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Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion gene. No targeted therapies have been established and multimodal therapy is ineffective with a 5-year survival rate of 15-25%. Many questions about the biology of DSRCT remain unanswered including the critical genes regulated by the EWSR1-WT1 fusion gene, the role of the - and +KTS fusion gene isoforms produced by alternative splicing, and the relationship between EWSR1-WT1 binding and gene expression alterations. To address these questions, we established four doxycycline-inducible DSRCT cell lines that selectively knockdown the expression of EWSR1-WT1. RNA-seq analysis on these cell lines with or without EWSR1-WT1 knockdown identified commonly altered genes and pathways. To illuminate the role of the EWSR1-WT1 -KTS and +KTS isoforms, the -KTS, +KTS, or both isoforms were overexpressed in a mesothelial cell line, a hypothesized cell of origin for DSRCT. Remarkably, different gene expression alterations were observed between the -KTS and +KTS isoforms. While overexpression of the -KTS isoform upregulated 765 genes and downregulated 366 genes, overexpression of the +KTS isoform upregulated 203 genes and downregulated 25 genes. Gene set enrichment analysis showed EWSR1-WT1 regulated gene signatures are recapitulated by overexpression of the -KTS isoform or both +/-KTS isoforms, but not the +KTS isoform alone, suggesting the -KTS isoform predominantly regulates gene expression in DSRCT. To interrogate the influence of direct and indirect gene regulation by EWSR1-WT1, ChIP-seq and CUT&RUN data were integrated into our RNA-seq analysis. Across each of our four DSRCT cell lines, approximately 25% of EWSR1-WT1 bound peaks led to upregulation, 10% led to downregulation, and 65% led to no gene expression alteration. EWSR1-WT1 binding accounted for 33% of EWSR1-WT1 commonly upregulated genes but only 4.2% of commonly downregulated genes. Upregulated EWSR1-WT1 bound genes were more likely to be bound in the promoter or first intron and coincide with Znf263 or Hoxb4 motifs. To evaluate the functional role of EWSR1-WT1 targets in DSRCT biology, a screen of the 24 druggable EWSR1-WT1 targets was conducted and a number of novel and known target genes were identified as important for DSRCT survival. Current work is verifying these targets with gene knockdown and evaluating their function in vivo. Citation Format: Justin W. Magrath, Ilon A. Goldberg, Alifiani B. Hartono, Sean B. Lee. EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3529.

Since the closure of Charity Hospital after Hurricane Katrina, New Orleans Student-Run Free Clinics have helped fill the resulting void in healthcare access for the underserved New Orleans population. To better understand the health insurance status and health outcomes of this patient population, 1036 patient records from seven New Orleans Student-Run Free Clinics were collected and analyzed between February 2017 and March 2020. Insurance status was significantly associated with gender, race, homelessness, and prior incarceration, but not with education. Substance use rehabilitation centers had low uninsured rates, while homeless shelters had higher uninsured rates. Patients on Non-Medicaid insurance were most likely to be prescribed a medication for diabetes (p = .01), hypertension (p = .21), and psychiatric conditions (p = .04), followed by those on Medicaid, and then those who were uninsured. This study demonstrates the benefits of health insurance and provides important data that can inform future health insurance enrollment efforts and health policy.

The purpose of this study is to provide a deeper analysis characterizing the current health status of Tulane University School of Medicine’s student-run free clinic patients. Only one prior study by Rebholz et al. (South Med J 106(3):217–223, https://doi.org/10.1097/SMJ.0b013e318287fe9a , 2013) has explored the demographics in this population. Ultimately, this study will allow for easy interpretation of the demographics of the student clinic system as a whole and for the individual clinics participating in this study. This information will allow clinics to better customize care for their respective populations and ultimately improve health outcomes. Patient demographic data was collected from five preceptor-based clinics from December 2016 to May 2019 and submitted via a REDCap survey. Survey fields included patient age, sex, gender, race, ethnicity, insurance status, chief complaints, past medical history, social history, and medications. Gross data was analyzed in Excel and subsequently stratified by clinic for inter-clinic comparison. A total of 772 patient responses were collected from five different student-run clinics; 56% were male and 44% were female. The three most common reasons for a clinic visit were complaint-free wellness visits (26%), musculoskeletal complaints (16%), and respiratory complaints (11%). The three most common comorbidities included smoking and/or tobacco abuse (28%), psychiatric conditions (19%), and illicit drug abuse (15%). Future applications derived from this study may include redistribution of resources for patient education, social services, medical inventory, and preventative health services based on patient clinical needs.

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive form of pediatric cancer caused by a translocation between chromosomes 11 and 22, creating the novel EWSR1-WT1 fusion gene. Current therapy including surgery and the P6 chemotherapy regimen is insufficient, leading to a 5-year survival rate of only 15-25%. One potential explanation for this poor prognosis is the existence of a cancer stem cell (CSC) population with the ability to resist chemotherapy and cause both tumor recurrence and metastasis. While a CSC population has been identified in the closely related Ewing Sarcoma, most commonly caused by the EWSR1-FLI1 fusion gene, no CSC population has yet been identified in DSRCT. In this study, novel culture conditions were developed that enabled the formation of tumorspheres in vitro in the two commonly available DSRCT cell lines: JN-DSRCT-1 and BER-DSRCT. These novel culture conditions led to upregulation of stemness genes at the RNA and protein level including NANOG and SOX2, two genes that are associated with poor survival outcomes in TCGA sarcomas data. CCK-8 assay demonstrated an increased chemoresistance for tumorspheres versus normal adherent culture especially for doxorubicin, which was confirmed by reduction in PARP cleavage. This chemoresistance may be partially explained by a more quiescent CSC state as shown by reduced cell proliferation and a lower percentage of cells in the S and G2/M phases. However, cells grown in tumorspheres still robustly formed tumors in vivo, thus demonstrating the two hallmark CSC traits of chemoresistance and tumor formation. RNA-seq was performed to elucidate key differences between the adherent and sphere culture conditions and identify potential targets for the therapeutically important CSC-like population. Overrepresentation analysis and gene set enrichment analysis both showed upregulation of pathways related to chromatin assembly and disassembly, suggesting epigenetic changes as DSRCT cells move to a more stem-like state. RNA-seq further revealed kinases upregulated in tumorspheres including B-lymphocyte kinase (BLK) which previous studies have demonstrated is oncogenic, regulated by EWSR1-WT1, and not necessary for normal survival. BLK knockdown reduced CSC-like properties including abrogation of tumorsphere formation and reduction in the levels of SOX2 and NANOG. Together, this work for the first time identifies a CSC-like population in DSRCT and BLK as a potential DSRCT CSC target. Citation Format: Justin W. Magrath, Alifiani B. Hartono, Sean B. Lee. Identification, characterization, and targeting of desmoplastic small round cell tumor cancer stem cell-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 902.

Since the closure of Charity Hospital after Hurricane Katrina, New Orleans Student-Run Free Clinics have helped fill the resulting void in healthcare access for the underserved New Orleans population. To better understand the health insurance status and health outcomes of this patient population, 1036 patient records from seven New Orleans Student-Run Free Clinics were collected and analyzed between February 2017 and March 2020. Insurance status was significantly associated with gender, race, homelessness, and prior incarceration, but not with education. Substance use rehabilitation centers had low uninsured rates, while homeless shelters had higher uninsured rates. Patients on Non-Medicaid insurance were most likely to be prescribed a medication for diabetes (p = .01), hypertension (p = .21), and psychiatric conditions (p = .04), followed by those on Medicaid, and then those who were uninsured. This study demonstrates the benefits of health insurance and provides important data that can inform future health insurance enrollment efforts and health policy.

Glioblastoma multiforme (GBM) is the most common and deadliest form of primary brain tumor. Despite treatment with surgery, radiotherapy, and chemotherapy with the drug temozolomide, the expected survival after diagnosis remains low. The median survival is only 14.6 months and the two-year survival is a mere 30%. One reason for this is the heterogeneity of GBM including the presence of glioblastoma cancer stem cells (GSCs). GSCs are a subset of cells with the unique ability to proliferate, differentiate, and create tumors. GSCs are resistant to chemotherapy and radiation and thought to play an important role in recurrence. In order to effectively treat GBM, a drug must be identified that can kill GSCs. The ionophore salinomycin has been shown to kill cancer stem cells and is therefore a promising future treatment for GBM. This study focuses on salinomycin's potential to treat GBM including its ability to reduce the CSC population, its toxicity to normal brain cells, its mechanism of action, and its potential for combination treatment.

Glioblastoma multiforme (GBM) is the deadliest and most common form of primary brain tumor. Conventional treatments are ineffective at treating GBM due to the heterogeneous cellular makeup of the tumors as well as the existence of drug‑resistant cells known as cancer stem cells (CSCs). CSCs have the ability to initiate tumorigenesis and self‑renew, which can lead to recurrence. Salinomycin, an antibiotic commonly used in agricultural feed, has been revealed to target CSCs in other cancer types. A few studies have suggested salinomycin can be effective at treating glioblastoma stem cells (GSCs); however, no study has examined the effect of salinomycin treatment on GSC markers. In the present study, flow cytometry, RT‑qPCR, and limiting dilution assays were used to further analyze the effects of salinomycin on GSCs. It was revealed that salinomycin decreased the expression of the GSC marker SOX2 at both the transcriptional and translational level. However, the effect of salinomycin on the GSC markers Nestin and CD133 was inconsistent between GBM subtypes. Additionally, the present findings provide initial evidence of caspase‑3‑dependent and independent apoptosis as the method by which salinomycin induces cell death in GBM. The present results indicated that salinomycin is an effective candidate as a chemotherapeutic agent that can treat GBM by targeting both bulk tumor cells as well as CSCs.

Glioblastoma stem cells (GSCs) are a unique subpopulation of cells within glioblastoma multiforme (GBM) brain tumors that possess the ability to self-renew and differentiate into bulk tumor cells. GSCs are resistant to currently available treatments and are the likely culprit behind tumor relapse in GBM patients. However, GSCs are currently inaccessible to the larger scientific community because obtaining a sufficient number of GSCs remains technically challenging and cost-prohibitive. Thus, the objective of this study was to develop a more efficient GSC culture strategy that results in a higher cell yield of GSCs at a lower cost. We observed that the basic fibroblast growth factor (bFGF) is indispensable in allowing GSCs to retain an optimal stem cell-like phenotype in vitro, but little change was seen in their stemness when grown with lower concentrations of bFGF than the established protocol. Interestingly, a dynamic fluctuation of GSC protein marker expression was observed that corresponded to the changes in the bFGF concentration during the culture period. This suggested that bFGF alone did not control stem cell-like phenotype; rather, it was linked to the fluctuations of both bFGF and media pH. We demonstrated that a high level of stem cell-like phenotype could be retained even when lowering bFGF to 8 ng/mL when the media pH was simultaneously lowered to 6.8. These results provide the proof-of-concept that GSC expansion costs could be lowered to a more economical level and warrant the use of pH- and bFGF-controlled bioprocessing methodologies to more optimally expand GSCs in the future.