR/TE1/TE2=7/0.06/2.46 ms, RF1/RF2 durations=40/520 μs, flip angle=12°, matrix size=2563, field of view=2803 mm3, voxel size=1.1 mm isotropic, number of radial spokes=25,000, and scan time=6 minutes. Segmentation of bone voxels was performed using ITK-SNAP in a semi-automatic fashion, leading to 3D renderings of the skull. For comparison, a CT scan was also performed in the human cadaveric skull with 1 mm isotropic resolution. Validation: The biometric accuracy was assessed by measuring eight anatomic distances: (1) Maximum craniocaudal aperture of the right orbit. (2) Maximum craniocaudal aperture of the left orbit. (3) Maximum height of the mandible from chin point in the midline. (4) Maximum cranial length (5) Maximum cranial width. (6) Maximum height of piriform aperture. (7) Distance between lateral most aspect of mandibular condyles. (8) Distance between lateral most aspect of posterior hard palate in both CT- and MRI-based 3D renderings of the human cadaveric skull using Mimics software (Materialise®, Ghent, Belgium). These distances were compared with those directly measured on the cadaveric skull. RESULTS/ANTICIPATED RESULTS: Compares CT with the proposed MRI method on cadaveric human skull images, along with corresponding 3D renderings. Compared with CT, the 3D rendered images maintain most features over the entire head (e.g., zygomatic arch), except for appearance of some artifacts in the mandibular region. In vivo head images in 2 adult subjects: axial magnitude images and 3D rendering. In the axial images, bone voxels as well as the inner table of the cranium are clearly visualized, and cranial and spinal bone structures are well depicted in the 3D renderings. Some voxels were erroneously included or excluded in the renderings. The mean difference in measurements of the 8 anatomic distances was 6, 4, and 2 mm when comparing MRI Versus CT, MRI Versus in situ, and CT Versus in situ, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Bone proton magnetization exhibits a substantial level of signal decay during the relatively long duration of RF2 due to its very short T2 relaxation time. In contrast, soft-tissue retains nearly the same level of signal intensities over all echoes. Thus, subtraction of ECHO22 from ECHO11, when compared with the difference between ECHO11 and ECHO12, enhances bone contrast from soft tissue. The proposed, dual-RF dual-echo 3D UTE imaging technique produces isotropic high-resolution bone-specified images in the whole head within a clinically feasible imaging time (6 min), leading to clear visualization of craniofacial skeletal structures. These are key components necessary for translation to the clinical setting. Optimization of postprocessing for more realistic 3D renderings and thus accurate anatomic measurements is currently being implemented. The proposed method’s potential as a nonradiative alternative to CT will then be thoroughly evaluated in pediatric patients.

OBJECTIVES/SPECIFIC AIMS: Exosomes are living nanoscale vesicles that can shuttle large amounts of bioactive cargo for intercellular communication. The potential of these nanovesicles to serve as both biomarkers for disease diagnosis and vehicles for delivery of therapeutics has only begun to be explored. To realize these potentials, molecular tools for effective exosome tracking and capturing must be invented in order to advance basic research and clinical translation. METHODS/STUDY POPULATION: We utilize a surface display strategy that enables exosome modification in living mammalian systems. By reconfiguring the surface protein CD63 or viral envelope glycoprotein VSV-G, we generate 3 topologically distinctive protein chimeras for exosome imaging and capture in mammalian systems. RESULTS/ANTICIPATED RESULTS: We have shown that these genetically encoded protein chimeras have the ability to correctly target and integrate into exosomes in cultured human cells. Furthermore, we have demonstrated that the secreted exosomes could be successfully captured by an affinity peptide intentionally displayed on the outer surface of exosomes. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study highlights the potential of these fusion proteins for exosome tracking and provides novel genetic tools for exosome research and translation, one of which is loading protein therapeutics for targeted delivery.

OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of the heavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxin type A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, which provide a novel multipurpose biologic with potential clinical applications in tumor detection/imaging, inhibition of tumor progression, and reduction of bioactive hormone secretion in metastatic neuroendocrine (NE) cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancer patient-derived tissues for prediction of patient response for recombinant HCR (rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in a NE cancer liver metastasis mouse models and in the NE patient-derived 3D MicroTumor system. (4) To collect preclinical data to design and conduct a clinical trial with NE cancer patients, a major goal toward translating our discoveries into much needed therapies. METHODS/STUDY POPULATION: Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducible expression vector in E. coli BL21. The rHCR was His-Tag purified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZ cells were plated at a density of 5000 cells/well in 96-well plates and incubated under standard conditions overnight. The next day, cells were treated with 10, 100, or 500 nmol/L of rHCR and incubated for 72 hours. Following incubation, cell viability was assessed by ATP quantification using the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated and injected into polydimethylsiloxane bioreactors in a matrigel and collagen suspension for 3D culture experiments. The viability of 3D cultures incubated with various doses of rHCR was assessed by measuring the uptake of the near-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT, and MZ cells were seeded in 6-well plates at a density of 3×105 cells/well for 24 hours followed by treatment with 100 nmol/L for 72 hours. Total cellular proteins were isolated and analyzed to assess the level of SV2A expression and the effect of rHCR on the expression levels of NET marker proteins. Immunohistochemistry: Deparaffinized tissue culture slides were incubated with SV2A primary antibody in 1% BSA and incubated overnight at 4°C. Slides were rinsed twice with TBS containing 0.025% Triton, followed by 0.3% H2O2 for 15 minutes. Slides were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Detection of protein-protein interaction: Precleared cell lysate was incubated with glutathione-agarose beads in the presence of 10 μg of GST-tagged rHCR for 2 h at 4°C with end-over end mixing. Samples were then centrifuged at 10,000 g for 2 minutes and the supernatant was analyzed by SDS-PAGE. Preclinical models: To allow rHCR testing on NET patient derived cells in a very novel 3D surrogates, sterile collected fresh NET tissues will be obtained from the UAB Tissue Procurement, dissociated into a single cell suspension and injected into a polydimethylsiloxane bioreactors containing extracellular matrix composed of bovine collagen and matrigel. Such 3D cell culture will be maintained in bioreactors with constant supply of media through the microchannels and treated with rHCR at concentrations ranging from 10 nM to 1 mM. Following histologic confirmation of growth and morphology of NET patient-derived 3D surrogates we will test the anticancer activity of rHCR in this system using the standard cytotoxicity assays as well as we will validate the NET hormone expression using immunohistochemistry assay. To create an animal model of NE cancer progression, we will perform intrasplenic injection of NET cell lines. In approximately 4 weeks, the animals should develop NE liver metastases based upon our previous experience. rHCR-iFPs accumulation in the tumor mass: rHCR-iFPs will be injected to the tumor bearing mice after 4 weeks of cells implantation in 1 week interval for total of 4 treatments at the concentrations of 0.125, 1.25, and 12.5 mg/kg. RESULTS/ANTICIPATED RESULTS: Based on the preliminary data, we expect to detect rHCR-iFPs in NE cancer xenografts and in patient derived 3D explants. Our preliminary data revealed that treating NETcells with rHCR significantly reduced NE peptide expression in 3 days. Thus, we expect to see the decrease of NE tumor markers even if the fluorescent detection method is not sensitive enough to monitor the signal. The reduction of the NET markers can be used as an indicator of the rHCR-iFPs uptake by the tumor mass. If HCR exhibit high binding affinity to SV2 receptors in NET models but moderate anticancer efficacy, we plan to use rHCR peptide to conjugate with the nanocarrier for targeted drug delivery. In this case rHCR peptide can be used as a ligand that specifically binds to NE cancer cells and delivers anticancer drug. In 3D NET patient derived explants we expect significant reduction of NET markers and hormones (serotonin and calcitonin) in NE cancer cells upon long-term rHCR-iFPs treatment. In addition, we will perform multiplex protein quantification assay using Luminex to assess the various hormones, cytokines, and growth factors to be repurposed into a diagnostic and detection reagent, or a drug delivery ligand for targeted therapies. DISCUSSION/SIGNIFICANCE OF IMPACT: NE cancers are highly metastatic: NE cancers such as carcinoid, islet cell tumors, and medullary thyroid cancer frequently metastasize to the liver. They are the second most prevalent GI malignancy. Ninety percent of patients with pancreatic carcinoid tumors and 50% of patients with islet cell tumors develop isolated hepatic metastases. Patients with untreated, isolated NE liver metastases have <30% 5-year survival. Thus, there is a critical need for new therapies for NE cancers. Surgery is the only curative therapy available for patients with NE cancers but most cannot be cured: surgical removal is the most effective treatment for NE cancers; however, a very high percentage of patients present with metastatic disease. While surgical resection can be potentially curative, many patients are not candidates for operative intervention due to widespread metastases or the degree of hepatic involvement by the NE cancers. Moreover, other forms of therapy including chemoembolization, radioembolization, cryoablation, and chemotherapy have had limited efficacy. We hope that our in vitro data on rHCR toxicity and specificity to NET, validated in the pre-clinical models will allow the first in-human application of this technology in clinical studies.

OBJECTIVES/SPECIFIC AIMS: Incomplete spinal cord injury typically results in life-long disability, often in the form of profound loss of locomotion capability. Individuals who have experienced incomplete spinal cord injury exhibit persistent eccentric motor deficits, which are particularly prevalent in the weight acceptance phase of gait and emphasized in sagittal plane knee motion and frontal plane hip motion. METHODS/STUDY POPULATION: Motion analysis can capture the kinematic and joint-level deficits of these individuals, but it is impossible to directly calculate the contributions of individual muscles to weight acceptance due to the complexity of the musculoskeletal system. Instead, those muscle contributions must be simulated in order to approximate muscle power during locomotion. RESULTS/ANTICIPATED RESULTS: The traditional method for driving these simulations with electromyography readings is unavailable for individuals who have neuromuscular deficits (e.g., spasticity or paralysis), due to the need to generate reliable maximum voluntary isometric contractions for baseline purposes. Instead, this research develops a novel method for using resting electromyography data to drive musculoskeletal simulations using a muscle activation threshold paradigm. DISCUSSION/SIGNIFICANCE OF IMPACT: The simulation results of this method more closely resemble experimental results, but further simulation refinement is needed to fully capture the true muscle activity.

OBJECTIVES/SPECIFIC AIMS: Prostate cancer (PCa) is the most common noncutaneous malignancy in men in the United States. A significant fraction of advanced PCa treated with androgen deprivation therapy experience relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). The PCa tumor microenvironment is comprised of a complex mixture of epithelial and stroma cell types engaged in multifaceted heterotypic interactions functioning to maintain tumor growth and immune evasion. We recently uncovered the important role played by myeloid-derived suppressor cells (MDSCs) to mediate tumor immune evasion in aggressive PCa (Wang, Lu et al., Cancer Discovery, 2016). Immune checkpoint blockade (ICB) has elicited durable therapeutic responses across a number of cancer types. However, the impact of ICB on mCRPC has been disappointing, which may signal the need to combine mechanistically-distinct ICB agents and/or override immunosuppression in the tumor microenvironment. Our objective is to determine if robust immunotherapy responses in mCRPC may be elicited by the combined actions of ICB agents together with targeted agents that neutralize MDSCs yet preserve T cell function. METHODS/STUDY POPULATION: We created a novel embryonic stem cell-based chimeric mouse model of mCRPC engineered with signature mutations to study the response to single and combination immunotherapy. The efficacy studies were followed with detailed mechanistic investigation and clinical validation. RESULTS/ANTICIPATED RESULTS: Consonant with early stage clinical trials experience, anti-CTLA4 or anti-PD1 monotherapy failed to impact disease progression. Similarly, modest anti-tumor activity was observed with combination ICB as well as monotherapy with targeted agents including Cabozantinib (tyrosine kinase inhibitor), Dactolisib (PI3K/mTOR inhibitor), and Dasatinib (tyrosine kinase inhibitor). In contrast, mCRPC primary and metastatic disease showed robust responses to dual ICB treatment together with either Cabozantinib or Dactolisib, but not with Dasatinib which impaired T cell infiltration in the tumor. Detailed intratumoral immune profiling with mass cytometry (CyTOF) showed that combined ICB and Cabozantinib or Dactolisib was associated with significant depletion of MDSCs. Cabozantinib and Dactolisib blocked the PI3K signaling activity in MDSCs and reduced their immunosupppresive activity. Mechanistically, the combination efficacy was due to the upregulation of IL-1RA and suppression of MDSC-promoting cytokines secreted by mCRPC cells. DISCUSSION/SIGNIFICANCE OF IMPACT: We demonstrated that an antibody cocktail targeting CTLA4 and PD1 was insufficient to generate effective anti-tumor response, but combination of ICB with targeted therapy that inactivates PI3K signaling displayed superior synergistic efficacy through impairing MDSCs in the tumor microenvironment. These observations illuminate a clinical path hypothesis for combining ICB with MDSC-targeted therapies in the treatment of mCRPC. Importantly, conclusions from the study on PCa may have implications in combination immunotherapy for aggressive breast cancer which is also largely resistant to immune checkpoint blockade and replete with immunosuppressive myeloid cells.

OBJECTIVES/SPECIFIC AIMS: (1) Determine the anti-proliferative effect of Copanlisib (α/δ PI3 kinase inhibitor) in CTCL cell lines and synergy with other anti-tumor drugs such as HDAC inhibitors. (2) Determine the effect of Copanlisib treatment on downstream targets of the PI3K/AKT/mTOR pathway. METHODS/STUDY POPULATION: We will test the anti-proliferative effect of Copanlisib on the cell lines H9 and HH, which are well characterized for evaluating new therapies for CTCL (Netchiporouk et al., 2017). We will also test the anti-proliferative effect of Copanlisib in combination with HDAC inhibitors on CTCL cell lines. Cell Titer Glo (Promega) will be used for the proliferation assay. Briefly, Cell-Titler Glo will be added after 24, 48, and 72 hours and luminescence will be measured as a % of maximal growth. Inhibitory effect will be determined by comparison to % growth of the control cultures without Copanlisib treatment. Our next objective is to determine the effect of Copanlisib treatment on downstream targets of the PI3K/AKT/mTOR pathway using Western blot analysis. In brief, 30 μg of each lysate will be subjected to 4%–12% gradient SDS-PAGE gel eletrophoresis. All primary antibodies were purchased from Cell Signaling Technology. Membranes will be washed with TBST and incubated with 1:10,000 dilution of IRDye-conjugated secondary antibody (Licor) for 1 hour. Results will be expressed as relative intensity: the intensity of each band adjusted to that of GAPDH. Experiments will be done in triplicate and 1-way analysis of variance followed by multiple comparison test will be applied to compare the cell proliferation between different treatment groups. RESULTS/ANTICIPATED RESULTS: Previous results from the Ai lab have demonstrated the importance of the PI3 kinase signaling cascade using high-throughput proliferation assays and siRNA knockdown of individual and double PI3 kinase isoforms (unpublished data). So far I have successfully established culture of HH and H9 cells in 96 well plates (100,000 cells/200 μL). We are titrating Copanlisib at doses from 20 nm to 20 μM. Initial results suggest a promising anti-proliferative effect and currently we are optimizing the most effective pharmacologic dose. Thus we anticipate that Copanlisib will exhibit a potent anti-proliferation activity in CTCL cell lines. H9 and HH cell lysates have been collected and preserved for Western blot analysis. We anticipate that Copanlisib treatment will significantly decrease the phosphorylation of AKT and 4EB-P1, both downstream targets of PI3 kinase signaling. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will elucidate the importance of the PI3 kinase/AKT/mTOR pathway in tumor proliferation in CTCL. Identifying the importance of specific isoforms of PI3 kinase in CTCL will allow for more targeted selection of treatment. Copanlisib targets α and δ isoforms of PI3K and is newly approved by the FDA for low grade B cell lymphoma. Our results seek to quantify the anti-proliferative effect of Copanlisib and determine an on-target mechanism of action by investigating the drug’s effects on downstream signaling molecules of the PI3 kinase pathway. This project will elucidate the disease process of CTCL and provide important insight for its management.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to test the hypothesis that the adherens junctions of colon epithelial cells regulate lncRNAs levels and function via the microprocessor and RISC complexes to suppress expression of pro-tumorigenic markers and aberrant cell behavior. METHODS/STUDY POPULATION: To test this hypothesis, we used colon epithelial cancer cell lines. We performed RNA-seq following knockdown of PLEKHA7, a key component of the adherens junctions, to identify changes in lncRNA expression and downstream mRNA levels. We confirmed junctional localization of affected lncRNAs from the RNA-seq and those that we found in our preliminary study by using in situ hybridization (ISH). RESULTS/ANTICIPATED RESULTS: RNA-seq identified junction-associated lncRNAs whose expression levels are regulated by PLEKHA7. The top upregulated lncRNA upon PLEKHA7 depletion was MIR17HG, an oncogenic host transcript of a cluster of miRNAs. These mature miRNAs also co-precipitate with PLEKHA7. PLEKHA7 knockdown results in increased levels of MIR17HG, but only a subset of its hosted miRNAs (miR-19a,b). Notably, miR-19a and mir-19b are highly upregulated in colon cancer. Our data suggest that 2 PLEKHA7-associated miRNAs, miR-203a and miR-372, mediate suppression MIR17HG. Re-expression of PLEKHA7 in aggressive colon cancer cells that lack PLEKHA7 suppressed expression of MIR17HG, as well as anchorage independent growth of these cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data point towards a novel mechanism of lncRNA regulation that tethers epithelial tissue integrity with pro-tumorigenic cell transformation. Reducing elevated MIR17HG levels, is a potential therapeutic approach to suppress the tumorigenic behavior of cells that have lost their junctional integrity and homeostasis. identify a network of miRNA-mRNA-lncRNA interactions that could be exploited for further mechanistic studies, as well as for diagnostic and therapeutic purposes in the future.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if trimethylamine N-oxide (TMAO) alone could acutely alter cardiac contractile function on a beat-to-beat basis. METHODS/STUDY POPULATION: CD1 adult mouse hearts were extracted, attached to a force transducer, oxygenated, and paced within an organ bath. Changes in contractility were measured after pipetting or reverse perfusing TMAO through the aorta via a modified Langendorff apparatus to facilitate TMAO delivery into the myocardium. To determine if our findings translated to the human heart, we performed contractility experiments using human right atrial appendage biopsy tissue retrieved during cardiopulmonary bypass procedures. To investigate whether TMAO alters contractile rate, in a separate series of experiments, the atria and sinoatrial node of isolated hearts were kept intact to allow for spontaneous beating without artificial pacing and were treated with TMAO or vehicle. In addition, intracellular calcium measurements were performed on spontaneously beating embryonic rat cardiomyocytes after TMAO or vehicle treatment. RESULTS/ANTICIPATED RESULTS: We found acute exposure to TMAO, diluted into the organ bath, increased average contraction amplitude 20% and 41% at 300 µM and 3000 µM, respectively (p<0.05, n=6). Langendorff reverse perfusion of mouse hearts ex vivo with 300 µM TMAO generated an even greater response than nonperfusion peripheral exposure and increased isometric force 32% (p<0.05, n=3). Consistent with what we observed in mouse hearts, incubation of human atrial muscle tissue with TMAO at 3000 µM increased isometric tension 31% compared with vehicle (p<0.05, n=4). TMAO treatment (3000 µM) also increased average beating frequency of ex vivo mouse hearts by 27% compared with vehicle (p<0.05, n=3) and increased the spontaneous beating frequency of primary rat cardiomyocytes by 13% compared with vehicle treatment (p<0.05, n=4). DISCUSSION/SIGNIFICANCE OF IMPACT: TMAO, at pathological concentrations, directly increases the force and rate of cardiac contractility. Initially, these inotropic and chronotropic effects may be adaptive during CKD; however, chronic increases in isometric tension and beating frequency can promote cardiac remodeling and heart failure. Further translational studies are needed to understand the intricate relationship between the microbiome, kidneys, and heart and to examine if TMAO represents a therapeutic target for reducing cardiovascular mortality in CKD patients.

OBJECTIVES/SPECIFIC AIMS: (1) To evaluate clinical outcomes in mechanically ventilated patients with and without fever. We hypothesize that, after adjusting for confounding factors such as age and severity of illness: (a) In septic patients, fever will be associated with improved clinical outcomes. (b) In nonseptic patients, fever will be associated with worse clinical outcomes. (2) To examine the relationship between antipyretics and mortality in mechanically ventilated patients at risk for an acute lung injury. We hypothesize that antipyretics will have no effect on clinical outcomes in mechanically ventilated patients with and without sepsis. METHODS/STUDY POPULATION: This is a retrospective study of a “before and after” observational cohort of 1705 patients with acute initiation of mechanical ventilation in the Emergency Department from September 2009 to March 2016. Data were collected retrospectively on the first 72 hours of temperature and antipyretic medication from the EHR. Temperatures measurements were adjusted based on route of measurement. Patients intubated for cardiac arrest or brain injury were excluded from our primary analysis due to the known damage of hyperthermia in these subsets. Cox proportional hazard models and multivariable linear regression analyzed time-to-event and continuous outcomes, respectively. Predetermined patient demographics were entered into each multivariable model using backward and forward stepwise regression. Models were assessed for collinearity and residual plots were used to assure each model met assumptions. RESULTS/ANTICIPATED RESULTS: Antipyretic administration is currently undergoing analysis. Initial temperature results are reported here. In the overall group, presence of hypothermia or fever within 72 hours of intubation compared with normothermia conferred a hazard ratio (HR) of 1.95 (95% CI: 1.48–2.56) and 1.31 (95% CI: 0.97–1.78), respectively. Presence of hypothermia and fever reduced hospital free days by 3.29 (95% CI: 2.15–4.42) and 2.34 (95% CI: 1.21–3.46), respectively. In our subgroup analysis of patients with sepsis, HR for 28-day mortality 2.57 (95% CI: 1.68–3.93) for hypothermia. Fever had no effect on mortality (HR 1.11, 95% CI: 0.694–1.76). Both hypothermia and fever reduced hospital free days by 5.39 (95% CI: 4.33–7.54) and 3.98 (95% CI: 2.46–5.32) days, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: As expected, both hypothermia and fever increased 28-day mortality and decreased hospital free days. In our sepsis subgroup, hypothermia again resulted in higher mortality and fewer hospital free days, while fever did not have a survival benefit or cost, but reduced hospital free days. Antipyretic administration complicates these findings, as medication may mask fever or exert an effect on survival. Fever may also affect mechanically ventilated septic patients differently than septic patients not on mechanical ventilation. Continued analysis of this data including antipyretic administration, ventilator free days and progression to ARDS will address these questions.

OBJECTIVES/SPECIFIC AIMS: The primary goal of this project is to verify findings from a murine prostatitis model in the human setting. METHODS/STUDY POPULATION: Methods include primary cell isolation and culture, FACS, adoptive transfer, 3D cell culture, histology, immunofluorescence, xenograft, and tissue recombination. The study population includes patients undergoing HoLEP or radical prostatectomy due to hyperplasia or adjacent bladder or prostate cancer. RESULTS/ANTICIPATED RESULTS: Having verified similar sensitivities to androgen receptor (AR) inhibitors between naive murine and human basal prostate stem cells, we anticipate that autoimmune inflammation in humans affects the response of basal prostate stem cells in a manner similar to the murine setting as well. This includes increased proliferation, increased differentiation, and decreased response to AR inhibitors. DISCUSSION/SIGNIFICANCE OF IMPACT: The identification of survival mechanisms used by basal prostate stem cells in an androgen deprived environment may give insight to the process by which prostate cancer becomes androgen independent. The effect of inflammation on proliferation, survival, and AR signaling in these cells may also provide information relevant to cancer initiation and progression.

OBJECTIVES/SPECIFIC AIMS: The Empower Lab was established in 2015 with the goal of providing students with hands-on research experience in sexual and gender-based violence and health. METHODS/STUDY POPULATION: The Empower Lab consists of 10–12 undergraduate, graduate, and medical students at a time. Students undergo a rigorous application process, and agree to volunteer 8 hours per week for at least 1 year. Students are assigned to teams, and learn research skills such as literature searches, systematic reviews, research question generation, study design, IRB procedures, database creation and management, data collection and analysis, oral and poster presentation, manuscript preparation, team collaboration and communication, advocacy, and leadership. Students start as research assistants, and can be promoted to team leader, and associate director of research. Students mentor and teach each other, and are supervised by the principal investigator (PI). A survey skill self-assessment is administered to lab members on entry to the lab, every 4–6 months, and upon exit. RESULTS/ANTICIPATED RESULTS: In total, 20 students have participated in the lab to date, and 12 are currently enrolled. Eighty percent of the lab members are women. The students are 45% undergraduates, 15% graduate (nursing, social work, public health), 20% medical students, and 10% not currently enrolled in school (gap year). Twenty students completed entry surveys, 11 students have completed interim surveys, and 5 students have completed exit surveys. Examination of current surveys indicates that students are gaining skills throughout the lab experience. Free-text feedback provided further insight. Currently, the lab has 5 IRB-approved studies actively recruiting participants, 4 manuscripts being written, and 3 studies in the development phase. Students have presented at three local and 2 national meetings to date. Changes have been made to the lab structure over time in order to provide clear expectations and feedback, and strengthen student performance. DISCUSSION/SIGNIFICANCE OF IMPACT: The Empower Lab is an innovative public health lab that provides opportunities for real-world research experience for students. The teamwork, collaboration, and structure of the lab permit mentoring, support, and teaching from peers, as well as from the PI. The Lab increases the PI’s productivity. Students are encouraged to develop and implement their own research ideas, further encouraging independence and initiative. Although the number of surveys is limited to date, they indicate improvement in skills and confidence among lab members. The predominance of women in the lab suggests that this is a strong model for recruitment and retention of women in STEM.

OBJECTIVES/SPECIFIC AIMS: A hallmark of progressive HIV-1 infection is the massive activation and depletion of the gut barrier protective CD4 T helper subsets (Th17 and Th22) in the intestinal mucosa. The loss of these cells is thought to contribute to microbial translocation and systemic immune activation that occurs during chronic infection. In addition to the loss of protective Th subsets, we previously showed that chronically HIV-1 infected individuals have an altered colonic mucosal microbiome, which is in part characterized by a lower relative abundance of bacteria that produce the short-chain fatty acid butyrate in conjunction with increased relative abundance of gram-negative pathobionts. This dysbiosis was linked to markers of mucosal and systemic immune activation in these individuals. Following up on these clinical observations, we sought to understand how a loss of butyrate might contribute to HIV-associated inflammation. Initial studies showed that the addition of butyrate to cultured lamina propria mononuclear cells (LPMC) resulted in decreased pathobiont-driven gut T cell activation, HIV-1 infection levels and production of IL-17 and IFNy. Since the gut barrier protective Th17 and Th22 subsets are preferentially infected and depleted, which is critical to HIV-1 pathogenesis, we wanted to determine the mechanism by which butyrate modulates activation of these important Th subsets in the gut. METHODS/STUDY POPULATION: Total LPMCs or purified LP CD4 T cells were isolated from human jejunal tissue (n=3–6), labeled with CFSE and cultured with TCR/CD28 beads to mimic APC driven T cell activation, with the addition of butyrate at physiologic doses(0–2 mM). Four days after culture, secreted cytokine(IL-17 and IFNy) levels were measured by ELISA. Cells were then short-term (4 hr) mitogenically stimulated (PMA/Ionomycin) in the presence of a golgi transport inhibitor. Total CD4 T cell activation (CD38+/HLA-DR+, CD25+), proliferation (CFSElow), and frequencies of intracellular cytokines were measured by multi-color flow cytometry. Paired t-tests were performed to determine statistical significance. RESULTS/ANTICIPATED RESULTS: Butyrate inhibited LP CD4 T cell activation (p=0.013) and proliferation (p=0.015) within total LPMCs stimulated with TCR/CD28 beads in a dose-dependent manner, with significant activity starting at 0.125 mM. Quantification of total secreted cytokines revealed that butyrate significantly decreased both IL-17 and IFNy production after 4 days of culture at 0.0625 mM and 0.25 mM of butyrate, respectively. Assays using purified LP CD4 T cells demonstrated that butyrate directly decreased LP CD4 T cell activation, proliferation and cytokine production in response to TCR/CD28 stimulation. Studies on specific T helper subsets revealed that butyrate inhibited proliferation of Th17 cells at lower concentrations (IC50:0.147 mM) compared with Th1 (IC50:0.229 mM) and Th22 (IC50:0.258 mM) and Th non-IL-22/IL-17/IFNy producing (IC50:2.14 mM) subsets. In addition, it appeared there was a paradoxical increase of HIV-1 infection levels at lower concentrations of butyrate (0.125 mM). DISCUSSION/SIGNIFICANCE OF IMPACT: The addition of butyrate to activated LP CD4 T cells decreases TCR-mediated activation in a dose-dependent manner, and butyrate acts directly on purified LP CD4 T cell populations independent of other cell populations. Butyrate differentially inhibited the proliferation of Th17, Th1, and Th22 subsets, with Th17 cells being the most sensitive to butyrate but increased the infection levels of all T helper subsets at low concentrations. Further studies are needed to determine the mechanism of butyrate’s actions on LP Th cells and the sensitivity of Th17 cells to the inhibitory effects of butyrate. These results could help direct targeted manipulation of the colonic microbiome of HIV-1 infected individuals to help resolve inflammation and limit the impact of the infection in the gut mucosa and systemically.

OBJECTIVES/SPECIFIC AIMS: Interleukin-23 (IL-23) promotes differentiation of naïve T-cells into Th17 cells, which drive the pathogenesis of autoinflammatory conditions such as psoriasis. IL-23-neutralizing antibody therapies are now in use for treatment of psoriasis, with promising results. Studies in mice have shown that IL-23 plays a role in inhibiting the growth, progression, and metastasis of melanomas. Thus, therapeutic neutralization of IL-23 in patients may inadvertently increase their susceptibility to development of melanoma. In this study, we aim to characterize expression of IL-23 receptors (IL-23R) in human melanocytes and melanoma cells and tissue and to study the effect of IL-23 on growth, proliferation, and tumorigenicity of these cells. METHODS/STUDY POPULATION: IL-23R expression was characterized using immunofluorescence staining, Western blot, and flow cytometric analysis. Response of melanoma and melanocytes to recombinant IL-23 treatment will be studied through similar methods in addition to assays of cell proliferation and tumorigenicity. RESULTS/ANTICIPATED RESULTS: Preliminary immunofluorescence staining and flow cytometry results indicate that both human melanoma and primary melanocytes express IL-23 receptors. Western blot analysis showed that melanoma cell line A375 expressed nearly twice the amount of IL-23R versus normal melanocytes (p<0.05). Based on previous studies, we anticipate that addition of recombinant IL-23 to cultures of melanoma will reduce proliferative potential, and we expect similar addition to normal melanocytes will increase DNA repair mechanisms. DISCUSSION/SIGNIFICANCE OF IMPACT: In showing that human melanocytes and melanoma cells express IL-23 receptors, and potentially showing the inhibitory effect of IL-23 in the development of melanocytic neoplasms, our findings imply that using IL-23 neutralizing therapies may increase risk of developing melanoma, especially in patients who are already susceptible. As such, these therapies must be used with great care in these patients.