Transcriptomic-Based Microenvironment Classification Reveals Precision Medicine Strategies for Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The complex tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) has hindered the development of reliable predictive biomarkers for targeted therapy and immunomodulatory strategies. A comprehensive characterization of the TME is necessary to advance precision therapeutics in PDAC. METHODS: A transcriptomic profiling platform for TME classification based on functional gene signatures was applied to 14 publicly available PDAC datasets (n = 1657) and validated in a clinically annotated independent cohort of patients with PDAC (n = 79). Four distinct subtypes were identified using unsupervised clustering and assessed to evaluate predictive and prognostic utility. RESULTS: TME classification using transcriptomic profiling identified 4 biologically distinct subtypes based on their TME immune composition: immune enriched (IE); immune enriched, fibrotic (IE/F); fibrotic (F); and immune depleted (D). The IE and IE/F subtypes demonstrated a more favorable prognosis and potential for response to immunotherapy compared with the F and D subtypes. Most lung metastases and liver metastases were subtypes IE and D, respectively, indicating the role of clonal phenotype and immune milieu in developing personalized therapeutic strategies. In addition, distinct TMEs with potential therapeutic implications were identified in treatment-naive primary tumors compared with tumors that underwent neoadjuvant therapy. CONCLUSIONS: This novel approach defines a distinct subgroup of PADC patients that may benefit from immunotherapeutic strategies based on their TME subtype and provides a framework to select patients for prospective clinical trials investigating precision immunotherapy in PDAC. Further, the predictive utility and real-world clinical applicability espoused by this transcriptomic-based TME classification approach will accelerate the advancement of precision medicine in PDAC.

Spatial mapping of human hematopoiesis at single-cell resolution reveals aging-associated topographic remodeling.

ABSTRACT: The spatial anatomy of hematopoiesis in the bone marrow (BM) has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized BM core biopsy tissue specimens, providing a rich resource for studying the intact human BM topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence-based digital whole slide image analysis and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of foci with increased lymphocytes, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human BM states using intact archival tissue specimens.

GATA4 Regulates Developing Endocardium Through Interaction With ETS1.

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.

Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling.

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.

Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo.

RATIONALE: Loss-of-function studies in cardiac myocytes (CMs) are currently limited by the need for appropriate conditional knockout alleles. The factors that regulate CM maturation are poorly understood. Previous studies on CM maturation have been confounded by heart dysfunction caused by whole organ gene inactivation. OBJECTIVE: To develop a new technical platform to rapidly characterize cell-autonomous gene function in postnatal murine CMs and apply it to identify genes that regulate transverse tubules (T-tubules), a hallmark of mature CMs. METHODS AND RESULTS: We developed CRISPR/Cas9/AAV9-based somatic mutagenesis, a platform in which AAV9 delivers tandem guide RNAs targeting a gene of interest and cardiac troponin-T promoter-driven Cre to RosaCas9GFP/Cas9GFP neonatal mice. When directed against junctophilin-2 (Jph2), a gene previously implicated in T-tubule maturation, we achieved efficient, rapid, and CM-specific JPH2 depletion. High-dose AAV9 ablated JPH2 in 64% CMs and caused lethal heart failure, whereas low-dose AAV9 ablated JPH2 in 22% CMs and preserved normal heart function. In the context of preserved heart function, CMs lacking JPH2 developed T-tubules that were nearly morphologically normal, indicating that JPH2 does not have a major, cell-autonomous role in T-tubule maturation. However, in hearts with severe dysfunction, both adeno-associated virus-transduced and nontransduced CMs exhibited T-tubule disruption, which was more severe in the transduced subset. These data indicate that cardiac dysfunction disrupts T-tubule structure and that JPH2 protects T-tubules in this context. We then used CRISPR/Cas9/AAV9-based somatic mutagenesis to screen 8 additional genes for required, cell-autonomous roles in T-tubule formation. We identified RYR2 (Ryanodine Receptor-2) as a novel, cell-autonomously required T-tubule maturation factor. CONCLUSIONS: CRISPR/Cas9/AAV9-based somatic mutagenesis is a powerful tool to study cell-autonomous gene functions. Genetic mosaics are invaluable to accurately define cell-autonomous gene function. JPH2 has a minor role in normal T-tubule maturation but is required to stabilize T-tubules in the failing heart. RYR2 is a novel T-tubule maturation factor.

Evolutionary re-wiring of p63 and the epigenomic regulatory landscape in keratinocytes and its potential implications on species-specific gene expression and phenotypes.

Although epidermal keratinocyte development and differentiation proceeds in similar fashion between humans and mice, evolutionary pressures have also wrought significant species-specific physiological differences. These differences between species could arise in part, by the rewiring of regulatory network due to changes in the global targets of lineage-specific transcriptional master regulators such as p63. Here we have performed a systematic and comparative analysis of the p63 target gene network within the integrated framework of the transcriptomic and epigenomic landscape of mouse and human keratinocytes. We determined that there exists a core set of ∼1600 genomic regions distributed among enhancers and super-enhancers, which are conserved and occupied by p63 in keratinocytes from both species. Notably, these DNA segments are typified by consensus p63 binding motifs under purifying selection and are associated with genes involved in key keratinocyte and skin-centric biological processes. However, the majority of the p63-bound mouse target regions consist of either murine-specific DNA elements that are not alignable to the human genome or exhibit no p63 binding in the orthologous syntenic regions, typifying an occupancy lost subset. Our results suggest that these evolutionarily divergent regions have undergone significant turnover of p63 binding sites and are associated with an underlying inactive and inaccessible chromatin state, indicative of their selective functional activity in the transcriptional regulatory network in mouse but not human. Furthermore, we demonstrate that this selective targeting of genes by p63 correlates with subtle, but measurable transcriptional differences in mouse and human keratinocytes that converges on major metabolic processes, which often exhibit species-specific trends. Collectively our study offers possible molecular explanation for the observable phenotypic differences between the mouse and human skin and broadly informs on the prevailing principles that govern the tug-of-war between evolutionary forces of rigidity and plasticity over transcriptional regulatory programs.

A global analysis of the complex landscape of isoforms and regulatory networks of p63 in human cells and tissues.

BACKGROUND: The transcription factor p63 belongs to the p53/p63/p73 family and plays key functional roles during normal epithelial development and differentiation and in pathological states such as squamous cell carcinomas. The human TP63 gene, located on chromosome 3q28 is driven by two promoters that generate the full-length transactivating (TA) and N-terminal truncated (ΔN) isoforms. Furthermore alternative splicing at the C-terminus gives rise to additional α, ß, γ and likely several other minor variants. Teasing out the expression and biological function of each p63 variant has been both the focus of, and a cause for contention in the p63 field. RESULTS: Here we have taken advantage of a burgeoning RNA-Seq based genomic data-sets to examine the global expression profiles of p63 isoforms across commonly utilized human cell-lines and major tissues and organs. Consistent with earlier studies, we find ΔNp63 transcripts, primarily that of the ΔNp63α isoforms, to be expressed in most cells of epithelial origin such as those of skin and oral tissues, mammary glands and squamous cell carcinomas. In contrast, TAp63 is not expressed in the majority of normal cell-types and tissues; rather it is selectively expressed at moderate to high levels in a subset of Burkitt's and diffuse large B-cell lymphoma cell lines. We verify this differential expression pattern of p63 isoforms by Western blot analysis, using newly developed ΔN and TA specific antibodies. Furthermore using unsupervised clustering of human cell lines, tissues and organs, we show that ΔNp63 and TAp63 driven transcriptional networks involve very distinct sets of molecular players, which may underlie their different biological functions. CONCLUSIONS: In this study we report comprehensive and global expression profiles of p63 isoforms and their relationship to p53/p73 and other potential transcriptional co-regulators. We curate publicly available data generated in part by consortiums such as ENCODE, FANTOM and Human Protein Atlas to delineate the vastly different transcriptomic landscapes of ΔNp63 and TAp63. Our studies help not only in dispelling prevailing myths and controversies on p63 expression in commonly used human cell lines but also augur new isoform- and cell type-specific activities of p63.

Role of chromatin and transcriptional co-regulators in mediating p63-genome interactions in keratinocytes.

BACKGROUND: The Transcription Factor (TF) p63 is a master regulator of epidermal development and differentiation as evident from the remarkable skin phenotype of p63 mouse knockouts. Furthermore, ectopic expression of p63 alone is sufficient to convert simple epithelium into stratified epithelial tissues in vivo and p63 is required for efficient transdifferentiation of fibroblasts into keratinocytes. However, little is known about the molecular mechanisms of p63 function, in particular how it selects its target sites in the genome. p63, which acts both as an activator and repressor of transcription, recognizes a canonical binding motif that occurs over 1 million times in the human genome. But, in human keratinocytes less than 12,000 of these sites are bound in vivo suggesting that underlying chromatin architecture and cooperating TFs mediate p63-genome interactions. RESULTS: We find that the chromatin architecture at p63-bound targets possess distinctive features and can be used to categorize p63 targets into proximal promoters (1%), enhancers (59%) and repressed or inactive (40%) regulatory elements. Our analysis shows that the chromatin modifications H3K4me1, H3K27me3, along with overall chromatin accessibility status can accurately predict bonafide p63-bound sites without a priori DNA sequence information. Interestingly, however there exists a qualitative correlation between the p63 binding motif and accessibility and H3K4me1 levels. Furthermore, we use a comprehensive in silico approach that leverages ENCODE data to identify several known TFs such as AP1, AP2 and novel TFs (RFX5 for e.g.) that can potentially cooperate with p63 to modulate its myriad biological functions in keratinocytes. CONCLUSIONS: Our analysis shows that p63 bound genomic locations in keratinocytes are accessible, marked by active histone modifications, and co-targeted by other developmentally important transcriptional regulators. Collectively, our results suggest that p63 might actively remodel and/or influence chromatin dynamics at its target sites and in the process dictate its own DNA binding and possibly that of adjacent TFs.

An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma.

Although numerous putative oncogenes have been associated with the etiology of head and neck squamous cell carcinoma (HNSCC), the mechanisms by which these oncogenes and their downstream targets mediate tumor progression have not been fully elucidated. We performed an integrative analysis to identify a crucial set of targets of the oncogenic transcription factor p63 that are common across multiple transcriptomic datasets obtained from HNSCC patients, and representative cell line models. Notably, our analysis revealed FST which encodes follistatin, a secreted glycoprotein that inhibits the transforming growth factor TGFß/activin signaling pathways, to be a direct transcriptional target of p63. In addition, we found that FST expression is also driven by epidermal growth factor receptor EGFR signaling, thus mediating a functional link between the TGF-ß and EGFR pathways. We show through loss- and gain-of-function studies that FST predominantly imparts a tumor-growth and migratory phenotype in HNSCC cells. Furthermore, analysis of single-cell RNA sequencing data from HNSCC patients unveiled cancer cells as the dominant source of FST within the tumor microenvironment and exposed a correlation between the expression of FST and its regulators with immune infiltrates. We propose FST as a prognostic biomarker for patient survival and a compelling candidate mediating the broad effects of p63 on the tumor and its associated microenvironment.

Dynamic changes in P300 enhancers and enhancer-promoter contacts control mouse cardiomyocyte maturation.

Cardiomyocyte differentiation continues throughout murine gestation and into the postnatal period, driven by temporally regulated expression changes in the transcriptome. The mechanisms that regulate these developmental changes remain incompletely defined. Here, we used cardiomyocyte-specific ChIP-seq of the activate enhancer marker P300 to identify 54,920 cardiomyocyte enhancers at seven stages of murine heart development. These data were matched to cardiomyocyte gene expression profiles at the same stages and to Hi-C and H3K27ac HiChIP chromatin conformation data at fetal, neonatal, and adult stages. Regions with dynamic P300 occupancy exhibited developmentally regulated enhancer activity, as measured by massively parallel reporter assays in cardiomyocytes in vivo, and identified key transcription factor-binding motifs. These dynamic enhancers interacted with temporal changes of the 3D genome architecture to specify developmentally regulated cardiomyocyte gene expressions. Our work provides a 3D genome-mediated enhancer activity landscape of murine cardiomyocyte development.

Blocking PI3K p110ß Attenuates Development of PTEN-Deficient Castration-Resistant Prostate Cancer.

A common outcome of androgen deprivation in prostate cancer therapy is disease relapse and progression to castration-resistant prostate cancer (CRPC) via multiple mechanisms. To gain insight into the recent clinical findings that highlighted genomic alterations leading to hyperactivation of PI3K, we examined the roles of the commonly expressed p110 catalytic isoforms of PI3K in a murine model of Pten-null invasive CRPC. While blocking p110α had negligible effects in the development of Pten-null invasive CRPC, either genetic or pharmacologic perturbation of p110ß dramatically slowed CRPC initiation and progression. Once fully established, CRPC tumors became partially resistant to p110ß inhibition, indicating the acquisition of new dependencies. Driven by our genomic analyses highlighting potential roles for the p110ß/RAC/PAK1 and ß-catenin pathways in CRPC, we found that combining p110ß with RAC/PAK1 or tankyrase inhibitors significantly reduced the growth of murine and human CRPC organoids in vitro and in vivo. Because p110ß activity is dispensable for most physiologic processes, our studies support novel therapeutic strategies both for preventing disease progression into CRPC and for treating CRPC. IMPLICATIONS: This work establishes p110ß as a promising target for preventing the progression of primary PTEN-deficient prostate tumors to CRPC, and for treating established CRPC in combination with RAC/PAK1 or tankyrase inhibitors.

Massively parallel in vivo CRISPR screening identifies RNF20/40 as epigenetic regulators of cardiomyocyte maturation.

The forward genetic screen is a powerful, unbiased method to gain insights into biological processes, yet this approach has infrequently been used in vivo in mammals because of high resource demands. Here, we use in vivo somatic Cas9 mutagenesis to perform an in vivo forward genetic screen in mice to identify regulators of cardiomyocyte (CM) maturation, the coordinated changes in phenotype and gene expression that occur in neonatal CMs. We discover and validate a number of transcriptional regulators of this process. Among these are RNF20 and RNF40, which form a complex that monoubiquitinates H2B on lysine 120. Mechanistic studies indicate that this epigenetic mark controls dynamic changes in gene expression required for CM maturation. These insights into CM maturation will inform efforts in cardiac regenerative medicine. More broadly, our approach will enable unbiased forward genetics across mammalian organ systems.

Reactivation of super-enhancers by KLF4 in human Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a disease of significant morbidity and mortality and rarely diagnosed in early stages. Despite extensive genetic and genomic characterization, targeted therapeutics and diagnostic markers of HNSCC are lacking due to the inherent heterogeneity and complexity of the disease. Herein, we have generated the global histone mark based epigenomic and transcriptomic cartogram of SCC25, a representative cell type of mesenchymal HNSCC and its normal oral keratinocyte counterpart. Examination of genomic regions marked by differential chromatin states and associated with misregulated gene expression led us to identify SCC25 enriched regulatory sequences and transcription factors (TF) motifs. These findings were further strengthened by ATAC-seq based open chromatin and TF footprint analysis which unearthed Krüppel-like Factor 4 (KLF4) as a potential key regulator of the SCC25 cistrome. We reaffirm the results obtained from in silico and chromatin studies in SCC25 by ChIP-seq of KLF4 and identify ΔNp63 as a co-oncogenic driver of the cancer-specific gene expression milieu. Taken together, our results lead us to propose a model where elevated KLF4 levels sustains the oncogenic state of HNSCC by reactivating repressed chromatin domains at key downstream genes, often by targeting super-enhancers.

Molecular Profiling Establishes Genetic Features Predictive of the Efficacy of the p110ß Inhibitor KIN-193.

Aberrant activation of the PI3K pathway is a common alteration in human cancers. Therapeutic intervention targeting the PI3K pathway has achieved limited success due to the intricate balance of its different components and isoforms. Here, we systematically investigated the genomic and transcriptomic signatures associated with response to KIN-193, a compound specifically targeting the p110ß isoform. By integrating genomic, transcriptomic, and drug response profiles from the Genomics of Drug Sensitivity in Cancer database, we identified mutational and transcriptomic signatures associated with KIN-193 and further created statistical models to predict the treatment effect of KIN-193 in cell lines, which may eventually be clinically valuable. These predictions were validated by analysis of the external Cancer Cell Line Encyclopedia dataset. These results may assist precise therapeutic intervention targeting the PI3K pathway. SIGNIFICANCE: These findings provide new insights into molecular signatures associated with sensitivity of the p110ß inhibitor KIN-193, which may provide a useful guide for developing precise treatment methods for cancer.

A reference map of murine cardiac transcription factor chromatin occupancy identifies dynamic and conserved enhancers.

Mapping the chromatin occupancy of transcription factors (TFs) is a key step in deciphering developmental transcriptional programs. Here we use biotinylated knockin alleles of seven key cardiac TFs (GATA4, NKX2-5, MEF2A, MEF2C, SRF, TBX5, TEAD1) to sensitively and reproducibly map their genome-wide occupancy in the fetal and adult mouse heart. These maps show that TF occupancy is dynamic between developmental stages and that multiple TFs often collaboratively occupy the same chromatin region through indirect cooperativity. Multi-TF regions exhibit features of functional regulatory elements, including evolutionary conservation, chromatin accessibility, and activity in transcriptional enhancer assays. H3K27ac, a feature of many enhancers, incompletely overlaps multi-TF regions, and multi-TF regions lacking H3K27ac retain conservation and enhancer activity. TEAD1 is a core component of the cardiac transcriptional network, co-occupying cardiac regulatory regions and controlling cardiomyocyte-specific gene functions. Our study provides a resource for deciphering the cardiac transcriptional regulatory network and gaining insights into the molecular mechanisms governing heart development.

Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor.

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.