Publications by authors named "Thomas C Whisenant"

13 Publications

  • Page 1 of 1

Toward Improved and Standardized Diagnostic Pipelines in Transplantation.

Transplantation 2021 01;105(1):12-13

Division of Organ Transplant and Bioinformatics and Biorepository, Scripps Clinic Bio-Repository and Bio-Informatics Core, La Jolla, CA.

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http://dx.doi.org/10.1097/TP.0000000000003438DOI Listing
January 2021

Systems biology approaches in solid organ transplantation.

Curr Opin Organ Transplant 2021 02;26(1):37-42

Scripps Clinic Bio-Repository and Bio-Informatics Core.

Purpose Of Review: Organ transplantation research has led to the discovery of several interesting individual mechanistic pathways, molecules and potential drug targets but there are still no comprehensive studies that have addressed how these varied mechanisms work in unison to regulate the posttransplant immune response that drives kidney rejection and dysfunction.

Recent Findings: Systems biology is a rapidly expanding field that aims to integrate existing knowledge of molecular concepts and large-scale genomic and clinical datasets into networks that can be used in cutting edge computational models to define disease mechanisms in a holistic manner. Systems biology approaches have brought a paradigm shift from a reductionist view of biology to a wider agnostic assessment of disease from several lines of evidence. Although the complex nature of the posttransplant immune response makes it difficult to pinpoint mechanisms, systems biology is enabling discovery of unknown biological interactions using the cumulative power of genomic data sets, clinical data and endpoints, and improved computational methods for the systematic deconvolution of this response.

Summary: An integrative systems biology approach that leverages genomic data from varied technologies, such as DNA sequencing, copy number variation, RNA sequencing, and methylation profiles along with long-term clinical follow-up data has the potential to define a framework that can be mined to provide novel insights for developing therapeutic interventions in organ transplantation.
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http://dx.doi.org/10.1097/MOT.0000000000000837DOI Listing
February 2021

Irreversible Electroporation Combined with Checkpoint Blockade and TLR7 Stimulation Induces Antitumor Immunity in a Murine Pancreatic Cancer Model.

Cancer Immunol Res 2019 10 13;7(10):1714-1726. Epub 2019 Aug 13.

Moores Cancer Center, University of California San Diego, San Diego, California.

Irreversible electroporation (IRE) is a nonthermal ablation technique that is used clinically in selected patients with locally advanced pancreatic cancer, but most patients develop recurrent distant metastatic disease. We hypothesize that IRE can induce an vaccination effect by releasing tumor neoantigens in an inflammatory context. Using an immunocompetent mouse model, we demonstrated that IRE alone produced complete regression of subcutaneous tumors in approximately 20% to 30% of mice. IRE was not effective in immunodeficient mice. Mice with complete response to IRE demonstrated prophylactic immunity and remained tumor free when rechallenged with secondary tumors on the contralateral flank. CD8 T cells from IRE-responsive mice were reactive against peptides representing model-inherent alloantigens and conferred protection against tumor challenge when adoptively transferred into immunocompromised, tumor-naïve mice. Combining IRE with intratumoral Toll-like receptor-7 (TLR7) agonist (1V270) and systemic anti-programmed death-1 receptor (PD)-1 checkpoint blockade resulted in improved treatment responses. This combination also resulted in elimination of untreated concomitant distant tumors (abscopal effects), an effect not seen with IRE alone. These results suggest that the systemic antitumor immune response triggered by IRE can be enhanced by stimulating the innate immune system with a TLR7 agonist and the adaptive immune system with anti-PD-1 checkpoint blockade simultaneously. Combinatorial approaches such as this may help overcome the immunosuppressive pancreatic cancer microenvironment.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774877PMC
October 2019

BAL Cell Gene Expression in Severe Asthma Reveals Mechanisms of Severe Disease and Influences of Medications.

Am J Respir Crit Care Med 2019 10;200(7):837-856

Division of Allergy, National Jewish Hospital, Denver, Colorado.

Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma. To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression. BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores. Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and β-agonist-naive subjects given a β-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes. Gene expression in BAL cells is influenced by factors seldomly considered. Notably, β-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.
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http://dx.doi.org/10.1164/rccm.201811-2221OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812436PMC
October 2019

Oncogenic mutations in IKKβ function through global changes induced by K63-linked ubiquitination and result in autocrine stimulation.

PLoS One 2018 18;13(10):e0206014. Epub 2018 Oct 18.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.

Mutations at position K171 in the kinase activation loop of Inhibitor of κB kinase beta (IKKβ) occur in multiple myeloma, spleen marginal zone lymphoma and mantle cell lymphoma. Previously, we demonstrated that these result in constitutive kinase activation and stimulate Signal Transducer and Activator of Transcription 3 (STAT3). This work also identified K147 as a site of K63-linked regulatory ubiquitination required for activation of signaling pathways. We now present a more detailed analysis of ubiquitination sites together with a comprehensive examination of the signaling pathways activated by IKKβ K171E mutants. Downstream activation of STAT3 is dependent upon the activity of: UBE2N, the E2 ubiquitin ligase involved in K63-linked ubiquitination; TAK1 (MAP3K7), or TGFβ Activated Kinase, which forms a complex required for NFκB activation; JAK kinases, involved proximally in the phosphorylation of STAT transcription factors in response to inflammatory cytokines; and gp130, or IL-6 Receptor Subunit Beta which, upon binding IL-6 or other specific cytokines, undergoes homodimerization leading to activation of associated JAKs, resulting in STAT activation. We further demonstrate, using an IL-6-responsive cell line, that IKKβ K171E mutants stimulate the release of IL-6 activity into conditioned media. These results show that IKKβ K171E mutants trigger an autocrine loop in which IL-6 is secreted and binds to the IL-6 receptor complex gp130, resulting in JAK activation. Lastly, by examining the differential abundance of proteins associated with K63-only-ubiquitinated IKKβ K171E, proteomic analysis demonstrates the global activation of proliferative responses. As cancers harboring K171-mutated IKKβ are likely to also exhibit activated STAT3 and p44/42 MAPK (Erk1/2), this suggests the possibility of using MAPK (Erk1/2) and JAK inhibitors, or specific ubiquitination inhibitors. K63-linked ubiquitination occurs in other kinases at sites homologous to K147 in IKKβ, including K578 in BRAF V600E, which serves as an oncogenic driver in melanoma and other cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193727PMC
April 2019

Development and clinical validity of a novel blood-based molecular biomarker for subclinical acute rejection following kidney transplant.

Am J Transplant 2019 01 31;19(1):98-109. Epub 2018 Aug 31.

Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Noninvasive biomarkers are needed to monitor stable patients after kidney transplant (KT), because subclinical acute rejection (subAR), currently detectable only with surveillance biopsies, can lead to chronic rejection and graft loss. We conducted a multicenter study to develop a blood-based molecular biomarker for subAR using peripheral blood paired with surveillance biopsies and strict clinical phenotyping algorithms for discovery and validation. At a predefined threshold, 72% to 75% of KT recipients achieved a negative biomarker test correlating with the absence of subAR (negative predictive value: 78%-88%), while a positive test was obtained in 25% to 28% correlating with the presence of subAR (positive predictive value: 47%-61%). The clinical phenotype and biomarker independently and statistically correlated with a composite clinical endpoint (renal function, biopsy-proved acute rejection, ≥grade 2 interstitial fibrosis, and tubular atrophy), as well as with de novo donor-specific antibodies. We also found that <50% showed histologic improvement of subAR on follow-up biopsies despite treatment and that the biomarker could predict this outcome. Our data suggest that a blood-based biomarker that reduces the need for the indiscriminate use of invasive surveillance biopsies and that correlates with transplant outcomes could be used to monitor KT recipients with stable renal function, including after treatment for subAR, potentially improving KT outcomes.
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http://dx.doi.org/10.1111/ajt.15011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6387870PMC
January 2019

Transcriptomic studies in tolerance: Lessons learned and the path forward.

Hum Immunol 2018 May 2;79(5):395-401. Epub 2018 Mar 2.

Department of Surgery, Comprehensive Transplant Center, Northwestern University, Chicago, IL, USA.

Immunosuppression after solid organ transplantation is a delicate balance of the immune response and is a complex phenomenon with many factors involved. Despite advances in the care of patients receiving organ transplants the adverse effects associated with immunosuppressive agents and the risks of long-term immunosuppression present a series of challenges and the need to weigh the risks and benefits of either over or under-immunosuppression. Ideally, if all transplant recipients could develop donor-specific immunological tolerance, it could drastically improve long-term graft survival without the need for immunosuppressive agents. In the absence of this ideal situation, the next best approach would be to develop tools to determine the adequacy of immunosuppression in each patient, in a manner that would individualize or personalize therapy. Despite current genomics-based studies of tolerance biomarkers in transplantation there are currently, no clinically validated tools to safely increase or decrease the level of IS that is beneficial to the patient. However, the successful identification of biomarkers and/or mechanisms of tolerance that have implications on long-term graft survival and outcomes depend on proper integration of study design, experimental protocols, and data-driven hypotheses. The objective of this article is to first, discuss the progress made on genomic biomarkers of immunological tolerance and the future avenues for the development of such biomarkers specifically in kidney transplantation. Secondly, we provide a set of guiding principles and identify the pitfalls, advantages, and drawbacks of studies that generate genomic data aimed at understanding transplant tolerance that is applicable to all solid transplants.
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http://dx.doi.org/10.1016/j.humimm.2018.02.011DOI Listing
May 2018

Gene expression profiling of U2AF2 dependent RNA-protein interactions during CD4 + T cell activation.

Genom Data 2017 Mar 14;11:77-80. Epub 2016 Dec 14.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, United States.

CD4 T cell activation is a central component of the mammalian adaptive immune response and is underscored by a dramatic change in the gene expression profile in these cells. The changes in gene expression that occur during T cell activation are regulated in multiple ways including post-transcriptionally by complexes of RNA-binding proteins. Recently, our study explored the role of the RNA-binding protein U2AF2 and its interacting proteins in mediating posttranscriptional changes in constitutive and alternative splicing during T cell activation. First, we used RNA-seq to identify the global changes in gene expression and splicing that occur with T cell activation. Next, we used RIP-seq to identify the specific genes bound to U2AF2 during T cell activation. After identification of the protein interacting partners of U2AF2, we used splicing sensitive microarrays to measure the effects on global gene expression of using siRNAs to knock down a sampling of these proteins. Finally, we used RIP-chip to measure the effects of the same siRNA knockdown on the transcripts specifically bound to U2AF2. Here we provide the experimental details and analysis of the gene expression data for each of these techniques, which have been deposited into Gene Expression Omnibus (GEO) with the Superseries ID: GSE62923.
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http://dx.doi.org/10.1016/j.gdata.2016.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192059PMC
March 2017

Genome-wide expression profiling in the peripheral blood of patients with fibromyalgia.

Clin Exp Rheumatol 2016 Mar-Apr;34(2 Suppl 96):S89-98. Epub 2016 Feb 12.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.

Objectives: Fibromyalgia (FM) is a common pain disorder characterized by nociceptive dysregulation. The basic biology of FM is poorly understood. Herein we have used agnostic gene expression as a potential probe for informing its underlying biology and the development of a proof-of-concept diagnostic gene expression signature.

Methods: We analyzed RNA expression in 70 FM patients and 70 healthy controls. The isolated RNA was amplified and hybridized to Affymetrix® Human Gene 1.1 ST Peg arrays. The data was analyzed using Partek Genomics Suite version 6.6.

Results: Fibromyalgia patients exhibited a differential expression of 421 genes (p<0.001), several relevant to pathways for pain processing, such as glutamine/glutamate signaling and axonal development. There was also an upregulation of several inflammatory pathways and downregulation of pathways related to hypersensitivity and allergy. Using rigorous diagnostic modeling strategies, we show "locked" gene signatures discovered on Training and Test cohorts, that have a mean Area Under the Curve (AUC) of 0.81 on randomized, independent external data cohorts. Lastly, we identified a subset of 10 probesets that provided a diagnostic sensitivity for FM of 95% and a specificity of 96%. We also show that the signatures for FM were very specific to FM rather than common FM comorbidities.

Conclusions: These findings provide new insights relevant to the pathogenesis of FM, and provide several testable hypotheses that warrant further exploration and also establish the foundation for a first blood-based molecular signature in FM that needs to be validated in larger cohorts of patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888802PMC
July 2016

The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

PLoS One 2015 7;10(12):e0144409. Epub 2015 Dec 7.

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs) has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs). Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs), that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation-induced assembly of RNA splicing interactomes that is important for understanding T cell activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144409PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671683PMC
June 2016

Combining docking site and phosphosite predictions to find new substrates: identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate.

Cell Signal 2013 Dec 24;25(12):2518-29. Epub 2013 Aug 24.

Department of Developmental and Cell Biology, University of California Irvine, United States; Center for Complex Biological Systems, University of California Irvine, United States.

Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180-193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells.
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http://dx.doi.org/10.1016/j.cellsig.2013.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132694PMC
December 2013

Computational prediction and experimental verification of new MAP kinase docking sites and substrates including Gli transcription factors.

PLoS Comput Biol 2010 Aug 26;6(8). Epub 2010 Aug 26.

Department of Developmental and Cell Biology, University of California, Irvine, California, United States of America.

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new 'D-site' class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.
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http://dx.doi.org/10.1371/journal.pcbi.1000908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928751PMC
August 2010

Kidney transplant rejection and tissue injury by gene profiling of biopsies and peripheral blood lymphocytes.

Am J Transplant 2004 Sep;4(9):1475-89

Section of Renal Transplantation, Transplant Center A110, Cleveland Clinic Foundation, Cleveland, OH, USA.

A major challenge for kidney transplantation is balancing the need for immunosuppression to prevent rejection, while minimizing drug-induced toxicities. We used DNA microarrays (HG-U95Av2 GeneChips, Affymetrix) to determine gene expression profiles for kidney biopsies and peripheral blood lymphocytes (PBLs) in transplant patients including normal donor kidneys, well-functioning transplants without rejection, kidneys undergoing acute rejection, and transplants with renal dysfunction without rejection. We developed a data analysis schema based on expression signal determination, class comparison and prediction, hierarchical clustering, statistical power analysis and real-time quantitative PCR validation. We identified distinct gene expression signatures for both biopsies and PBLs that correlated significantly with each of the different classes of transplant patients. This is the most complete report to date using commercial arrays to identify unique expression signatures in transplant biopsies distinguishing acute rejection, acute dysfunction without rejection and well-functioning transplants with no rejection history. We demonstrate for the first time the successful application of high density DNA chip analysis of PBL as a diagnostic tool for transplantation. The significance of these results, if validated in a multicenter prospective trial, would be the establishment of a metric based on gene expression signatures for monitoring the immune status and immunosuppression of transplanted patients.
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http://dx.doi.org/10.1111/j.1600-6143.2004.00526.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2041877PMC
September 2004
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