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AMA Research Challenge 2022

In silico modeling of LAT1 efflux agonists for the treatment of pancreatic cancer

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Ricardo Correa, MD, EdD
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Stuart M. Greenstein, MD
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Stephanie Strohbeen, MD
 
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Jessica Li, MD/PhD Candidate
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Charles Lopresto, DO

### Sponsor
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Welcome to the largest national, multi-specialty research event featuring research from 750 medical students, residents and fellows, and international medical graduates.


poster

Neuroblastoma is the most common extracranial solid tumor in children. It accounts for more than 7% of malignancies in patients less than fifteen years of age and makes up 15% of oncology deaths. Neuroblastoma is a cancer derived from primordial neural crest cells under conditions of inappropriate migration, maturation, and or differentiation. These extracranial tumors are formed in the adrenal medulla, sympathetic ganglia, and abdomen. Current treatments for neuroblastoma include surgery, chemotherapy, radiotherapy, and biotherapy. Interestingly, neuroblastoma tumors are heterogenous with some children having low-risk tumors that regress entirely while others have high-risk tumors with widespread metastasis and poor outcomes. The expression of MYCN, a gene in a family of oncogenic drivers, in neuroblastoma tumors has been associated with high-risk neuroblastoma and worse outcomes. Children with high-risk neuroblastoma account for approximately half of all patients diagnosed with neuroblastoma. Despite current interventions and aggressive treatment, children with high-risk neuroblastoma have a long-term survival rate of less than 50%. Currently, there are not many targeted therapies for high-risk neuroblastoma as there is little know about the mechanism driving this cancer. By identifying the mechanism driving tumor progression, a treatment to specifically target the different steps of this cancer can be created. Signaling through neuronal nicotinic acetylcholine receptors (nAChRs) is involved in many neuronal processes of neural tissue, including the proliferation of neural crest cells. These receptors are a diverse group of ligand-gated pentametric channels can exist as homopentamers or heteropentamers of α or β subunits. The only mammalian homopentamer is the α7 receptor that when activated is suggested to promote neural cell survival. Furthermore, expression of nAChR has been shown to be regulated by MYCN. Our lab is interested to see if MYCN-expressing neuroblastoma tumors upregulate the expression of nAChRs leading to an overall drive in tumor progression. Therefore, targeting this interaction between nAChRs and MYCN can become a potential therapy of high-risk neuroblastoma. 


N-MYC Transcription Factor Increases the Expression of Alpha-7 Nicotinic Acetylcholine Receptor Promoting Cell Viability in Neuroblastoma Cell Line

Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) shows an initial response but followed by a drug resistance, increase in tumor relapse and distant metastases. Immunotherapy against programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients. However, it shows resistance in other patients due to new immune checkpoints such as VISTA, TIGIT, and LAG3. 

eIF4A1 is a mRNA helicase known to translate many oncogenic mRNAs including survivin, c-MYC, ROCK1, STAT1, ARF6 etc., We previously established that eIF4A1 is a vulnerable target in drug-resistant TNBC. Interestingly, we observed a reduction in levels of PD-L1 when eIF4A1 is targeted. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 are indeed upstream regulators of PD-L1. This premise formed the basis to co-target eIF4A1 and PD-L1. Before that, we need to establish the clinical relevance of these targets in human TNBC biospecimens. This will ascertain the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC.

Approach: Human biospecimens obtained from CHTN NIH repositories (more than 100 TNBC primary tumor and metastatic biospecimens) will be analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3 via immunohistochemistry (after deglycosylation for effective detection of PD-L1) and also by immunoblotting. 

Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level were elevated in drug-resistant tumors than normal breast tissue samples as assesses by immunohistochemistry. 

Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating other immune checkpoints such as VISTA, TIGIT, and LAG3. 


Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

Background
Given basic advances in cancer immunotherapy, further understanding of potential antigenic targets will improve treatment protocols. Cancer testes antigens (CTAs) are relatively novel proteins to consider in the overlap of the breast cancer and immunotherapy setting. CTAs are normally expressed in benign testis and placenta; and show aberrant expression in many malignancies, including breast cancer. This work indicated that the CTA, ARMC3 (Armadillo repeat-containing protein 3) is commonly expressed in breast cancer, and when evaluated for its chemical relationship to breast cancer resident T-cell receptor (TCR) chemical features, there was an indication of T-cell response to ARMC3. This result was substantially informed by survival data and granzyme expression.
Methods
The software used for the recovery of TCR recombination reads is available at https://github.com/bchobrut-USF/blanck_group. TCR alpha and TCR beta recombination data were extracted from the National Institutes of Health (NIH) genomics datasets. Chemical complementarity scores (CSs) were determined using the software available at https://github.com/bchobrut/brca_swcs. Linkage of chemical CSs to survival rates, and to RNAseq values (for assessing gene expression), was done with a web tool designed for these purposes termed, adaptivematch.com. Kaplan-Meier analysis conducted to compare survival probability for the upper and lower 50th percentiles of CSs. Multivariate analysis of age, race, menopause, cancer stage was performed for ARMC3-based CSs. 
Results 
Among a set of approximately 280 CTAs, ARMC3 expression stood out as representing a strong distinction for survival rates for both OS and DFS (OS p=0.0147, DFS p=0.0105). We next considered the possibility that a TCR CDR3-ARMC3 interaction would be consistent with the expression of immune biomarkers. Results indicated higher RNA expression of certain immune markers with increasing CS values. There was a survival distinction for the upper and lower 50th percentiles of CSs, with the upper 50th percentile representing a greater disease-free survival probability. Only the tumor stage and the ARMC3 based CS represented independent factors associated with survival distinctions.
Conclusion
For the ARMC3 expression; the higher the expression level, the better the survival probability. CS assessments of TCR CDR3-CTA pairs may indicate targetable antigens and may be useful for risk stratification and guiding therapy in breast cancer.


T-cell Receptor (TCR) Chemistry And Survival Data Support The Consideration Of ARMC3 As A Breast Cancer Antigen

Metastatic triple-negative breast cancer (mTNBC) has poor prognosis. Neoadjuvant chemotherapy (NACT) such as taxanes showed an initial treatment response but followed by drug resistance, tumor relapse, and metastases. The chemoresistance is even observed in targeted PARP1 therapy. Hence, there is an unmet need for. developing effective combination therapies against mTNBC. eIF4A1 is an mRNA helicase involved in translation of a subset of oncogenic mRNAs including c-MYC, cyclins D1 and D3, STAT1 etc., Previously, we discovered that eIF4A1 may regulate cancer stemness and drug resistance in TNBC. Furthermore, we found that protein levels of eIF4A1 and STAT1 were upregulated in NACT-resistant than therapy-naïve human TNBC biospecimens. STAT1 is an upstream regulator of PD-L1. Additionally, when eIF4A1 is targeted in paclitaxel-resistant human TNBC cells, PD-L1 was downregulated. Based on this premise, we targeted eIF4A1 with a small molecule inhibitor Rocaglamide A (RocA) and anti-PD-L1 immunotherapy as an effective therapeutic strategy in a murine TNBC model.

Targeting of eIF4A1 along with anti-PD-L1 therapy limits lung metastases efficaciously in triple-negative breast cancer

Background
TCEP is an organophosphorus flame-retardant (OPFR) widely used in polyurethane foams and household products. From these materials, TCEP emits into the surrounding environment, which humans encounter through inhalation and dermal absorption. Previous studies have shown OPFRs to have endocrine disrupting actions, and thus, influence diseases such as cancer. Breast cancer is the second most common cancer among women in the United States, and because of this prevalence, it is imperative to investigate possible carcinogens. Due to the endocrine disrupting nature of OPFRs, we are investigating the effects of TCEP on hormone-dependent breast cancer cell lines MCF-7 and T-47D.

Methods
Our study examines the effects of TCEP, alone and in combination with hormones and anti- hormones, on ERα and BRCA-1 expression in MCF-7 and T-47D breast cancer cell lines by utilizing western blot analyses, cellular viability assays, confocal microscopy, and RT-qPCR analyses. In order to deplete any endogenous steroids or effectors, breast cancer cells were cultured in a medium containing 5% charcoal-stripped fetal bovine serum for six days.

Results
Western blot analysis revealed alterations in ERα and BRCA-1 expression after 24 hours of treatment with varying TCEP concentrations (1μM-2mM). Analysis revealed a concentration- dependent increase in BRCA-1 expression in both cell lines, and a concentration-dependent decrease in ERα expression in the T-47D cell line. During hormonal studies, cells were treated with an optimum concentration of TCEP along with combinations of hormones and anti- hormones. After 24-hour treatment with a combination of E2 + TCEP, a significant decrease in ERα expression was observed in both cell lines. A significant increase of BRCA-1 expression occurred after 24-hour treatment of TCEP + E2 in both cell lines. These effects were sensitive to the presence of antiestrogens. Cellular viability assays revealed a significant increase in cellular viability in both cell lines when treated with a combination of E2 + TCEP.

Conclusion
Our results suggest that TECP acts as an endocrine disrupting compound on the steroid receptors and tumor suppressor genes in breast cancer cells. Further studies are warranted to determine the exact mechanism of action and influence of TCEP on breast cancer progression.

The Effects of Tris(2-Chloroethyl) phosphate (TCEP) on the Expression of Estrogen Receptor α (ERα) and Tumor Suppressor BRCA-1 in MCF-7 and T-47D Breast Cancer Cell Lines

Abstract Title: A national inpatient study: Risk factor analysis of venous thromboembolism in cancer.
Author name(s) and title(s): Satya Rijal MD, Gagan Kaur MD, Chia Chi Loh MD, Sowmya Sagireddy MD
Background: Venous thromboembolism is a condition that is associated with significant morbidity and mortality in cancer and its prevalence has increased proportionally in cancer inpatients. There has been a limited study in the past to evaluate the all-cause mortality and risk factor analysis of VTE in cancer. The goal of our study is to evaluate the mortality risk of cancer patients in VTE and establish the relationship between demographic characteristics and medical comorbidities in cancer inpatients with the risk of venous thromboembolism.
Methods: We conducted a retrospective cohort study using the nationwide inpatient sample (NIS, 2012-2014) total of 339,395 patients which included hospitalized patients with a primary diagnosis of cancer and divided into groups with VTE (N = 7,700) and without VTE (N = 331,695). We applied the binomial logistic regression model in our study to measure the odds ratio (OR) of demographic characteristics, comorbidities, and in-patient mortality rate associated with cancer patients with VTE.
Results: The highest majority of VTE in cancer inpatients were middle-aged adults of 36-50 years (83.1%), males (50%), blacks (19.3%), and followed by Hispanics (17.2%). While comparing the medical comorbidities, VTE risk was prevalent in cancer inpatients with CHF (OR 2.68, 95%CI: 2.30-3.12), followed by hypertension (OR 1.23, 95% CI: 1.16-1.29), diabetes (OR 1.16, 95%CI: 1.07-1.26), and chronic pulmonary disease (OR 1.13, 95%CI: 1.05-1.22). The prevalence of all-cause mortality was significantly higher with VTE (12%) carrying an increased risk of in-hospital mortality rate approximately four-fold (OR 3.87, 95% CI: 3.58-4.18).
Conclusion: The substantial difference in cause and effect of the current thromboprophylaxis use made it important to study the prevalence and in-hospital mortality risk factors of VTE in cancer which allows us for improving medical management by identifying patients at risk and formulating treatment and disease prevention strategies in healthcare settings. Our study could not include some comorbidities and lacks detailed patient-oriented clinical data that might affect the relationship between the risk of VTE in cancer, so we might require a large clinical trial including other comorbidities in the future to better explain the usefulness and outcomes related to risk factors of VTE in cancer inpatients.



A national inpatient study: Risk factor analysis of venous thromboembolism in cancer

Background: Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is caused by mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied dual AAV-mediated gene therapy to a mouse model of USH1B, the shaker-1 (Myo7a4626SB/4626SB) mouse. The shaker-1 mouse has a nonsense mutation in Myo7a. It is profoundly deaf throughout life, and it has significant vestibular dysfunction, similar to patients with USH1B. 

Methods: Neonatal (P0 to P5) shaker-1 (Myo7a4626SB/4626SB) mutant mice were used in this study. Due to the ~6.7 kb of the Myo7a cDNA, the dual AAV approach was used for gene delivery via the posterior semicircular canal. Auditory brainstem response (ABR) was used to assess auditory function. Vestibular evoked potential (VsEP) and circling behavior recording were used to assess vestibular function. Immunohistochemistry was used to evaluate viral transduction efficiency, sensory hair cell viability, and stereocilia morphology. 

Results: The shaker-1 mice are profoundly deaf due to significant stereocilia disorganization and rapidly progressive cochlear hair cell loss. Even though vestibular hair cells do not undergo degeneration in shaker-1 mice, they also exhibit significant vestibular dysfunction, due to stereocilia disorganization of the vestibular hair cells as well. When Myo7a cDNA was delivered to shaker-1 inner ears using the dual AAV approach, cochlear hair cell survival was improved. However, stereocilia organization and auditory function were not improved. In the vestibular system, dual AAV-mediated Myo7a delivery caused an improvement in stereocilia organization. In addition, the treated shaker-1 mice also had improved vestibular function, reflected by reduced circling behavior and improved VsEP thresholds. 

Conclusions: Our results showed that dual AAV gene therapy was able to improve the vestibular function in the shaker-1 mutant mice.

Medical Genetics and Genomics - Dual AAV-Mediated Gene Replacement Therapy Improves the Vestibular Function in a Mouse Model of USH1B - Medical Genetics and Genomics

Background
FMR1-related disorders is a classification of X-linked trinucleotide repeat conditions which includes Fragile X syndrome. Preimplantation genetic testing for monogenic diseases (PGT-M) is classically considered a linkage-based test for determination of the allele present in an at-risk embryo. FMR1 expansion size modifies interpretation of embryo disease risk, where >200 CGG repeats is consistent with a diagnosis of Fragile X syndrome. This study is the first collection of embryo outcomes that includes triplet repeat size determination.

Methods
Trophectoderm biopsies were sent to CooperSurgical for PGT-M for FMR1 between January 2018 and February 2021. Samples underwent linkage analysis via Karyomapping (lllumina, USA) and triplet repeat size determination. Patients with FMR1 expansions on both alleles and those with a full expansion (>200 repeats) on one allele were removed. A total of 1130 blastocysts from 318 IVF cycles underwent triplet repeat size determination and linkage analysis. 

Results
48.6% (549/1130) of embryos had the abnormal FMR1 allele. 69.4% (381/549) blastocyst samples inherited the abnormal allele but did not experience a significant expansion, with repeats remaining < 200. 202/381 of samples were euploid. 17.9% of the total study population (202/1130) was euploid, abnormal allele present, and expansion <200.

Conclusion
Embryo triplet repeat size determination increased the number of embryos that could be considered for transfer compared to linkage-only methods by 17.9%. Complete information about CGG sizes in embryos is an asset for genetic counseling about selection for embryo transfer, especially in a patient population where FMR1 carrier status confers a risk for premature ovarian insufficiency. Expanded information of embryo status may reduce the number of total IVF cycles a patient pursues if they choose to transfer a carrier embryo with a repeat size < 200.

Medical Genetics and Genomics - Observed outcomes of FMR1 PGT-M analysis with incorporation of CGG repeat expansion - Medical Genetics and Genomics

Background: Arteriovenous Fistula (AVF) stenosis is responsible for a very significant morbidity, mortality and economic cost. Despite the magnitude of the clinical problem there are currently no effective therapies for AVF stenosis. The focus of this project was therefore to identify uremia specific functional and histological differences in AVF stenosis in a mouse model.

Methods: Mice were made uremic through the removal of the upper and lower poles of one kidney followed by a nephrectomy of the contralateral kidney. AVFs were created through an end (vein) to side (artery) anastomosis using standard techniques. Functional parameters included flow mediated dilation (FMD) and the area enclosed within the elastic lamina at 100 micron intervals x 12 from the AV anastomosis. The histological end point was the ratio of thrombus area/perimeter at 100 micron intervals x 12 from the AV anastomosis (with the first 600 microns being considered proximal and the second 600 microns as distal).

Results: A total of 22 mice were used in these experiments. The mean +/- SE for BUN in the uremic animals was 109+/-4.8 mg/dL as compared to 17.4 +/- 1.0 mg/dL in the control animals (p<0.05). FMD at 60 seconds was decreased significantly in uremic animals (13.4 +/- 1.5%) as compared to the control animals (19.5 +/- 1.1%), although there was no difference at the 90 second peak. There was a trend towards a smaller area enclosed by the elastic lamina (an indicator of inward remodeling) in the uremic animals, although this achieved statistical significance only in the distal segments (at 1000-1200 microns beyond the AV anastomosis). Finally, uremic animals had significantly more attached thrombus (thrombus area/perimeter) in the distal portions of the AVF (9414.7 +/- 7187.5) as compared to the proximal portions (1055.0 +/- 529.5).

Conclusion: These data document impairment of endothelial linked, functional (FMD and inward remodeling) and histological (thrombus formation) pathways in a mouse model of AVF stenosis. Further elucidation of the uremia specific mechanistic pathways responsible for these differences, could result in the development of novel drugs and devices for the treatment of AVF stenosis in patients with CKD and ESRD.

Uremia Induces Functional and Histological Changes in a Mouse Model of AVF Stenosis

Traumatic brain injury (TBI) is characterized by diffuse axonal injury that leads to neurodegeneration mediated by glial-driven neuroinflammation and neurotoxicity. Several chronic morbidities associated with TBI are psychiatric and stress-related, such as post-traumatic stress disorder. The basolateral (BLA) and central amygdala (CeA) are regulators of emotion, behavior, and autonomic function, and disruption of the amygdala due to experimental TBI has been shown to induce chronic anxiety-like behaviors, dysfunction of neurotransmission, hypertrophy of glutamatergic neurons, and dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Beyond their role in stress and inflammation, changes in glucocorticoid receptor (GR) expression can mediate behavior, neurotransmission, neuroplasticity, and reflect dysregulation of the HPA axis. To evaluate these processes, 64 young adult, age-matched male and naturally cycling female Sprague-Dawley underwent sham surgery or midline fluid percussion injury (mFPI), and were sacrificed at 7-, 56-, or 168-days post injury (DPI). Image processing was used to analyze ionized calcium binding adaptor molecule 1 (IBA1) and glial fibrillary acid protein (GFAP)-stained photomicrographs to quantify microglia and astrocyte activation, respectively. Visual interpretation of amino-cupric-silver (AmCuAg) staining patterns were subjectively done to assess neuropathology. Simple western blotting was performed in 58 animals (25 from histology cohort) using whole amygdala biopsies to evaluate GR protein at 7- and 56-DPI. Time post-surgery, despite sporadic sex and nuclei-specific instances, was a primary driver of microglia activation and astrogliosis by 168-DPI within the BLA/CeA. The chronic, mild increase in AmCuAg staining intensity visually observed in all groups indicated that age-related neurodegeneration was likely behind histopathologic findings. Midline FPI decreased GR protein in males only at 7-DPI. These data indicate that age and HPA axis dysregulation may be primarily responsible for previously reported behavioral, neurochemical, cellular, and molecular outcomes, ruling out TBI-induced axonal injury and chronic neurodegeneration in the BLA/CeA underlying the pathophysiology of affective-like symptoms after mFPI.

Next from AMA Research Challenge 2022

N-MYC Transcription Factor Increases the Expression of Alpha-7 Nicotinic Acetylcholine Receptor Promoting Cell Viability in Neuroblastoma Cell Line

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Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

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