Head and neck squamous cell carcinoma (HNSCC) is a prevalent and often fatal malignancy associated with significant treatment-related toxicity. There is an urgent need for a preclinical model to assess therapeutic options and guide clinical decision-making. To define conditions for establishing patient-derived organoid (PDO) models that faithfully recapitulate morphological, histopathological, and genomic characteristics of HNSCC patients and can predict radiation and chemotherapy responses in patients, PDOs were generated from a group of HNSCC patients. The morphological, histological, mutational, and biological characteristics and treatment responses were evaluated. We demonstrate that the PDOs closely resemble resected tumors from which they were derived with respect to histopathology, differentiation state markers, p16 status, and mutation profiling. We observe patient-to-patient variation in cell proliferation rates. Additionally, they exhibit differential responses to radiotherapy and chemotherapy, which were examined using a cell viability assay. This methodology offers potential for drug screening in a pre-clinical context with the potential to mirror clinical outcomes. Our WNT-free growth conditions maintained the differentiation status of PDOs and enabled rapid assessment of drug response and the development of new models to identify new treatment options for head and neck cancer patients.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)-induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow-sense heritability was estimated at 0.22-0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7-12% of the variance in OXY locomotion and anxiety-like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near Gabra2 (alpha-2 subunit of GABA-A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163-181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). Transcriptome analysis identified five, localized striatal cis-eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9 codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder.

In previous work, we used a SomaLogic platform targeting approximately 5000 proteins to generate a serum protein signature of centenarians that we validated in independent studies that used the same technology. We set here to validate and possibly expand the results by profiling the serum proteome of a subset of individuals included in the original study using liquid chromatography tandem mass spectrometry (LC-MS/MS). Following pre-processing, the LC-MS/MS data provided quantification of 398 proteins, with only 266 proteins shared by both platforms. At 1% FDR statistical significance threshold, the analysis of LC-MS/MS data detected 44 proteins associated with extreme old age, including 23 of the original analysis. To identify proteins for which associations between expression and extreme-old age were conserved across platforms, we performed inter-study conservation testing of the 266 proteins quantified by both platforms using a method that accounts for the correlation between the results. From these tests, a total of 80 proteins reached 5% FDR statistical significance, and 26 of these proteins had concordant pattern of gene expression in whole blood generated in an independent set. This signature of 80 proteins points to blood coagulation, IGF signaling, extracellular matrix (ECM) organization, and complement cascade as important pathways whose protein level changes provide evidence for age-related adjustments that distinguish centenarians from younger individuals. The comparison with blood transcriptomics also highlights a possible role for neutrophil degranulation in aging.

SUMMARYLocal immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in variousin vitroandin vivomodels of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immunefingerprintfor each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues.Ex vivoanalyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines theirin situlocalization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.HIGHLIGHTSAging tissues are associated with tissue-autonomousimmunefingerprints, primarily driven by interactions of tissue stromal and myeloid populations.STING shapes these immune fingerprints of aging tissues in unexpected ways.Loss of STING alters the location, numbers, and viability of tissue resident macrophages.STING signaling is critical for longer lifespans and maintenance of tissue architecture.

False discovery is an ever-present concern in omics research, especially for burgeoning technologies with unvetted specificity of their biomolecular measurements, as such unknowns obscure the ability to characterize biologically informative features from studies performed with any single platform. Accordingly, performing replication studies of the same samples using different omics platforms is a viable strategy for identifying high-confidence molecular associations that are conserved across studies. However, an important caveat of replication studies that include the same samples is that they are inherently non-independent, leading to overestimating conservation if studies are treated otherwise. Strategies for accounting for such inter-study dependencies have been proposed for meta-analysis methods devised to increase statistical power to detect molecular associations in one or more studies. Still, they are not immediately suited for identifying conserved molecular associations across multiple studies. Here, we present a unifying strategy for performing inter-study conservation analysis as an alternative to meta-analysis strategies for aggregating summary statistical results of shared features across complementary studies while accounting for inter-study dependency. This method, which we call "adjusted maximum p-value" (AdjMaxP), is easy to implement with inter-study dependency and conservation estimated directly from the p-values from each study's molecular feature-level association testing results. Through simulation-based assessment, we demonstrate AdjMaxP's improved performance for accurately identifying conserved features over a related meta-analysis strategy for non-independent studies. AdjMaxP offers an easily implementable strategy for improving the precision of analyses for biomarker discovery from cross-platform omics study designs, thereby facilitating the adoption of such protocols for robust inference from emerging omics technologies.

Abstract Oral squamous cell carcinoma (OSCC), a primary subtype of head and neck squamous cell carcinoma (HNSCC), is a complex malignancy comprising multiple anatomical sites. OSCC ranks among the deadliest cancers globally, with a 5-year survival rate of ~65%. In previous studies, we have shown that pharmacological blockade of Wnt/β-catenin/CBP activity with small molecule inhibitors effectively abolished oncogenic cell phenotypes in OSCC. However, the underlying mechanisms promoting changes in OSCC cell identities remain unknown. To address this knowledge gap, we used an immunocompetent mouse model of OSCC induced by a tobacco-derived carcinogen, 4-nitroquinoline-1-oxide (4NQO), to interrogate cell states by single-cell RNA sequencing (scRNAseq) of tongue tissues from healthy mice (n=2), 4NQO-derived mouse tongue OSCC (n=2), and from 4NQO-derived mouse tongue tissues treated with an inhibitor of β-catenin/CBP (n=4), and generated a high-quality dataset comprising ~50K cells across all conditions. We performed multiple analyses of the generated data to catalogue the cell type repertoire and its changes among conditions. We observed significant changes in cellular composition between the 4NQO-induced and inhibitor-treated groups. The proportion of epithelial cells decreased upon treatment consistent with greatly diminished tumor volumes, while endothelial and fibroblast populations increased compared to the 4NQO control group. Epithelial sub-typing using known markers revealed a decrease in basal cancer stem-like cells (Krt5+, Krt14+) concomitant with an increase in cycling cells (Top2a+, Cdc20+). In addition, we identified a decrease in a stress cell phenotype associated with the AP-1 complex (Jun+, Fos+). The latter subgroup exhibited a positive enrichment for the “stress” module (n=100 genes) derived from human HNSCC patients described by Puram et al. To further validate the relationship between the stress subtype with β-catenin/CBP activity and its relation to the AP-1 complex, we projected the Puram stress module and the down-regulated signature from the β-catenin/CBP inhibitor-treated group compared with the 4NQO-control group onto independent cohorts of human subjects with HPV-negative HNSCC. We used both the bulk TCGA RNA-seq subset (n=367 patients) and a previously published scRNAseq dataset from Choi et al., specifically profiling the epithelial compartment (3K cells, n = 16 patients). We observed highly significant positive association of the stress program and β-catenin/CBP activity in both datasets (pearson ρ = 0.74 and 0.70, respectively), indicating that inhibition of β-catenin/CBP activity reduces the stress response. Our results suggest that mitigation of tumorigenic profiles in 4NQO-induced tumors upon inhibition of β-catenin/CBP signaling in OSCC may serve as an effective treatment strategy for the benefit of human patients and shed further light into the molecular mechanisms driving treatment response. Citation Format: Mohammed Muzamil Khan, Eric Reed, Lina Kroehling, Kenichi Nomoto, Junji Matsui, Manish Bais, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Reducing the effect of cellular stress in murine oral tumors with pharmacological blockade of β-catenin/CBP activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB288.

In previous studies, we have shown that pharmacological blockade of Wnt/β-catenin/CBP activity with small molecule inhibitors is effective in abolishing oncogenic phenotypes in oral squamous cell carcinoma (OSCC). To further characterize changes in Wnt/β-catenin activity during malignant transformation, we used an immunocompetent mouse model of oral cancer induced by a tobacco-derived carcinogen, 4-nitroquinoline-1-oxide (4NQO), which recapitulates the human OSCC mutational landscape and tumor immune environment. We performed single-cell RNA sequencing (scRNAseq) on tongue tissues from healthy mice (n=2), 4NQO-derived mouse OSCC (n=2), and from 4NQO-derived mouse OSCC treated with a pharmacologic grade 𝛽-catenin/CBP inhibitor (n=2). The experiment yielded ~50K cells across all conditions and identified multiple cell types and states, including epithelial and immune cells, as well as endothelia, fibroblasts, and glial cells. We found significant cellular composition changes between 4NQO and 4NQO inhibitor-treated groups, with the proportion of epithelial cells decreasing upon treatment with the 𝛽-catenin/CBP inhibitor, and the endothelial and fibroblast populations increasing in the 𝛽-catenin/CBP inhibitor group. Analysis within the immune compartment showed significant cell type proportion changes between the 4NQO and 4NQO inhibitor-treated groups, including changes in neutrophils population decreasing upon treatment to inhibitor, macrophages, and DCs increasing in the treatment group, along with T- and B-cells. ~50% of the cellular composition in each group comprised of neutrophil population. Further subtyping of the neutrophil population into early and late “neutrotime” subclasses based on published signatures showed enrichment of the early neutrotime class in the inhibitor-treated group and of the late neutrotime class in the 4NQO group. Analysis within the epithelial compartment identified several cell type proportions, including basal (Krt5+, Krt15+), proliferating (Krt5+, Krt14+), and cycling (Top2a, Cdc20) cells increased upon treatment with the inhibitor, and acinar cell-types (Muc5b, Aqp5, Smgc) decreasing upon treatment in comparison with the 4NQO group. The inhibitor-treated group also showed lower proportions of malignant transforming cell states compared to the 4NQO group. A cell-cell communication analysis was performed between epithelial and immune compartments to identify ligand-receptor (LR) interactions indicative of treatment response. Among the significant interactions, the LRs related to collagen organization (Cdh1, Icam1, Lamc1) and major histocompatibility complex genes such as H2-d1, H2-k1, H2-q4, etc., were enriched in the treatment group indicating cellular plasticity and immune regulation processes in comparison with the 4NQO group. Taken together, our results indicate mitigation of cellular heterogeneity and plasticity profiles in 4NQO-induced tumors upon treatment with 𝛽-catenin/CBP inhibitor in OSCC. Citation Format: Mohammed Muzamil Khan, Eric Reed, Lina Kroehling, Manish Bais, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Targeting murine oral tumors by pharmacological inhibition of b-catenin/CBP epigenetic activity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-017.

Age-related changes in immune cell composition and functionality are associated with multimorbidity and mortality. However, many centenarians delay the onset of aging-related disease suggesting the presence of elite immunity that remains highly functional at extreme old age. To identify immune-specific patterns of aging and extreme human longevity, we analyzed novel single cell profiles from the peripheral blood mononuclear cells (PBMCs) of 7 centenarians (mean age 106) and publicly available single cell RNA-sequencing (scRNA-seq) datasets that included an additional 7 centenarians as well as 52 people at younger ages (20-89 years). The analysis confirmed known shifts in the ratio of lymphocytes to myeloid cells, and noncytotoxic to cytotoxic cell distributions with aging, but also identified significant shifts from CD4 + T cell to B cell populations in centenarians suggesting a history of exposure to natural and environmental immunogens. Our transcriptional analysis identified cell type signatures specific to exceptional longevity that included genes with age-related changes (e.g., increased expression of STK17A , a gene known to be involved in DNA damage response ) as well as genes expressed uniquely in centenarians’ PBMCs (e.g., S100A4 , part of the S100 protein family studied in age-related disease and connected to longevity and metabolic regulation ) . Collectively, these data suggest that centenarians harbor unique, highly functional immune systems that have successfully adapted to a history of insults allowing for the achievement of exceptional longevity.

Head and neck cancers, which include oral squamous cell carcinoma (OSCC) as a major subsite, exhibit cellular plasticity that includes features of an epithelial-mesenchymal transition (EMT), referred to as partial-EMT (p-EMT). To identify molecular mechanisms contributing to OSCC plasticity, we performed a multiphase analysis of single cell RNA sequencing (scRNAseq) data from human OSCC. This included a multiresolution characterization of cancer cell subgroups to identify pathways and cell states that are heterogeneously represented, followed by casual inference analysis to elucidate activating and inhibitory relationships between these pathways and cell states. This approach revealed signaling networks associated with hierarchical cell state transitions, which notably included an association between β-catenin-driven CREB-binding protein (CBP) activity and mTORC1 signaling. This network was associated with subpopulations of cancer cells that were enriched for markers of the p-EMT state and poor patient survival. Functional analyses revealed that β-catenin/CBP induced mTORC1 activity in part through the transcriptional regulation of a raptor-interacting protein, chaperonin containing TCP1 subunit 5 (CCT5). Inhibition of β-catenin-CBP activity through the use of the orally active small molecule, E7386, reduced the expression of CCT5 and mTORC1 activity in vitro, and inhibited p-EMT-associated markers and tumor development in a murine model of OSCC. Our study highlights the use of multiresolution network analyses of scRNAseq data to identify targetable signals for therapeutic benefit, thus defining an underappreciated association between β-catenin/CBP and mTORC1 signaling in head and neck cancer plasticity.

The ever-increasing availability of publicly available cancer omics datasets makes it possible to perform in-silico studies aimed at elucidating disease mechanisms of action (MOAs) that may aid in therapeutic development. To this end, we recently developed Structure Learning for Hierarchical Networks (SHiNe) to model and distinguish direct and indirect signaling interactions from gene expression data to identify key signaling “hubs” and “bottlenecks”, associated crosstalk, and downstream targets that we used to identify potential effectors of head and neck squamous carcinoma (HNSC). SHiNe is an advanced gene regulatory network reconstruction approach optimized for learning multiple Markov networks in the “large p, small n” settings (i.e., large number of genes, small number of samples) typical of omics data. We applied SHiNe to the analysis of TCGA-HNSC RNA-seq data to learn HPV-negative and subtype- specific networks for previously validated molecular subtypes (Atypical, Basal, Classical, and Mesenchymal). SHiNe learned networks were sparser, with higher clustering coefficients, and more significantly enriched for protein-protein interactions than randomly simulated networks. Further annotation of the learned networks with multiple layers of omics information revealed several highly eigen-central genes in the HPV-negative network to be characterized by additional omics features. Examples included EP300, ranked 2nd by eigen-centrality with a single somatic mutation (SSM) rate of 7.87% (versus 4.59% in all other TCGA tumors), MYH9 (rank=12, SSM=6.1%), RPS6KA4 (rank=39, CNV Gain=16.5%), GNAI3 (rank=45, CNV Loss=10.55%), and CFL1 (rank=51, CNV Gain=20.72%). Community detection in the HPV(-) network structure using the Walktrap algorithm yielded 33 signaling communities, with the highest centrality community encompassing most of the central genes within the network (e.g., KLHL11, EP300, TAOK1, ADAM17, APAF1) and including genes strongly enriched for DNA repair and maintenance pathways. Co-localization analysis of omics features in the HPV-negative network identified within the most central community revealed a cluster of genes with copy number variation gain ≥ 25%, including ABCC5, ACTL6A, AP2M1, ATP11B, MAP3K13, NCBP2, PAK2, PIK3CA, PLD1, PRKCI, SKIL, TBL1XR1, and ZMAT3, with most of these genes located on the q arm of chromosome 3. While amplification of 3q26-29 is a known characteristic of HNSCC associated with poorer patient outcome, the MOA remains unclear, and thus our network analysis offers an opportunity to identify meaningful signaling interactions for therapeutic intervention of 3q26-29 mutated tumors. Indeed, we have identified candidate associations with the 3q26-29 amplicon genes that we are currently validating. We are also developing a graphical interface for the interactive inspection of the inferred networks. Taken together, these results highlight the potential for network-based analysis to support the study and identification of novel candidate regulators of head and neck tumorigenesis. Citation Format: Anthony Federico, Eric Reed, Maria Kukuruzinska, Xaralabos Varelas, Stefano Monti. Gene regulatory network connectivity analysis identifies novel candidate effectors of HNSC tumorigenesis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-020.

As high-throughput genomics assays become more efficient and cost effective, their utilization has become standard in large-scale biomedical projects. These studies are often explorative, in that relationships between samples are not explicitly defined a priori, but rather emerge from data-driven discovery and annotation of molecular subtypes, thereby informing hypotheses and independent evaluation. Here, we present K2Taxonomer, a novel unsupervised recursive partitioning algorithm and associated R package that utilize ensemble learning to identify robust subgroups in a 'taxonomy-like' structure. K2Taxonomer was devised to accommodate different data paradigms, and is suitable for the analysis of both bulk and single-cell transcriptomics, and other '-omics', data. For each of these data types, we demonstrate the power of K2Taxonomer to discover known relationships in both simulated and human tissue data. We conclude with a practical application on breast cancer tumor infiltrating lymphocyte (TIL) single-cell profiles, in which we identified co-expression of translational machinery genes as a dominant transcriptional program shared by T cells subtypes, associated with better prognosis in breast cancer tissue bulk expression data.

ABSTRACTPsychostimulant (methamphetamine, cocaine) use disorders have a genetic component that remains mostly unknown. Here, we conducted genome-wide quantitative trait locus (QTL) analysis of methamphetamine stimulant sensitivity. To facilitate gene identification, we employed a Reduced Complexity Cross between closely related C57BL/6 mouse substrains and examined maximum speed and distance traveled over 30 min following methamphetamine (2 mg/kg, i.p.). For maximum methamphetamine-induced speed following the second and third administration, we identified a single genome-wide significant QTL on chromosome 11 that peaked near theCyfip2locus [LOD = 3.5, 4.2; peak = 21 cM (36 Mb)]. For methamphetamine-induced distance traveled, we identified a single genome-wide significant QTL on chromosome 5 that peaked near a functional intronic indel inGabra2that codes for the alpha-2 subunit of the GABA-A receptor [LOD = 5.2; peak = 35 cM (67 Mb)]. Striatalcis-expression QTL mapping corroboratedGabra2as a functional candidate gene underlying methamphetamine-induced distance traveled. CRISPR/Cas9-mediated correction of the mutant intronic deletion on the C57BL/6J background to the wild-type C57BL/6NJ allele was sufficient to reduce methamphetamine-induced locomotor activity toward the wild-type C57BL/6NJ-like level, thus validating the quantitative trait variant (QTV). These studies demonstrate the power and efficiency of Reduced Complexity Crosses in identifying causal genes and variants underlying complex traits. Functionally restoringGabra2expression decreased methamphetamine stimulant sensitivity and supports preclinical and human genetic studies implicating the GABA-A receptor in psychostimulant addiction-relevant traits. Importantly, our findings have major implications for investigators studying psychostimulants in the C57BL/6J strain - the gold standard strain in biomedical research.

Background Chemicals in disparate structural classes activate specific subsets of PPARγ’s transcriptional programs to generate adipocytes with distinct phenotypes. Objectives Our objectives were to 1) establish a novel classification method to predict PPARγ ligands and modifying chemicals, and 2) create a taxonomy to group chemicals based on their effects on PPARγ’s transcriptome and downstream metabolic functions. We tested the hypothesis that environmental adipogens highly ranked by the taxonomy, but segregated from therapeutic PPARγ ligands, would induce white but not brite adipogenesis. Methods 3T3-L1 cells were differentiated in the presence of 76 chemicals (negative controls, nuclear receptor ligands known to influence adipocyte biology, potential environmental PPARγ ligands). Differentiation was assessed by measuring lipid accumulation. mRNA expression was determined by RNA-Seq and validated by RT-qPCR. A novel classification model was developed using an amended random forest procedure. A subset of environmental contaminants identified as strong PPARγ agonists were analyzed by their effects on lipid handling, mitochondrial biogenesis and cellular respiration in 3T3-L1 cells and human preadipocytes. Results We used lipid accumulation and RNA sequencing data to develop a classification system that 1) identified PPARγ agonists, and 2) sorted chemicals into likely white or brite adipogens. Expression of Cidec was the most efficacious indicator of strong PPARγ activation. Two known environmental PPARγ ligands, tetrabromobisphenol A and triphenyl phosphate, which sorted distinctly from therapeutic ligands, induced white adipocyte genes but failed to induce Pgc1a and Ucp1 , and induced fatty acid uptake but not mitochondrial biogenesis in 3T3-L1 cells. Moreover, two chemicals identified as highly ranked PPARγ agonists, tonalide and quinoxyfen, induced white adipogenesis without the concomitant health-promoting characteristics of brite adipocytes in mouse and human preadipocytes. Discussion A novel classification procedure accurately identified environmental chemicals as PPARγ ligands distinct from known PPARγ-activating therapeutics. The computational and experimental framework has general applicability to the classification of as-yet uncharacterized chemicals.

ABSTRACT We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes ( Hnrnph1, Rufy1 ) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a small deletion in the first coding exon supported Hnrnph1 as a quantitative trait gene. We have since shown that Hnrnph1 mutants also exhibit reduced methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants ( QTVs ) that modulate Hnrnph1 function at the molecular level are not known. Nine single nucleotide polymorphisms and seven indels distinguish C57BL/6J from DBA/2J within Hnrnph1 , including four variants within the 5’ untranslated region (UTR) . Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to cause a decrease in MA-induced locomotor activity. Gene-level transcriptome analysis of striatal tissue from 114 kb congenics versus Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression for those differentially expressed genes that were common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Exon-level analysis (including noncoding exons) revealed decreased 5’ UTR usage of Hnrnph1 and immunoblot analysis identified a corresponding decrease in hnRNP H protein in 114 kb congenic mice. Molecular cloning of the Hnrnph1 5’ UTR containing all four variants (but none of them individually) upstream of a reporter induced a decrease in reporter signal in both HEK293 and N2a cells, thus identifying a set of QTVs underlying molecular regulation of Hnrnph1 .

We previously identified a 210 kb region on chromosome 11 (50.37-50.58 Mb, mm10) containing two protein-coding genes (Hnrnph1, Rufy1) that was necessary for reduced methamphetamine-induced locomotor activity in C57BL/6J congenic mice harboring DBA/2J polymorphisms. Gene editing of a deletion in the first coding exon of each gene supported Hnrnph1 as a quantitative trait gene. We since showed that Hnrnph1 mutants also exhibit a reduction in methamphetamine-induced reward, reinforcement, and dopamine release. However, the quantitative trait variants (QTVs) that modulate Hnrnph1 at the molecular level are not known. There are nine SNPs and seven indels that distinguish C57BL/6J from DBA/2J within Hnrnph1, including four variants within the 5UTR. Here, we show that a 114 kb introgressed region containing Hnrnph1 and Rufy1 was sufficient to induce a decrease in MA-induced locomotor activity. Transcriptome analysis of 114 kb congenics versus Hnrnph1 mutants identified a nearly perfect correlation of fold-change in expression in differentially expressed genes common to both mouse lines, indicating functionally similar effects on the transcriptome and behavior. Ten overlapping genes showed differential exon usage, including Hnrnph1 and Ppp3ca. Differential 5UTR exon usage of Hnrnph1 and Ppp3ca were validated using real-time quantitative PCR. Cloning of the Hnrnph1 5UTR containing all four variants together (but none of them individually) induced a functional decrease in reporter expression in both HEK293 and N2a cells, thus identifying a set of QTVs underlying molecular regulation of Hnrnph1.

Abstract Single-cell RNA-seq (scRNA-seq) is an emerging platform for high-throughput profiling of individual cells in a sample and is routinely employed to investigate the transcriptional landscapes of the cellular constituents of tumors. To this end, many scRNA-seq specific clustering algorithms have emerged to analytically partition cells into modules of comparatively similar profiles. To facilitate data driven molecular subtyping of such scRNA-seq clustering results and other large-scale-omics studies, we have developed K2 Taxonomer. K2 Taxonomer is an R package built around a novel top-down hierarchical clustering algorithm, utilizing repeated perturbations of the data to generate robust taxonomical partitions of observations. The software runs additional analyses to define gene co-expression signatures of these modules, as well as to integrate user-input annotations of genes and/or observations. An interactive web portal has been generated to assist in the interrogation of the full compendium of results. We applied K2 Taxonomer to publicly available HNSCC scRNA-seq data, identifying pertinent tumor cell subtypes, distinguished by cell cycle and epithelial-to-mesenchymal transition, and with analytical projection of this signature onto TCGA-HNSCC bulk RNA-seq data exhibiting association with worse survival in TCGA-HNSCC patients. A transcriptional signature corresponding to suppression of Mtorc1 and Wnt/β-catenin signaling was also identified in a sub-population of HNSCCs, and shown to be associated with improved patient survival. Of notice, highly ranked markers of this signature are significantly associated with gene expression changes altered by E7386 - a novel β-catenin/CBP modulator with an activity profile that closely overlaps with that of ICG-001, but exhibits ~50-100-fold lower EC50 values - suggesting a role for this signaling axis in subsets of HNSCC. In conclusion, taxonomical subtyping with K2 Taxonomer provides a novel framework to expand the scope of applicability of scRNA-seq clustering results, and has revealed potentially novel HNSCC subtypes that offer directions for future studies. Citation Format: Eric Reed, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Subtyping of HNSCC single-cell RNA-seq identifies transcriptional programs characterized by suppression of Mtorc1 and Wnt signaling pathways and better patient prognosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4419.

The need to reduce per sample cost of RNA-seq profiling for scalable data generation has led to the emergence of highly multiplexed RNA-seq. These technologies utilize barcoding of cDNA sequences in order to combine multiple samples into a single sequencing lane to be separated during data processing. In this study, we report the performance of one such technique denoted as sparse full length sequencing (SFL), a ribosomal RNA depletion-based RNA sequencing approach that allows for the simultaneous sequencing of 96 samples and higher. We offer comparisons to well established single-sample techniques, including: full coverage Poly-A capture RNA-seq, microarrays, as well as another low-cost highly multiplexed technique known as 3' digital gene expression (3'DGE). Data was generated for a set of exposure experiments on immortalized human lung epithelial (AALE) cells in a two-by-two study design, in which samples received both genetic and chemical perturbations of known oncogenes/tumor suppressors and lung carcinogens. SFL demonstrated improved performance over 3'DGE in terms of coverage, power to detect differential gene expression, and biological recapitulation of patterns of differential gene expression from in vivo lung cancer mutation signatures.