Unique Transcriptional Programs Research Articles

Single-cell RNA-seq of Drosophila miranda testis reveals the evolution and trajectory of germline sex chromosome regulation.

Although sex chromosomes have evolved from autosomes, they often have unusual regulatory regimes that are sex- and cell-type-specific such as dosage compensation (DC) and meiotic sex chromosome inactivation (MSCI). The molecular mechanisms and evolutionary forces driving these unique transcriptional programs are critical for genome evolution but have been, in the case of MSCI in Drosophila, subject to continuous debate. Here, we take advantage of the younger sex chromosomes in D. miranda (XR and the neo-X) to infer how former autosomes acquire sex-chromosome-specific regulatory programs using single-cell and bulk RNA sequencing and ribosome profiling, in a comparative evolutionary context. We show that contrary to mammals and worms, the X down-regulation through germline progression is most consistent with the shutdown of DC instead of MSCI, resulting in half gene dosage at the end of meiosis for all 3 X's. Moreover, lowly expressed germline and meiotic genes on the neo-X are ancestrally lowly expressed, instead of acquired suppression after sex linkage. For the young neo-X, DC is incomplete across all tissue and cell types and this dosage imbalance is rescued by contributions from Y-linked gametologs which produce transcripts that are translated to compensate both gene and protein dosage. We find an excess of previously autosomal testis genes becoming Y-specific, showing that the neo-Y and its masculinization likely resolve sexual antagonism. Multicopy neo-sex genes are predominantly expressed during meiotic stages of spermatogenesis, consistent with their amplification being driven to interfere with mendelian segregation. Altogether, this study reveals germline regulation of evolving sex chromosomes and elucidates the consequences these unique regulatory mechanisms have on the evolution of sex chromosome architecture.

Genome-wide p63-Target Gene Analyses Reveal TAp63/NRF2-Dependent Oxidative Stress Responses.

The p63 isoforms, TAp63 and ΔNp63, control epithelial morphogenesis and tumorigenesis through the interaction with distinct transcription factors and the subsequent regulation of unique transcriptional programs.

Molecular Classification of Relapsed DLBCL Reveals Novel Biologic Subgroups

Background Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults. Though standard immunochemotherapy regimens can result in clinical remission and cure in a majority of patients, approximately 30% of patients are primary treatment resistant or eventually relapse (rrDLBCL), and their prognosis is very poor. Recent molecular classification by large scale genomic and transcriptomic profiling of newly diagnosed DLBCL tumors has resulted in a deeper understanding of disease drivers, however, the molecular heterogeneity of rrDLBCL is yet to be fully explored. Therefore, in this study, we used an unsupervised approach to better define transcriptional and genetic programs in rrDLBCL samples hypothesizing that molecular stratification within this high-risk group may lead to a better understanding of this disease, which remains an unmet need, and a more personalized therapeutic approach. Methods Data used in this study included RNA (n=143) and whole exome (n=126) sequencing data from available FFPE tumor samples at the time of a relapse (any line of treatment, r1-r10 relapse timepoints included in analysis, one per patient), consented to the Molecular Epidemiology Resource (n=61), banked in the Mayo Lymphoma Biobank (n=50), or consented to the CC-122-ST-001 clinical trial (n=32, NCT01421524). Unsupervised clustering was performed on protein coding gene expression values using the non-negative matrix factorization (NMF) approach. NMF was carried out by performing 200 runs on cluster sizes from 2 to 6. Cell of origin (COO) was determined using the method described by Reddy et al. The tumor microenvironment was analyzed using CIBERSORTx and Lymphoma Microenvironment Classification (LME). Genetic classification was done using LymphGen. Results In order to identify unique transcriptional programs in rrDLBCL, we implemented NMF and consensus clustering to define an ideal quantity of stable patient clusters. We identified 4 rrDLBCL patient clusters (rrC1-rrC4) as the most stable solution, based on consensus clustering selection criteria of iterative NMF runs. While relapse timepoint was not associated with individual clusters, we did observe significant differences in tumor characteristics between the clusters. COO classification found that rrC1 and rrC4 were enriched for GCB-DLBCL while rrC3 was enriched for ABC-DLBCL classification (P < .001). To explore the biologic programs in each cluster, we used pathFindR, which leverages protein-protein interaction networks to identify disease related pathways, rrC1 and rrC4 were defined by pathways involved in cell cycle and mismatch repair; oxidative phosphorylation in rrC2; and MAPK and NF-kB in rrC3. Next, LME classification was done to define patterns in the tumor microenvironment and we found a significant difference between groups (P < .001), with rrC2 and rrC4 tumors enriched for a depleted TME, while rrC1 and rrC3 had a more immune rich or inflammatory TME. This was supported by CIBERSORTx analysis identifying significant differences (P > .001) in abundance of CD8 T cells, memory CD4 T cells, and M1 macrophages between clusters, with rrC4 having the lowest abundance. We then explored genetic patterns in each cluster. Gene level mutation enrichment analysis showed BCL7A mutation exclusively in C4 (6/6, P = .03). The clusters were not associated with LymphGen classification. Lastly, our recently published high risk signature classification that is associated with early clinical failure and is characterized by metabolic dysregulation, a depleted TME, T53 alterations, and poor outcome, was significantly different between the groups (P = .03) with enrichment for high-risk cases in rrC4. In summary, we show for the first time that rrDLBCL patients can be classified into four gene expression clusters that are associated with distinct pathway, TME, and genetic programs. These clusters should now be tested to learn if they can help select patients for newer therapies for rrDLBCL such as CAR-T and bispecific antibodies.

From Genotype to Phenotype: How Enhancers Control Gene Expression and Cell Identity in Hematopoiesis.

Blood comprises a wide array of specialized cells, all of which share the same genetic information and ultimately derive from the same precursor, the hematopoietic stem cell (HSC). This diversity of phenotypes is underpinned by unique transcriptional programs gradually acquired in the process known as hematopoiesis. Spatiotemporal regulation of gene expression depends on many factors, but critical among them are enhancers-sequences of DNA that bind transcription factors and increase transcription of genes under their control. Thus, hematopoiesis involves the activation of specific enhancer repertoires in HSCs and their progeny, driving the expression of sets of genes that collectively determine morphology and function. Disruption of this tightly regulated process can have catastrophic consequences: in hematopoietic malignancies, dysregulation of transcriptional control by enhancers leads to misexpression of oncogenes that ultimately drive transformation. This review attempts to provide a basic understanding of enhancers and their role in transcriptional regulation, with a focus on normal and malignant hematopoiesis. We present examples of enhancers controlling master regulators of hematopoiesis and discuss the main mechanisms leading to enhancer dysregulation in leukemia and lymphoma.

P02.14.B SPATIAL TRANSCRIPTOMIC ARCHITECTURE OF TUMOURS OF THE PERIPHERAL NERVOUS SYSTEM

Abstract BACKGROUND Studies into the genetics of peripheral nerve tumors have been focused on their association with known gene mutations and syndromes as well as on the tumor microenvironment of malignant peripheral nerve sheath tumors. Still, not many advances have been made into clarifying the structure and diverse pathophysiology of these tumors. Here we present spatially resolved transcriptomic profiling of benign and malignant entities and describe the topographical architecture of the neoplastic and microenvironmental diversity. MATERIAL AND METHODS We employed spatially resolved transcriptomics using the 10X Visium technology from paraffine embedded specimens. We included hybrid peripheral nerve sheath tumors (HPNSTs) plexiform neurofibromas and malignant peripheral nerve sheath tumors (MPNST). Computational analysis was employed by the SPATA2 toolbox. Transcriptomic niches were identified by graph-based spatial clustering. Correlation of histomorphological features and transcriptomic programs was performed by a co-expression network-based analysis. RESULTS We identified hub genes associated with neurofibroma and schwannoma histopathology across primary pathologies and discovered that neurofibroma regions of HPNSTs exhibited unique transcriptional programs marked by a significant higher local accumulation of bone-derived macrophages and lymphoid cells compared to primary neurofibromas. A novel marker, Apolipoprotein D (APOD), was found to accurately identify neurofibroma subregions in HPNSTs, which was confirmed by immunostaining in a control cohort. In MPNSTs, we observed a high grade of inter- and intrapatient heterogeneity driven by unique transcriptional niches and genetic variability. Subsequent pathway analysis revealed distinct oncogenic signaling driver across regional niches along with variance in immune infiltration. Our findings highlight the disbalance between histological annotation and actual tumor cell abundance in MPNSTs indicated by inferred copy number alterations. CONCLUSION Our integrated analysis pipeline revealed distinct transcriptional signatures and cellular interactions within benign PNSTs, including neurofibromas and hybrid peripheral nerve sheath tumors (HPNSTs) and sought to expose the tumor heterogeneity of MPNST and identify various cancer niches. Our study underscores the potential of spatially resolved transcriptomics as a powerful tool for advancing our understanding of PNST biology, and ultimately improving diagnosis, prognosis, and treatment strategies.

Transcriptomic analysis reveals differential adaptation of colorectal cancer cells to low and acute doses of cisplatin

Over the years, the landscape of cisplatin-based cancer treatment options has undergone continuous transitions. Currently, there is much debate over the optimum dose of cisplatin to be administered to cancer patients. In clinical practice, it can extend from repeated low sub-toxic doses to a few cycles of acute high drug doses. Herein, the molecular understanding of the overall cellular response to such differential doses of cisplatin becomes crucial before any decision making; and it has been a grey area of research. In this study, colorectal cancer (CRC) cells were treated with either- a low sub-toxic dose (LD; 30 µM) or a ten times higher acute dose (HD; 300 µM) of cisplatin, and thereafter, the cellular response was mapped through RNA sequencing followed by transcriptomic analysis. Interestingly, we observed that the tumor cells’ response to varying doses of cisplatin is distinctly different, and they activate unique transcriptional programs. The analysis of differentially regulated or uniquely expressed transcripts and corresponding pathways revealed a preferential enrichment of genes associated with chromatin organization, oxidative stress, senescence-associated signaling, and developmentally-active signaling pathways in HD; whereas, modulation of autophagy, protein homeostasis, or differential expression of ABC transporters was primarily enriched in LD. This study is the first of its kind to highlight cellular transcriptomic adaptations to different doses of cisplatin in CRC cells. Consequently, since, protein homeostasis was found to be deeply affected after cisplatin treatment, we further analyzed one of the primary cellular protein homeostatic mechanisms- autophagy. It was activated upon LD, but not HD, and served as a pro-survival strategy through the regulation of oxidative stress. Inhibition of autophagy improved sensitivity to LD. Overall, our study provides a holistic understanding of the distinct molecular signatures induced in CRC cells in response to differential cisplatin doses. These findings might facilitate the design of tailored therapy or appropriate drug dose for enhanced efficacy against CRCs.

753 Preferential recruitment of immature neutrophils enables robust skin regeneration

After severe injury, adult mammalian wound healing occurs by formation of fibrotic scar and loss of skin appendages resulting in permanent functional impairment. In mice, severe skin injuries exhibit partial regeneration, including formation of new hair follicles (HFs) in the center of the wound, whereas the periphery remains fibrotic. We previously showed that fibroblasts residing within the regenerative zone exhibit activation of unique transcriptional programs reminiscent of early skin development (Abassi et al, Cell Stem Cell).

The plasma cell proteome initiates early in B cell activation in advance of Blimp-1.

Abstract Plasma cell (PC) differentiation is a highly regulated process that radically changes the morphology and function of a B lymphocyte to that of a professional protein secretor. It is becoming clear that this process precedes increase in immunoglobulin synthesis rather than reacting to it. We have shown previously that large segments of the PC transcriptome are initiated in an mTORC1-dependent manner prior to both Blimp-1 and Xbp1s expression. Still, the kinetics of induction of the PC proteome remain largely unknown. We performed a whole proteome kinetic analysis of activated Blimp-1-reporter follicular B cells in PC-inductive conditions every 12 hours for three days and observed distinct patterns in induction of the PC proteome. To elucidate transcriptional networks, we performed single cell RNA-seq with the same conditions and correlated transcription factor expression with genes coding for these proteome modules within single cells. We found distinct subgroups within proteome modules corresponding to transcriptional patterns each with a highly correlated transcription factor set. These included robust, transient, Myc-associated factors required for translation initiation; rapid, sustained expression of protein synthesis machinery; and gradual, sustained increases in proteins associated with ER function and secretion. Each of these preceded the induction of Blimp-1 and increase in immunoglobulin protein. Finally, using inducible deletion of Blimp-1 in conjunction with the reporter allele we show that these unique transcriptional and proteomic programs are initiated independently of Blimp-1 expression. Thus, activated B cells prepare for protein secretion by initiating the PC proteome prior to antibody secretion. Supported by grants from the NIH: R01 AI113545, AI139123, AI154932, R21 AI133998 (PI:Allman) and BTG was supported by training grant T32 CA009140 (PI:Pear)

Molecular Characterization of HOXA2 and HOXA3 Binding Properties.

The highly conserved HOX homeodomain (HD) transcription factors (TFs) establish the identity of different body parts along the antero–posterior axis of bilaterian animals. Segment diversification and the morphogenesis of different structures is achieved by generating precise patterns of HOX expression along the antero–posterior axis and by the ability of different HOX TFs to instruct unique and specific transcriptional programs. However, HOX binding properties in vitro, characterised by the recognition of similar AT-rich binding sequences, do not account for the ability of different HOX to instruct segment-specific transcriptional programs. To address this problem, we previously compared HOXA2 and HOXA3 binding in vivo. Here, we explore if sequence motif enrichments observed in vivo are explained by binding affinities in vitro. Unexpectedly, we found that the highest enriched motif in HOXA2 peaks was not recognised by HOXA2 in vitro, highlighting the importance of investigating HOX binding in its physiological context. We also report the ability of HOXA2 and HOXA3 to heterodimerise, which may have functional consequences for the HOX patterning function in vivo.

Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis

SummaryFibrosis-driven solid organ failure is an enormous burden on global health. Spiny mice (Acomys) are terrestrial mammals that can regenerate severe skin wounds without scars to avoid predation. Whether spiny mice also regenerate internal organ injuries is unknown. Here, we show that despite equivalent acute obstructive or ischemic kidney injury, spiny mice fully regenerate nephron structure and organ function without fibrosis, whereas C57Bl/6 or CD1 mice progress to complete organ failure with extensive renal fibrosis. Two mechanisms for vertebrate regeneration have been proposed that emphasize either extrinsic (pro-regenerative macrophages) or intrinsic (surviving cells of the organ itself) controls. Comparative transcriptome analysis revealed that the Acomys genome appears poised at the time of injury to initiate regeneration by surviving kidney cells, whereas macrophage accumulation was not detected until about day 7. Thus, we provide evidence for rapid activation of a gene expression signature for regenerative wound healing in the spiny mouse kidney.

PEG10 amplification at 7q21.3 potentiates large-cell transformation in cutaneous T-cell lymphoma

AbstractMycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, undergo large-cell transformation (LCT) in the late stage, manifesting aggressive behavior, resistance to treatments, and poor prognosis, but the mechanisms involved remain unclear. To identify the molecular driver of LCT, we collected tumor samples from 133 MF patients and performed whole-transcriptome sequencing on 49 advanced-stage MF patients, followed by integrated copy number inference and genomic hybridization. Tumors with LCT showed unique transcriptional programs and enriched expressions of genes at chr7q. Paternally expressed gene 10 (PEG10), an imprinted gene at 7q21.3, was ectopically expressed in malignant T cells from LCT, driven by 7q21.3 amplification. Mechanistically, aberrant PEG10 expression increased cell size, promoted cell proliferation, and conferred treatment resistance by a PEG10/KLF2/NF-κB axis in in vitro and in vivo models. Pharmacologically targeting PEG10 reversed the phenotypes of proliferation and treatment resistance in LCT. Our findings reveal new molecular mechanisms underlying LCT and suggest that PEG10 inhibition may serve as a promising therapeutic approach in late-stage aggressive T-cell lymphoma.

Ras-p53 genomic cooperativity as a model to investigate mechanisms of innate immune regulation in gastrointestinal cancers.

Despite increasingly thorough mechanistic understanding of the dominant genetic drivers of gastrointestinal (GI) tumorigenesis (e.g., Ras/Raf, TP53, etc.), only a small proportion of these molecular alterations are therapeutically actionable. In an attempt to address this therapeutic impasse, our group has proposed an innovative extreme outlier model to identify novel cooperative molecular vulnerabilities in high-risk GI cancers which dictate prognosis, correlate with distinct patterns of metastasis, and define therapeutic sensitivity or resistance. Our model also proposes comprehensive investigation of their downstream transcriptomic, immunomic, metabolic, or upstream epigenomic cellular consequences to reveal novel therapeutic targets in previously “undruggable” tumors with high-risk genomic features. Leveraging this methodology, our and others’ data reveal that the genomic cooperativity between Ras and p53 alterations is not only prognostically relevant in GI malignancy, but may also represent the incipient molecular events that initiate and sustain innate immunoregulatory signaling networks within the GI tumor microenvironment, driving T-cell exclusion and therapeutic resistance in these cancers. As such, deciphering the unique transcriptional programs encoded by Ras-p53 cooperativity that promote innate immune trafficking and chronic inflammatory tumor-stromal-immune crosstalk may uncover immunologic vulnerabilities that could be exploited to develop novel therapeutic strategies for these difficult-to-treat malignancies.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Molecular Profiling of the Cardiac Conduction System: the Dawn of a New Era.

Recent technological advances have led to an increased ability to define the gene expression profile of the cardiac conduction system (CCS). Here, we review the most salient studies to emerge in recent years and discuss existing gaps in our knowledge as well as future areas of investigation. Molecular profiling of the CCS spans several decades. However, the advent of high-throughput sequencing strategies has allowed for the discovery of unique transcriptional programs of the many diverse CCS cell types. The CCS, a diverse structure with significant inter- and intra-component cellular heterogeneity, is essential to the normal function of the heart. Progress in transcriptomic profiling has improved the resolution and depth of characterization of these unique and clinically relevant CCS cell types. Future studies leveraging this big data will play a crucial role in improving our understanding of CCS development and function as well as translating these findings into tangible translational tools for the improved detection, prevention, and treatment of cardiac arrhythmias.

Abstract 37: Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples

Abstract Determining patterns and mechanisms of drug resistance is fundamentally required for improving clinical outcome of cancer treatments. The ability to study multiple samples from different metastatic sites of the same patient is a clinically challenging task, which has become possible with the advent of “rapid” autopsy procedures (<6 hours from death) conducted on the patients deceased from cancer. We have obtained and analyzed whole-exome, whole genome and transcriptome sequencing data from advanced breast cancer samples derived from multiple cancer lesions of the same patient post-treatment through the Massachusetts General Hospital Rapid Autopsy program. We have integrated genomic and transcriptomic data through advanced clonal reconstruction methods (PhylogicNDT) to investigate how the patients developed resistance to multiple lines of anti-cancer therapy. Additionally, we have used previously collected cell-free DNA samples to establish both a spatial and temporal picture of cancer drug resistance and progression. We have examined clonal heterogeneity and resistance to targeted therapies in 10 metastatic ER+ breast cancer patients over 120 distinct metastatic sites. The majority of patients in this cohort were treated with CDK4/6 cell cycle inhibitors in addition to hormone therapy. We discovered that multiple metastatic sites often share similar clonal structure, but overall progeny of several sibling clones spreads throughout the body, creating “families” of more closely related lesions. These distinct lesion populations often develop independent resistance mechanisms to the many lines of treatment that the patents have received. At least 8 out of 10 patients showed multiple known and potentially novel resistance mechanisms in separate branches of the phylogenetic tree, often converging on distinct mutations in the same resistance genes. Notably we have identified ESR1, KRAS and chromatin modifier recurrent and convergent mutations across the cohort. Temporal analysis using cfDNA taken over the course of treatment allows us to identify clones that expanded in response to a given treatment. Using RNA expression and pathway analysis, we identify unique transcriptional programs, differentially expressed genes and ER-signaling changes between distinct clones within one patient, as well as compare genetically similar clones across patents. Citation Format: Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Elizaveta Leshchiner, Thomas Zhang, Christopher Pinto, Filippo Utro, Kahn Rhrissorrakrai, Chaya Levovitz, Brian P. Danysh, Kara Slowik, Raquel A. Jacobs, Laxmi Parida, Gad Getz, Dejan Juric. Convergence of resistance patterns in breast cancer after multiple lines of treatment through analysis of rapid autopsy samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 37.

Distinct Transcriptional Signatures Distinguish the Emergence of Multipotent Progenitors and Hematopoietic Stem Cells from Endothelial Precursors in the Murine Embryo

During embryonic development, blood cells emerge from hemogenic endothelium (HE), producing waves of hematopoietic progenitors prior to the emergence of rare hematopoietic stem cells (HSCs), which have the unique ability to self-renew and generate all cell types of the adult hematopoietic system. HSCs have significant potential for use in cellular therapies and disease modeling. However, efforts to generate HSCs in vitro from pluripotent stem cells (PSCs) have been limited by an incomplete understanding of the unique phenotypic markers and transcriptional programs that distinguish HE with HSC potential. Previous studies have demonstrated that yolk sac-derived erythromyeloid progenitors and HSCs originate from distinct populations of HE. However, it is not known whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from HE with common phenotypic and transcriptional properties. To investigate this, we combined index sorting of single hemogenic precursors with stromal co-culture that enables simultaneous detection of HSC and multilineage hematopoietic potential, to functionally validate surface markers that may distinguish hemogenic precursors with different hematopoietic fates. We previously found that the co-expression of two markers, CD61 and EPCR, identifies a subset of VE-Cadherin+ endothelial cells from the mouse P-Sp/AGM region (para-aortic splanchnopleura/aorta-gonad-mesonephros, where the first HSCs are generated from HE between E9 and E11 in development) enriched phenotypically for arterial endothelial surface markers (e.g. Dll4, CD44) and functionally for hemogenic precursors with HSC potential. However, this population remains heterogeneous, containing clonal hemogenic precursors with the potential for HSC as well as multilineage progenitor-restricted fates. Here, we report that expression of arterial marker CXCR4 further enriched for functional HSC potential in hemogenic precursors in the P-Sp/AGM between E9 and E10, when the first clonal HSC precursors are detected at rare frequency. In contrast, we detected more abundant clonal HE with multilineage hematopoietic potential (producing lymphoid, erythroid, and myeloid progeny in vitro but lacking HSC potential) at the same stage, which are distinguished by comparatively lower CXCR4 expression. To investigate transcriptional differences between HE populations differentially expressing CXCR4, we performed single-cell RNA sequencing of E9 P-Sp-derived VE-Cadherin+CD61+EPCR+ cells. Using an unbiased gene module analysis based on graph autocorrelation in the Monocle 3 platform to identify genes that co-vary over pseudotime, we found that Cxcr4 is uniquely expressed in a subset of cells simultaneously enriched for arterial-specific genes (including Dll4, Efnb2, Hey2, Sox17, Cd44) and genes with established roles in HSC maintenance and self-renewal (including Mecom, Cdkn1c, H19, Txnip, Kmt2a). Conversely, expression of these genes is decreased in cells undergoing the endothelial to hematopoietic transition at this stage based on pseudotemporal ordering, concomitant with increasing expression of hematopoietic-specifying transcription factors Runx1 and Gfi1, and other genes associated with definitive hematopoiesis (egs. Myb, Kit, Hlf, Gata2, Mpl, Lyl1). We also examined the aggregate expression of established HSC-specific signature genes from published data sets across pseudotime, and found that they exhibit similar expression dynamics to that of Cxcr4 and Dll4, reaching peak expression prior to the initiation of Runx1 and Gfi1 expression. Altogether, our studies support a model in which the initial populations of multipotent progenitors and HSCs emerge independently from HE in the P-Sp/AGM. Furthermore, our findings suggest that HE with HSC competence is uniquely defined by co-expression of arterial endothelial genes and genes encoding HSC self-renewal factors, providing insight into the earliest transcriptional programs that must be coordinated to drive HSC fate from endothelial precursors. Future studies will focus on identifying the signal pathways whose integration promotes expression of these HSC-defining transcriptional programs in endothelial cells, with the goal of advancing methods for HSC generation in vitro. Disclosures Bernstein: Lyell Immunopharma: Current equity holder in publicly-traded company, Other: Laboratory Support; Deverra Therapeutics: Current equity holder in publicly-traded company.

Abstract PO-29: Conserved and unique transcriptional programs in human and canine non-Hodgkin lymphomas inform the judicious applications for the spontaneous canine model of disease

Abstract Non-Hodgkin lymphomas (NHL) represent a diverse group of tumors that are common in both humans and dogs. Extensive work has documented driver genes and inferred the cell of origin for many subtypes of human NHL, and emerging data suggest that the driver events are distinct for the corresponding subtypes of canine NHL. This raises important questions about how and when spontaneous canine NHLs can or should be considered as models for human NHLs. Here, we show the application of bioinformatics tools that allow us to systematically reduce the dimensionality of complex transcriptional patterns across independent datasets, platforms, and species, and that are capable of identifying conserved and unique traits, as well as outcome associations. The application of these tools to human and canine NHL datasets shows that, for example, the gene expression cluster that defines the germinal center-B-cell (GCB)-like subtype is exclusively present in human samples, but not in dog samples. Conversely, we confirm that non-GCB subtypes (activated B-cell [ABC] or ABC-like) include a heterogeneous group of subtypes defined by expression of shared and species-specific gene clusters, including resident or infiltrating cells that mold the stromal and immune microenvironments. Moreover, we are able to expand this approach to other subtypes of NHL to discern the contributions from stromal transcriptional programs and from lymphocyte transcriptional programs by including samples in the analyses where stromal cells were depleted. Overall, our comparative genomic approach will enhance our understanding of the conserved and species-specific events that underlie the molecular etiology of NHL, informing when spontaneous canine NHL subtypes should be considered as independent, unrelated conditions and improving and refining when and how they can be used as a relevant spontaneous model of human NHL. Citation Format: Aaron L. Sarver, Jaime F. Modiano. Conserved and unique transcriptional programs in human and canine non-Hodgkin lymphomas inform the judicious applications for the spontaneous canine model of disease [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-29.

Abstract IA08: Understanding programs of invasion and metastasis in head and neck cancer

Abstract Despite advances in our understanding of tumor biology as well as radiation, chemotherapy, and surgical treatments, head and neck squamous cell carcinoma (HNSCC) remains the sixth leading cause of cancer-related mortality and a major cause of morbidity, with over 550,000 new cases per year. Although HNSCC mortality has slightly declined with improved tolerance of therapy, the majority of deaths in HNSCC are related to advanced disease with locoregional lymph node (LN) or distant metastasis. Indeed, the presence of LN metastases reduces survival by approximately 50% in all HNSCC patients. Unfortunately, a firm biologic understanding of pathways that underlie metastasis in HNSCC (and many other solid tumors) remains elusive. Tumor heterogeneity in HNSCC is a major challenge in clinical management and can manifest as variability among tumors (intertumoral heterogeneity) or among cells harboring distinct mutations or expressing unique transcriptional programs from the same tumor (intratumoral heterogeneity). Prior studies have suggested that intratumoral heterogeneity is present in HNSCC based on comparative genomic hybridization assays and DNA ploidy analyses, with recent studies validating intratumoral heterogeneity using whole-genome sequencing. In addition, there is growing interest in subpopulations of HNSCC cells, which may have unique properties such as the ability to self-renew or invade, contributing to recurrence or metastasis, respectively. The clinical significance of intratumoral heterogeneity has recently been demonstrated with a quantitative measure of genetic heterogeneity based on whole-exome sequencing (WES). This measure, termed mutant-allele tumor heterogeneity (MATH), suggests that intratumoral heterogeneity is associated with tumor progression and poor responses to treatment, supporting the idea that greater genetic variability predicts a worse clinical outcome in HNSCC. However, whether intratumoral transcriptional heterogeneity also contributes to clinical outcomes remained an open question. We recently utilized single cell RNA-sequencing (scRNA-seq), a powerful new approach to characterize expression heterogeneity present within oral cavity HNSCC (Puram et al., Cell 2017). The strength of these analyses is the ability to capture the transcriptional profile of each individual cell without contamination from other cells that may be present within the sample. As a result, single-cell sequencing offers 1) more detailed, nuanced expression data than analyses of tumor fragments and 2) captures the transcriptional profile of every cell, even rare populations, that might be overlooked in pooled RNA. We sequenced ~6000 single cells from 18 samples, including five matched primary/lymph node pairs. Our analyses reveal that stromal and immune populations have well-defined and consistent expression programs across tumors. In contrast, malignant cells are highly heterogeneous with variable gene signatures related to cell cycle, stress, hypoxia, epithelial structures, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors and in further in vitro analyses, we found that p-EMT cells were more invasive. Integration of single-cell transcriptomes with bulk expression profiles for >500 tumors from The Cancer Genome Atlas using a deconvolution approach identified a malignant cell-specific signature for each of these samples. Remarkably, this analysis identified the p-EMT program as the primary source of variability in HNSCC, while enabling us to redefine HNSCC subtypes by their malignant and stromal composition. Further integration with clinical annotations uncovered a significant association between the p-EMT program and nodal metastasis, grade, and adverse pathologic features including lymphovascular invasion and extracapsular extension. Consistent with these findings, the p-EMT score was a significant predictor of occult nodal metastases in clinically N0 patients, with predictive power equivalent to tumor thickness/size. Our studies provide a detailed map of expression heterogeneity in the HNSCC ecosystem and define an in vivo p-EMT program associated with metastasis and disease progression. Citation Format: Sidharth V. Puram. Understanding programs of invasion and metastasis in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA08.

Regulatory T cells suppress novel skeletal muscle macrophages identified by single-cell RNA sequencing

Abstract Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder that results from mutations in the dystrophin gene causing necrosis, inflammation and ultimately fibrosis. In DMD, muscle macrophages are multifaceted effector immune cells that regulate multiple pathogenic processes, including myofiber injury, inflammation and fibrosis, but also promote regeneration. Previously, we demonstrated that regulatory T cells (Tregs) ameliorated the severity of muscular dystrophy by regulating M1-like and M2-like macrophage activation, and more recently, a subset of undefined macrophages. Herein, we used single-cell RNA sequencing (scRNAseq) approaches to define the phenotypic complexity and transcriptional profile of muscle macrophages in healthy and dystrophic muscle. A T-distributed Stochastic Neighbor Embedding (t-SNE) analysis of gene expression data revealed that muscle macrophages clustered into six novel populations defined by unique transcriptional programs. We observed that a population expressing high levels of galectin-3 was absent in wildtype and dystrophic muscle before the onset of disease, but was increased at the onset of acute pathology and further expanded when Tregs were depleted in the mdx mouse model of DMD. In light of emerging evidence in the literature suggesting a role for galectin-3 and Tregs in fibrosis, we performed in vivo functional assays to underscore the role of the Treg-Macrophages axis in the control of fibrosis in DMD. Our data support a new paradigm, in which unique transcriptional programs define novel macrophage populations likely adapted to the diseased muscle milieu, and suggest that therapies focused on augmenting Tregs function in dystrophic muscle may be beneficial to treat fibrosis.

Transcriptional Profiling of the Adult Hair Follicle Mesenchyme Reveals R-spondin as a Novel Regulator of Dermal Progenitor Function.

SummaryThe adult hair follicle (HF) undergoes successive regeneration driven by resident epithelial stem cells and neighboring mesenchyme. Recent work described the existence of HF dermal stem cells (hfDSCs), but the genetic regulation of hfDSCs and their daughter cell lineages in HF regeneration remains unknown. Here we prospectively isolate functionally distinct mesenchymal compartment in the HF (dermal cup [DC; includes hfDSCs] and dermal papilla) and define the transcriptional programs involved in hfDSC function and acquisition of divergent mesenchymal fates. From this, we demonstrate cross-compartment mesenchymal signaling within the HF niche, whereby DP-derived R-spondins act to stimulate proliferation of both hfDSCs and epithelial progenitors during HF regeneration. Our findings describe unique transcriptional programs that underlie the functional heterogeneity among specialized fibroblasts within the adult HF and identify a novel regulator of mesenchymal progenitor function during tissue regeneration.