United States

Background: Cancer is a leading health concern and the second highest cause of death in the United States. When considering cancer from a genomic standpoint, genes can be divided into two main categories. One category includes oncogenes, which, when mutated, develop a gain of function. The second category consists of tumor suppressor genes, which in mutated state lose their ability to protect the cell. Both oncogenes and tumor suppressor genes are known as cancer-critical genes. This analysis evaluates 50 cancer-critical genes for presence of hotspot mutations affecting various signaling pathways. Specifically, we focused on nine pathways: RTK/RAS/MAP, TGFβ, PI3K, Wnt, GPCR, p53, JAK/STAT, Notch, and the cell cycle pathway. Our study aims to enhance understanding of the impact of hotspot mutations in these 50 cancer-critical genes. Additionally, the study will aim in integration of artificial intelligence (AI) to predict personalized treatments based on the identified mutations.

Methods: We conducted an analysis of previously identified mutations in 35 breast cancer patients and 39 lung cancer patients from the Prisma Health Cancer Institute. We utilized genomic databases, including ClinVar, to gather available information and published literature about these mutations. We analyzed and recorded characteristics of each mutation, including predicted changes in protein folding, clinical significance and associated pathological conditions, possible variations, and affected signaling pathways.

Results: Among the 50 cancer-critical genes previously sequenced for hotspot mutations, we identified a total of 79 different hotspot mutations in the studied group of 74 cancer patients. Notably, for 9 of these mutations we did not find any available information. Furthermore, one mutation (KDR p.Gln472His) exhibited a very high prevalence among cancer patients. We suggest further investigation of this group of mutations for potential clinical significance.

Conclusion: The identified unknown mutations in cancer-critical genes may have clinical significance and require further investigation. The information collected in this study will contribute to the development of a comprehensive cancer patient database accessible to clinicians and researchers. Moreover, the data will be further utilized in AI applications to predict personalized treatment approaches for cancer patients.


AMA Research Challenge 2023

Improving Diagnosis and Prognosis: Analysis of Gene Mutations in Breast and Lung Cancer Patients

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**Abstract:**



The Impact of the Tumor Microbiome on Anti-tumor Immunity in Mucosal Melanoma

Background 
Cancer-induced cachexia is a complex multifactorial syndrome characterized by loss of muscle mass and function that affects up to 80% of cancer patients which contributes to the morbidity and mortality of the disease. Despite the extensive research on cancer cachexia, its exact etiology remains unknown. There is also no consensus on effective treatment for the loss of muscle mass, which subsequently impacts the tolerance and response to anti-cancer therapies. Furthermore, little is known about the effect of muscle wasting on the neuromuscular junction (NMJ), the vital synaptic connection between the motor neuron and skeletal muscle. The primary purpose of this study is to analyze the stability of the NMJs from cachectic muscles during the initiation and progression of the muscle-wasting event. To this end, we use the genetically engineered mouse model of colorectal cancer (Apc) Min/+ that has shown tumor growth and cachexia. 

Methods 
Sternomastoid, gastrocnemius, extensor digitorum longus, soleus, and diaphragm muscles were extracted from (Apc) Min/+ and age-matched controls at 16 and 24 weeks of age. The muscles were then fixed in 4% paraformaldehyde and bathed with Alexa488™ alpha-bungarotoxin to specifically label nicotinic acetylcholine receptors (nAChRs) at the postsynaptic junctional folds. Fluorescently labeled muscles were then imaged at the confocal microscope to analyze the synapse morphology, or with an inverted fluorescence microscope to quantify fluorescence intensity. The area and the density of nAChRs were then analyzed using ImageJ. To capture relevant changes in the NMJs, we performed in vivo experiments to determine the turnover rate of nAChRs at the synapse before severe weight loss occurred. To this end, we labeled sternomastoid muscles in vivo and assessed fluorescence intensity decay over time. 

Results 
At early stages of cancer-induced cachexia (16 weeks), all muscles except sternomastoid showed a significant decrease in nAChRs density in (APC) Min/+ mice compared to controls. In advanced stages of cachexia (24 weeks), all muscles from (APC) Min/+ mice showed a significant decrease in nAChRs density. All muscles showed a significant decline in the total NMJ area in (APC) Min/+ at 16 weeks and 24 weeks. 

Conclusion 
The decreased nAChR density and NMJ area in both fast and slow twitch muscles demonstrates postsynaptic NMJ alteration in cancer-induced cachectic mice. Analysis of presynaptic cells (motor neuron and Schwann cell) will be performed to further look at the pathology of the NMJ in cancer-induced cachexia, as well as younger mice to determine when the degeneration starts. 


The Impact of Cancer-induced Cachexia on the Neuromuscular Synapse

Background
Chimeric antigen receptor (CAR) T cells have shown remarkable clinical efficacy in hematological cancers but still face significant obstacles in the treatment of solid tumors, such as pancreatic ductal adenocarcinoma (PDAC). Two major hurdles in developing effective CAR T therapies for PDAC are the identification of tumor-associated antigens (TAA) and overcoming a complex immunosuppressive tumor microenvironment (TME). We hypothesize that the optimal CAR T cell to treat PDAC recognizes an ideal TAA and protects itself from TME immunosuppression. We propose a novel CAR T cell to target the Muc16-ectodomain (Muc16CD) combined with antagonism of the vasoactive intestinal peptide (VIP) checkpoint pathway in the PDAC TME as a viable therapy to treat pancreatic tumors. 

Methods
Patient data was assessed based upon data generated by the TCGA Research Network. Primary human T cells were retrovirally transduced to express Muc16CD-directed CARs with or without secretion of VIPR antagonists. PDAC PDX tumors and CAR T cells assayed by flow cytometry for phenotype and functional profile. In vivo, Panc1-Muc16CD-GFPffLuc tumors were engrafted into SCID/Beige mice and treated with CAR T cells.

Results
Muc16 expression is significantly increased in PDAC patient tissues compared to normal pancreas and associated with decreased overall survival. Congruently, we found that PDAC PDX tumors demonstrate robust expression of both Muc16CD and VIP and are vulnerable to Muc16CD-directed CAR T cell cytotoxicity. Additionally, VIP limits the proliferative capacity of CAR T cells, which is reversed by VIPR antagonist peptides (antVIPR). Therefore, we engineered anti-Muc16CD CARs to secrete antVIPR peptides to provide continuous and localized delivery within the TME. We find that antVIPR expression by CAR T cells enhances viability, activation profile, and demonstrate less-differentiated and less exhaustive phenotypes. Functionally, antVIPR CAR T cells have enhanced activation and proliferative advantage after chronic antigen-stimulation. In a xenograft model of PDAC, antVIPR CAR T cells reduce tumor burden and improve overall survival in mice bearing human VIP-expressing PDAC tumors. At 118 days, antVIPR CAR T-treated mice have more circulating CAR T cells than parental CAR T-treated mice. 

Conclusion
Muc16CD is a clinically relevant target for CAR T therapy in PDAC. Furthermore, antagonizing the VIP checkpoint pathway in CAR T cells produces enhanced phenotypic and functional profiles. Collectively, this data demonstrates that the combination of targeting Muc16CD and VIP with a novel CAR T cell can improve anti-tumor efficacy against PDAC. The long-term goal of this work is translating antVIPR-secreting CAR T cells for the treatment of PDAC.


Using CAR T Cell-Secreted Antagonistic Peptides to Target Distinct Immune Checkpoint Pathway in Pancreatic Tumors

Background
High-throughput single-cell RNA-seq (scRNA-seq) is used to describe complex tissues by characterizing transcriptional states of individual cells. Defining a cell's position, both in regard to tissue margins and its social context, is essential for understanding the intrinsic and extrinsic variables that effect the transcriptional identity of individual cells. Conventional high-throughput scRNA-seq assays, however, decouple cells from their original locations within tissues. In situ hybridization readouts of gene expression and in situ sequencing preserve spatial information in tissues, but currently have a lower total read threshold than NextGen sequencing – imposing a restriction on either cell throughput or transcriptional breadth. Combining the above methods or using regional barcodes to define 2D positions have spatially-reconstructed tissue regions but are seldom employed in unspecialized laboratories.

Methods
We describe SEgmentation by Exogenous Perfusion (SEEP), a rapid, integrated, method for providing 3D spatial-segmentation to scRNA-seq data. Tissues are divided into layers based on accessibility of a fluorescent dye allowing sorted cells to be characterized by transcriptomic and regional identity.

Results
We use SEEP to explore how the transcriptional states of cells in high-grade serous ovarian cancer vary with respect to intratumoral position. We describe an integrated method for correlating 3D radial-spatial cell positions with scRNA-seq data. By employing a basic stain-and-sort method using an off-the-shelf live-dead stain, we are able to correlate transcriptional profiles with radial-spatial positions in both radially symmetric tissues (e.g., spheroids, organoids, spherical tumor masses) and linear tissue samples (e.g., punch biopsies). SEEP uses an imaging-based calibration step to inform the parameters of a FACS and sequencing based measurement step.

Conclusion
Across spheroid, organoid, and PDX models of high-grade serous ovarian cancer, we find common positional transcriptomic profiles, ranging from single-gene proclivities to multi-gene-set enrichments across tissue layers. The results validate long-standing beliefs regarding the relationship between cell state and position while also revealing new concepts on how the spatially unique microenvironment of individual cells within tumors influences cell identity.

High-throughput spatial reconstruction of cell states within ovarian cancer structures

Background
Breast cancer (BC) is the most common cancer affecting women and is associated with multiple genetic and environmental factors. To better understand individual’s genetic risk for disease, polygenic risk scores (PRSs) were developed using genome-wide association studies (GWASs). BC PRS have been developed with the hopes of improving risk stratification, but their replication is often limited. We worked with a population of genetically similar Dutch and American women to investigate the use of BC PRSs.

Methods
Two published PRSs utilizing 313 and 3280 single nucleotide polymorphisms (SNPs) were used to score 468 BC cases and 4,337 cancer negative controls from the integrated Cancer Repository for Cancer Research and cancer negative controls from the Netherland Twin Register. Blood samples were used to extract DNA and subjects were genotyped using the Illumina Global Screening Array and underwent standardized quality control, and 20 averaged runs of imputation, and polygenic scoring. Percentile scores were then compared via ANOVA testing, variability assessment, and area under the curve analysis.

Results
Results showed that the PRSs of BC cases were significantly greater than the controls at 52.81 and 48.07 (p<0.001), respectively, for the 313 PRS and 55.45 and 49.81 (p<0.001) for the 3820 PRS, respectively. The AUC was 0.596 (95% CI: 0.569-.624) for the 313 PRS and 0.603 (95% CI: 0.576-0.630) for the 3820 PRS, with both slightly underperforming the originally published AUC of 0.639 and 0.646 for the 313 and 3820 PRS, respectively. Variability of PRSs across imputation was 2.17% for the 313 PRS and 2.99% for the 3820 PRS, with a subset with increased score ranges.

Conclusion
Our study provides further evidence that BC PRSs are reproducible in our study populations with slightly less predictive capacity. In addition, we found that scores varied 2-3% between various imputation runs with some increased variability in subjects carrying many rare alleles, indicating that imputation methodology can have an impact an individual’s reported PRS. This may affect the clinical implementation of PRSs and may be worth considering as this new tool enters clinical use.

Exploration of breast cancer polygenic risk scores in a population of breast cancer patients

Background
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, largely due to its high rate of early metastasis. PDAC remains almost uniformly refractory to T cell-based immunotherapies that have transformed treatment in many other cancers, and there is a critical need to identify new therapeutic approaches. The abundance and functions of eosinophils in PDAC have not been explored despite increasing evidence for important roles of eosinophils in other tumor types. Here, we quantified tumor eosinophilia in preclinical PDAC models and a large PDAC patient cohort, and we assessed whether modulation of eosinophils impacts primary tumor growth or metastasis. 

Methods
We used flow cytometry and immunohistochemistry to quantify eosinophil abundance in PDAC patient samples and in subcutaneous, orthotopic, and genetically engineered mouse models (GEMMs) of PDAC. We used single cell RNA-sequencing (scRNA-seq) and cytokine arrays to study the mechanism by which eosinophils are recruited into pancreatic tumors. Using an eosinophil-deficient mouse model (dblGATA), genetically engineered cytokine-overexpressing cell lines, and systemic injection of exogenous interleukin-33 (IL-33), we studied the effects of eosinophil depletion, expansion, and activation on primary tumor growth and metastasis in murine PDAC. We used scRNA-seq to study mechanisms by which intratumoral eosinophils may impact PDAC metastasis. 

Results
Eosinophils were abundant in PDAC samples from human patients and murine models. They are likely recruited into tumors via local production of eotaxin-1 and eotaxin-2 by distinct populations of cancer associated fibroblasts and macrophages. Eosinophil expansion or depletion does not impact the growth of primary pancreatic tumors, but mice lacking eosinophils are more likely to develop spontaneous lung metastases. Treating mice with recombinant IL-33 expands eosinophils systemically and reduces rates of metastasis. Wild-type and eosinophil-deficient mice have similar metastasis burdens after intravenous injection of tumor cells, suggesting that eosinophils do not limit metastasis by impacting the pre-metastatic niche. Rather, eosinophils may interact with other intratumoral cell types to limit the entry of cancer cells into circulation. Indeed, scRNA-seq reveals that eosinophil deficiency is associated with a significant shift in the proportions of intratumoral endothelial cell (EC) subpopulations from tip-like to stalk-like ECs. 

Conclusion
Eosinophils are recruited by locally produced cytokines into the PDAC TME, where they shape the endothelial compartment and regulate the metastatic spread of tumor cells. Further studies are warranted to better delineate the mechanisms underlying this phenotype and to determine whether tumor eosinophilia can serve as a useful therapeutic target or prognostic biomarker in PDAC patients.

Eosinophils regulate metastasis in pancreatic cancer

In order to function correctly, cells must regulate gene expression; chemical modification of DNA is an important method by which they achieve this. For example, cytosine can be methylated to form 5-methylcytosine (5mC), a repressive epigenetic marker. 5mC can be oxidized by Ten-Eleven-Translocation (TET) enzymes to produce additional forms of cytosine. One of these derivatives, 5-hydroxymethylcytosine (5hmC), is an activator of gene expression and ͏can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Subsequently, 5caC is excised and replaced with unmodified cytosine. This process by which 5mC is converted back to cytosine is known as active demethylation (AD).
Improper control of methylation and demethylation can impair development and is a hallmark of many cancers. Ubiquitin-like Modifier with PHD and Ring Finger Domain 2 (UHRF2) is a DNA-interacting protein that plays an important but poorly characterized role in active demethylation. Previous work suggests that UHRF2 is required for the maintenance of 5hmC levels in somatic cells but does not demonstrate a mechanism. Furthermore, a paralog of this protein, UHRF1, binds to 5mC via its Set and Ring Associated (SRA) domain to preserve methylation levels in DNA. Thus, we proposed a related model where UHRF2 binds to 5hmC via its SRA domain and prevents further oxidation by TET enzymes. 
In this study, we overexpressed UHRF2 and TET2 in wild-type U2OS (human osteosarcoma) cells and U2OS cells with endogenous UHRF2 expression knocked out. We also conducted domain-deletion experiments to determine which domains of UHRF2 are required to preserve 5hmC levels.
As expected, we demonstrated that excess TET2 increased 5hmC levels in both cell lines, but concurrent overexpression of UHRF2 appeared to amplify this effect. We also determined that the plant homeodomain (PHD), tandem Tudor domain (TTD), and SRA domain are each necessary for UHRF2 to preserve 5hmC levels. Furthermore, we found that simultaneous overexpression of TET2 and UHRF2 increased levels of 5fC more than overexpressing TET2 alone, suggesting that UHRF2 may have a more complex role in regulating the AD cycle. Eventually, a better understanding of UHRF2 function will elucidate its role in cancer and may guide future research and therapeutics.

The epigenetic modifier UHRF2 has a complex role in 5hmC maintenance

Background
Neurofibromatosis type 2 (NF2) is cancer predisposition syndrome characterized by the development of bilateral vestibular (VS) and spinal schwannomas secondary to loss of heterozygosity of NF2 in Schwann cells or their precursors. Largely benign, tumors may result in deafness, vertigo, facial muscle weakness, chronic neuropathic pain, and death. There is an urgent need to develop pharmaceutical approaches for NF2 patients. Receptor tyrosine kinase inhibitors, brigatinib and dasatinib, were identified as potentially efficacious agents for the treatment of VS and shown to target the Ephrin A2 receptor (EPHA2). EPHA2 is a transmembrane receptor tyrosine kinase involved in cell contact-mediated motility, adhesion, and migration. 

Methods
We analyzed RNA Sequencing data and found EPHA2 expression to be increased in NF2-deficient Schwann cells. Data obtained from the TCGA Pancancer database demonstrated increased EPHA2 expression in NF2-/- cancers. We hypothesized that EPHA2 could be a potential target for the treatment of NF2-associated VS. Immunohistochemistry confirmed increased EphA2 expression in NF2-deficient murine vestibular schwannoma. 

ProteomicsDB was used to identify drugs predicted to target EPHA2 based on kinome analysis. We identified ponatinib, an ABL1 inhibitor, as an agent that targets EPHA2. Dose response curves obtained by cell titer glo luminescence assay and colony forming assays performed in human NF2-deficient schwann cells in vitro demonstrated impaired viability following treatment with ponatinib. Kinome profiling identified EPHA2 as the most significantly inhibited kinase following 24hour treatment with 100nM ponatinib. Western blot analysis was performed and EPHA2 protein expression was significantly decreased following ponatinib treatment.

We performed selective pharmacologic and genetic inhibition of EPHA2 with ALW-II-41-27 and siRNA in human NF2-deficient schwann cells in vitro. Dose response curves were obtained by cell titer glo luminescence assay and colony forming assays performed in human NF2-deficient schwann cells in vitro. 


Results

We demonstrate that EPHA2 expression is increased in NF2-/- Schwann cells and NF2-/- cancers. We identify ponatinib, a RTK inhibitor targeting ABL1 for CML, as an agent that targets EPHA2. We demonstrate that ponatinib treatment impairs the viability of both human and murine NF2-/- Schwann cells in vitro and decreases EPHA2 protein expression. Pharmacologic and siRNA-mediated inhibition of EPHA2 also impaired the growth of human NF2-/- Schwann cells in vitro. Lastly, we demonstrate that both ponatinib and EPHA2 inhibition induce morphological changes in NF2-/- Schwann cells. 

Conclusion
Our findings suggest that ponatinib or the direct targeting of EPHA2 may be efficacious for the treatment of NF2-associated vestibular schwannoma. Future in vivo efficacy studies are warranted. 


EPHA2 is a Potential Target for the Treatment of NF2-/- Vestibular Schwannoma
Kéyana Foster1, Dana K. Mitchell1, Alyssa Flint1, Brooke Rodriguez1, Henry Mang1, Chris Davis1, Steven P. Angus1, D. Wade Clapp1, Charles Yates2
1Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
2Department of Otolaryngology, Indiana University School of Medicine, Indianapolis, IN, USA


Single cell RNA sequencing (scRNA-seq) can detect gene expression and its dynamics in groups of cells. Its application to time-series samples can thus identify temporal gene expression programs active in different cell types. However, current scRNA-seq analysis needs improvement with regards to low gene detection, low replicate count, and inflated statistics when using cells as individual sampling data points. To address these issues, we explore whether “metacells” can be used as “replicates” through time. Toward this, we applied the use of metacells to a time-series scRNA-seq dataset from PBMCs after SARS-Cov-2 infection, which were then used in maSigPro for quadratic regression to find significantly differentially expressed genes (DEGs) over time, followed by clustering analysis of the expression velocity trends. Results demonstrated that this strategy could overcome the limitation of 1-2 replicates, as it correctly identified the known ISG15 interferon response program in almost all PBMC cell types. Its application further led to the uncovering of additional gene expression programs that potentially modulate different levels of host response after infection.

Identifying cell type transcriptome changes in COVID-19 patient PBMCs using metacells

Background 
Exosomes are extracellular vesicle (EV) nanocarriers responsible for delivering biologics including lipids, proteins, and nucleic acids. EVs are an attractive therapeutic weapon for delivering drugs and biologics to cells and tissues. Utilizing EVs for clinical applications is based on their low toxicity, ability to target to specific cells and tissues, and can be bioengineered to enhance their activities. Significant challenges remain before exosomes can be widely used in clinical settings. These include developing improved methods for purifying exosomes, enhancing the targeting of exosomes to diseased tissue, and boosting the efficiency of engineering exosomes for disease-specific applications. 
Exosomes need to be engineered in a more efficient way for broad therapeutic application. The lack of efficient intracellular bioengineering technologies to modify produced exosomes is a major obstacle to the widespread use of EVs for therapeutic applications. 
Advances in mammalian synthetic chromosome design and engineering offer a potential improvement in EV manufacturing. Our aim is to adopt the Artificial Chromosome Expression System (ACE System), an autonomous chromosome-based “circuit-board” designed to carry single or multiple copies of genes or DNA elements of interest for EV engineering.

Methods 
We have demonstrated that human miRNAs expressed from the ACE platform residing in a CHO-based cell line failed to process human miRNAs into CHO exosomes. This suggests that the processing of miRNAs from primary gene transcripts may be regulated in a species-specific manner. Thus, the ACE chromosome has been transferred into human HEK293 cells. Engineered miRNAs have been cloned downstream of a modified green fluorescent protein (AcGFP1) expressed from a doxycycline inducible promoter system retrofitted to contain ACE platform recombination system. 

Results 
Our results indicated that the ACE synthetic chromosome platform can be successfully engineered to express miRNAs as part of an initial doxycycline inducible AcGFP1 transcript. Furthermore, isolation of resulting EVs from the ACE engineered HEK293 cells indicated the presence of the expressed miRNAs. 

Conclusion 
From our work, we propose that the ACE synthetic platform has the potential to serve as a genetic circuit-board for modifying EVs of therapeutic value. In addition, the ACE synthetic chromosome is autonomous to the host cellular genome, i.e., non-integrating and non-mutagenic, thus would provide an increasing safety profile for using our developed synthetic chromosome technology in an ex vivo cellular therapy format. Future efforts will focus on enhancing the efficiency of our bioengineering approach for a specific disease indication.

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