Investigative Techniques Commons^™

Validation Of Next‑Generation Sequencing‑Derived Circulating Mrna Variants As Potential Diagnostic Tools In Hepatocellular Carcinoma And Cholangiocarcinoma, Daniel Zezulinski, Maarouf A. Hoteit, David E. Kaplan, Tingting Zhan, Cataldo Doria, Timothy M. Block, Aejaz Sayeed

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Liquid biopsy can transform oncology practice through early detection and real‑time monitoring of cancer progression and response to treatment. Circulating cell‑free tumor RNA offers promise as a novel and emerging alternative to circulating cell‑free tumor DNA for detecting the presence and understanding the biology of liver tumors. Both circulating DNA and RNA, which predominantly reside in extracellular vesicles, carry evidence of vital germline and somatic mutations. High impact single nucleotide changes, insertions and deletions in circulating RNA (ctRNA) serve a key role in cancer development and progression. Tumor cells exploit the RNA splicing machinery to induce structural and functional changes …

Stat-Independent Functions Of Janus Kinases 1 And 2 Are Obligatory For The Postnatal Development Of Mammary Epithelial Ducts, Rayane Dennaoui, Madison N. Wicker, Carson Moen, Michaela Schlederer, Kerry Vistisen, Aleata A. Triplett, Thomas Rülicke, Hallgeir Rui, Lukas Kenner, Emilio Casanova, Kay-Uwe Wagner

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Janus kinases 1 and 2 and STAT transcription factors are critical signaling nodes for numerous growth factors. In the mammary gland, JAK2 and STAT5a/b are essential for alveolar cell differentiation and lactation, but little is known about the cooperative roles of JAKs and STATs before pregnancy. We examined female mice conditionally deficient in JAK1/2 and discovered that both kinases jointly regulate epithelial cell proliferation and ductal morphogenesis. To assess the role of downstream STATs, we generated genetic models co-deficient in STAT3/5a/5b with or without STAT1 or JAK1. Although loss of STAT3/5a/5b leads to a JAK1-dependent upregulation of STAT1, the formation …

Real-World Impact Of Amiodarone On Apixaban Population Pharmacokinetics In Hospitalized Patients, Samantha Kolowrat, Chazmyn Riley, Kevin Lam, Lynda Thomson, Douglas F. Stickle, Walter K. Kraft

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Despite common coadministration in nonvalvular atrial fibrillation (NVAF), there is minimal evidence regarding the impact of concomitant amiodarone use on apixaban pharmacokinetics (PK). We described the population PK of apixaban (2.5 and 5mg twice daily) in 106 hospitalized patients with and without concomitant amiodarone administration at steady state. Apixaban PK was reasonably described by a one-compartment model with first-order absorption and linear elimination. Aside from the receipt of amiodarone, age was retained as a statistically significant covariate on apparent clearance. Model-based simulations depicted concomitant amiodarone use increasing AUCT for both 2.5mg (1.58, 90% CI [1.28, 1.87]) and 5mg (1.12, 90% …

Evaluating The Potential And Limitations Of Nanopore Adaptive Sampling For Targeted Transcriptome Sequencing, Nicole Debruyne, Feng Wang, Yang Xu, Lan Lin

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Long-read RNA sequencing is a powerful technology for transcriptomics, but low throughput and high cost pose challenges. Adaptive sampling, a feature of Oxford Nanopore Technologies, offers real-time enrichment by selectively ejecting non-target molecules. We evaluate adaptive sampling for human transcriptome analysis. Adaptive sampling modestly enriches target transcripts (1.3 × for cDNA sequencing, 1.9 × for direct RNA sequencing) while preserving gene expression and splicing profiles, but is significantly less effective than cDNA hybridization capture. Short read lengths and low sequencing quality limit performance. Adaptive sampling on direct RNA sequencing can boost target yield (~ 20%) within fixed run times, potentially …

Quantile Index Predictors Using R Package Hyper.Gam, Tingting Zhan, Misung Yi, Inna Chervoneva

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

MOTIVATION: Evaluation of single-cell protein expression from immunohistochemistry images is used increasingly in biomedical research. Many proteins are used solely for phenotyping cells in the tumor microenvironment. Other proteins with meaningfully quantitative expression levels provide so-called functional protein biomarkers. There is still a limited number of methods and software tools available for utilizing the entire distributions of single-cell expression levels.

RESULTS: We present the R package hyper.gam, providing a supervised learning framework for deriving biomarkers based on single-cell distribution quantiles. The single-cell data are first converted into sample quantile functions, which are then used as predictors in scalar-on-function regression models …

Elevated Nr2f1 Underlies The Persistence Of Invasive Disease After Treatment Of Braf-Mutant Melanoma, Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Oliveira Da Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L. F. Teh, Rama Kadamb, Weijia Cai, Sheera Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Despite the success of targeted inhibitors in cutaneous melanoma, therapeutic responses are limited by the aged tumor microenvironment and drug-tolerant residual cells. Given the similarities between drug tolerance and cellular dormancy, we studied the dormancy marker, nuclear receptor subfamily 2 group F member 1 (NR2F1), in response to BRAF-V600E inhibitors (BRAFi) plus MEK inhibitors (MEKi) in BRAF-mutant melanoma models. Transcriptomic analysis of melanoma patient samples treated with BRAFi + MEKi showed increased NR2F1. NR2F1 was highly expressed in the drug-tolerant invasive cell state of minimal residual disease in patient-derived and mouse-derived xenografts on BRAFi + MEKi. NR2F1 over-expression was sufficient …

5-Hydroxymethylcytosine Sequencing Of Plasma Cell-Free Dna Identifies Epigenomic Features In Prostate Cancer Patients Receiving Androgen Deprivation Therapies, Qianxia Li, Chiang-Ching Huang, Shane Huang, Yijun Tian, Jinyong Huang, Amirreza Bitaraf, Xiaowei Dong, Marja T. Nevalainen, Manishkumar Patel, Jodie Wong, Jingsong Zhang, Brandon J Manley, Jong Y. Park, Manish Kohli, Elizabeth M. Gore, Deepak Kilari, Liang Wang

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

BACKGROUND: We evaluated whether 5hmC signatures in cell-free DNA (cfDNA) are associated with treatment failure to androgen-deprivation therapies (ADT) among men with hormone-naive prostate cancer.

METHODS: We collected a total of 139 serial plasma samples from 55 prostate cancer patients receiving ADT at 3 time points including baseline (before initiating ADT, n = 55); 3 months (after initiating ADT, n = 55); and disease progression (n = 15) within 24 months or 24 months if no progression was detected (n = 14). We used selective chemical labeling sequencing to quantify 5hmC abundance across the genome and Kaplan-Meier analysis to assess …

Bayesian Population Pharmacokinetic Modeling Of Ondansetron For Neonatal Opioid Withdrawal Syndrome, Kevin Lam, John Mondick, Gary Peltz, Manhong Wu, Walter Kraft

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Ondansetron is an anti-emetic 5-HT3 receptor antagonist being investigated for treating neonatal opioid withdrawal syndrome (NOWS). Sparse PK data were analyzed from a multicenter, double-blind clinical trial with 98 mother/neonate dyads. Pregnant women with opioid use disorder were randomized to receive either placebo or ondansetron 8 mg intravenously within 4 h of delivery. Neonates born to mothers who were randomized to ondansetron received 0.07 mg/kg orally once every 24 h for up to five doses. Using current PK data, model parameters from a two-compartmental structural model from the literature (i.e., a priori model) were updated with the Metropolis-Hastings Markov-chain Monte …

Genetic Ancestry Concordant Rna Splicing In Prostate Cancer Involves Oncogenic Genes And Associates With Recurrence, Muthana Al Abo, Wen-Chi Foo, Lauren Howard, Shannon Mcgue, Bonnie Lacroix, Julie Kephart, Angela Clayton, Blair Thornburg, Monika Anand, Michael Rothberg, Shannon Mccall, Jiaoti Huang, Thomas Esther, Judd Moul, Michael Ferrandino, Thomas Polascik, Cary Robertson, Brant Inman, Andrew Armstrong, Yuan Wu, Terry Hyslop, Daniel George, Steven Patierno, Jennifer Freedman

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Black men suffer disproportionately from prostate cancer (PCa) compared to men of other races and ethnicities. Comparing the molecular landscape of PCa among Black and White patients has the potential to identify targets for development of new precision medicine interventions. Herein, we conducted transcriptomic analysis of prostate tumors and paired tumor-adjacent normals from self-reported Black and White PCa patients and estimated patient genetic ancestry. Clinical follow-up revealed increased biochemical recurrence (BCR) among Black patients compared to White patients with high-grade PCa. Transcriptomic analysis identified differential alternative RNA splicing events (ARSs) between Black and White PCa patients. Genes undergoing genetic ancestry-concordant …

The C-Terminal Phdvc5hch Tandem Domain Of Nsd2 Is A Combinatorial Reader Of Modified H3k4 And Tri-Methylated H3k27 That Regulates Transcription Of Cell Adhesion Genes In Multiple Myeloma, Andrea Berardi, Charlotte Leonie Kaestner, Michela Ghitti, Giacomo Quilici, Paolo Cocomazzi, Jianping Li, Federico Ballabio, Chiara Zucchelli, Stefan Knapp, Jonathan Licht, Giovanna Musco

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Histone methyltransferase NSD2 (MMSET) overexpression in multiple myeloma (MM) patients plays an important role in the development of this disease subtype. Through the expansion of transcriptional activating H3K36me2 and the suppression of repressive H3K27me3 marks, NSD2 activates an aberrant set of genes that contribute to myeloma growth, adhesive and invasive activities. NSD2 transcriptional activity also depends on its non-catalytic domains, which facilitate its recruitment to chromatin through histone binding. In this study, using NMR, ITC and molecular dynamics simulations, we show that the tandem PHD domain of NSD2 (PHDVC5HCHNSD2) is a combinatorial reader of unmodified histone H3K4 and tri-methylated H3K27 …

Cd8Α Structural Domains Enhance Gucy2c Car-T Cell Efficacy, Trevor R. Baybutt, Ariana A. Entezari, Adi Caspi, Ross E. Staudt, Robert D. Carlson, Scott A. Waldman, Adam E. Snook

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

Despite success in treating some hematological malignancies, CAR-T cells have not yet produced similar outcomes in solid tumors due, in part, to the tumor microenvironment, poor persistence, and a paucity of suitable target antigens. Importantly, the impact of the CAR components on these challenges remains focused on the intracellular signaling and antigen-binding domains. In contrast, the flexible hinge and transmembrane domains have been commoditized and are the least studied components of the CAR. Here, we compared the hinge and transmembrane domains derived from either the CD8ɑ or CD28 molecule in identical GUCY2C-targeted third-generation designs for colorectal cancer. While these structural …