Correlation of WNT signature genes with SMAD4 in human colorectal adenocarcinoma.

Differentiated lineages within the ectopic crypts

Absence of ectopic crypts in the single mutants

Epithelial-to-mesenchymal transition (EMT) is associated with tumor initiation, metastasis, and drug resistance. However, the mechanisms underlying these associations are largely unknown. We studied several tumor types to identify the source of EMT gene expression signals and a potential mechanism of resistance to immuno-oncology treatment. Across tumor types, EMT-related gene expression was strongly associated with expression of stroma-related genes. Based on RNA sequencing of multiple patient-derived xenograft models, EMT-related gene expression was enriched in the stroma versus parenchyma. EMT-related markers were predominantly expressed by cancer-associated fibroblasts (CAFs), cells of mesenchymal origin which produce a variety of matrix proteins and growth factors. Scores derived from a 3-gene CAF transcriptional signature ( COL1A1 , COL1A2 , COL3A1 ) were sufficient to reproduce association between EMT-related markers and disease prognosis. Our results suggest that CAFs are the primary source of EMT signaling and have potential roles as biomarkers and targets for immuno-oncology therapies.

Single-cell technologies, particularly single-cell RNA sequencing (scRNA-seq) methods, together with associated computational tools and the growing availability of public data resources, are transforming drug discovery and development. New opportunities are emerging in target identification owing to improved disease understanding through cell subtyping, and highly multiplexed functional genomics screens incorporating scRNA-seq are enhancing target credentialling and prioritization. ScRNA-seq is also aiding the selection of relevant preclinical disease models and providing new insights into drug mechanisms of action. In clinical development, scRNA-seq can inform decision-making via improved biomarker identification for patient stratification and more precise monitoring of drug response and disease progression. Here, we illustrate how scRNA-seq methods are being applied in key steps in drug discovery and development, and discuss ongoing challenges for their implementation in the pharmaceutical industry.

Phenotype of b-catenin gain of function mutants

Primary COAD tumor samples selected from TCGA RNA expression data

Loss of Smad4 in the intestinal epithelium

Correlation between SMAD4 and CTNNB1, NFκB and Rb protein expression in human colorectal adenocarcinoma

Immune checkpoint blockade (ICB) has led to durable clinical responses in multiple cancer types. However, biomarkers that identify which patients are most likely to respond to ICB are not well defined. Many putative biomarkers developed from a small number of samples often fail to maintain their predictive status in larger validation cohorts. We show across multiple human malignancies and syngeneic murine tumor models that neither pretreatment T cell receptor (TCR) clonality nor changes in clonality after ICB correlate with response. Dissection of tumor infiltrating lymphocytes pre- and post-ICB by paired single-cell RNA sequencing and single-cell TCR sequencing reveals conserved and distinct transcriptomic features in expanded TCR clonotypes between anti-PD1 responder and nonresponder murine tumor models. Overall, our results indicate a productive anti-tumor response is agnostic of TCR clonal expansion. Further, we used single-cell transcriptomics to develop a CD8+ T cell specific gene signature for a productive anti-tumor response and show the response signature to be associated with overall survival (OS) on nivolumab monotherapy in CheckMate-067, a phase 3 clinical trial in metastatic melanoma. These results highlight the value of leveraging single-cell assays to dissect heterogeneous tumor and immune subsets and define cell-type specific transcriptomic biomarkers of ICB response.

Background Single-cell (sc) sequencing performs unbiased profiling of individual cells and enables evaluation of less prevalent cellular populations, often missed using bulk sequencing. However, the scale and the complexity of the sc datasets poses a great challenge in its utility and this problem is further exacerbated when working with larger datasets typically generated by consortium efforts. As the scale of single cell datasets continues to increase exponentially, there is an unmet technological need to develop database platforms that can evaluate key biological hypotheses by querying extensive single-cell datasets. Large single-cell datasets like Human Cell Atlas and COVID-19 cell atlas (collection of annotated sc datasets from various human organs) are excellent resources for profiling target genes involved in human diseases and disorders ranging from oncology, auto-immunity, as well as infectious diseases like COVID-19 caused by SARS-CoV-2 virus. SARS-CoV-2 infections have led to a worldwide pandemic with massive loss of lives, infections exceeding 7 million cases. The virus uses ACE2 and TMPRSS2 as key viral entry associated proteins expressed in human cells for infections. Evaluating the expression profile of key genes in large single-cell datasets can facilitate testing for diagnostics, therapeutics, and vaccine targets, as the world struggles to cope with the on-going spread of COVID-19 infections. Main body In this manuscript we describe REVEAL: SingleCell, which enables storage, retrieval, and rapid query of single-cell datasets inclusive of millions of cells. The array native database described here enables selecting and analyzing cells across multiple studies. Cells can be selected using individual metadata tags, more complex hierarchical ontology filtering, and gene expression threshold ranges, including co-expression of multiple genes. The tags on selected cells can be further evaluated for testing biological hypotheses. One such example includes identifying the most prevalent cell type annotation tag on returned cells. We used REVEAL: SingleCell to evaluate the expression of key SARS-CoV-2 entry associated genes, and queried the current database (2.2 Million cells, 32 projects) to obtain the results in < 60 s. We highlighted cells expressing COVID-19 associated genes are expressed on multiple tissue types, thus in part explains the multi-organ involvement in infected patients observed worldwide during the on-going COVID-19 pandemic. Conclusion In this paper, we introduce the REVEAL: SingleCell database that addresses immediate needs for SARS-CoV-2 research and has the potential to be used more broadly for many precision medicine applications. We used the REVEAL: SingleCell database as a reference to ask questions relevant to drug development and precision medicine regarding cell type and co-expression for genes that encode proteins necessary for SARS-CoV-2 to enter and reproduce in cells.

A field experiment was conducted during kharif 2018 at Rice and Wheat Research Centre (RWRC), Malan to study the physiological response of different rice cultivars to varying fertility levels in mid-hills of Himachal Pradesh. The experiment consisted of 5 main-plot treatments comprising varying fertility levels {50% recommended dose of fertilizer (RDF), 100% RDF (90:40:40 kg N:P:K ha-1), 150% RDF, 50% RDF + Azolla and 100% RDF + Azolla} and 4 cultivars as sub-plot treatments {Vivekdhan 65, HPR 2143, HPR 2720 (red rice) and AZ 6508 (hybrid)}. The soil of the experimental site was silty clay loam in texture, acidic in reaction, medium in available nitrogen, phosphorus, and potassium. The increase in fertility level and Azolla application increased the dry matter accumulation and leaf area index of rice. The increasing trend in crop growth rate (CGR) during the active tillering and flowering stage was observed while the leaf area ratio reduced from 60 to 90 days after transplanting (DAT). Contrary to these the relative growth rate (RGR) and net assimilation rate (NAR) remained almost equal during both the time intervals between 30 and 60 days and 60 and 90 days after transplanting though it varied with varying fertility levels as well as between different cultivars. Additional azolla application at both the fertilizer doses of 50 and 100 % RDF showed a significant increase in CGR from 30 to 60 DAT as compared to the when it was not applied, the increase being 2.70 and 2.13 g m-2 day-1with the azolla application at 50 % and 100 % RDF, respectively. Further CGR recorded between 30 and 60 DAT with the application of azolla with 50 % RDF was statistically similar to the application of 100 % RDF alone, indicating a saving of 50 % RDF with the azolla application. Also the CGR recorded between 30 and 60 DAT with the application of 150 % RDF was also similar to the application of 100 % RDF along with azolla application. Similarly RGR increased with increasing fertilizer application as well as with azolla application at 30 to 60 and 60 to 90 DAT. LAR decreased during the flowering stage due to more contribution of flag leaf in net assimilation. Among the cultivars tested ‘Vivekdhan 65’ and ‘AZ 6508’ showed higher values of CGR as well as RGR, particularly between 60 to 90 DAT while ‘AZ 6508’ had significantly higher LAR. Initially, azolla applied to the crop was not much effective but during the later stages, its contribution increased the growth rate. These findings suggest that growth indices could be enhanced by applying higher doses of fertilizesr but the application of azolla can result in the saving of fertilizers.

Lineage-restricted transcription factors, such as the intestine-specifying factor CDX2, often have dual requirements across developmental time. Embryonic loss of CDX2 triggers homeotic transformation of intestinal fate, whereas adult-onset loss compromises crucial physiological functions but preserves intestinal identity. It is unclear how such diverse requirements are executed across the developmental continuum. Using primary and engineered human tissues, mouse genetics, and a multi-omics approach, we demonstrate that divergent CDX2 loss-of-function phenotypes in embryonic versus adult intestines correspond to divergent CDX2 chromatin-binding profiles in embryonic versus adult stages. CDX2 binds and activates distinct target genes in developing versus adult mouse and human intestinal cells. We find that temporal shifts in chromatin accessibility correspond to these context-specific CDX2 activities. Thus, CDX2 is not sufficient to activate a mature intestinal program; rather, CDX2 responds to its environment, targeting stage-specific genes to contribute to either intestinal patterning or mature intestinal function. This study provides insights into the mechanisms through which lineage-specific regulatory factors achieve divergent functions over developmental time.

Abstract Recent advances in single cell genomic technologies have enabled unbiased quantification of cellular states within complex tissues, mechanisms of differentiation, biomarker presentation in cellular subsets; yet their applications in evaluating the response to therapeutic interventions are still underexplored. In our study, we apply single cell RNA sequencing (scRNA-seq) to uncover mechanisms and drug targets for Multiple Myeloma(MM), a malignancy of plasma cells with estimates of 30,770 new cases and 12,770 deaths in 2018 within US alone. Using the 10x Genomics scRNA-seq platform, we profiled ten clinical MM patients, pre- and post-treatment with four standard of care chemotherapy regimens (VAD, VCD, VMP, MP; A=Adriamycin; C=Cyclophosphamide; D=Dexamethasone; M=Melphalan; P=Prednisone; V=Velcade). We sequenced a total of 106,000 cells and determined differential gene expression upon treatment at single-cell resolution comparing 37,926 pre- vs 44,842 post-treatment cells. We identify activation of immune cells with increased expression of IL1B, CD14, CD52, CCL4, IFITM3, PSMA4; and repression of translational machinery with reduced expression of PABPC1, PABPC4, FUS, POLR2A in post-treatment (abs(log2 FC >0.25) and P-value < 10-5). To understand the functional implication of gene expression changes, we identified that the upregulated genes are enriched for ontologies regulating protein ubiquitination pathway, communication between innate and adaptive immune cells, TREM1 signaling, dendritic cell maturation (P-value 3.16x10-17, 3.47x10-7, 3.09 x10-5, 4.07x10-5 respectively). Downregulated genes are enriched for EIF2 signaling, regulation of eIF4 and p70S6K signaling, mTOR signaling (P-value 3.16x10-13, 1.86x10-8, 2.24x10-6 respectively). Additionally, we performed a comparative analysis of differentially expressed genes in response to each drug regimen. We identified genes upregulated upon MP drug regimen are comparatively enriched for IL-8 signaling, while treatment with VMP enhances B-cell receptor signaling (P-value 5.5x10-12 and 9.0x10-9 respectively). Further, we have used our unbiased transcriptomic interrogation of MM clinical patients to identify a panel of 100+ biomarkers that are being evaluated at a proteomics level using our recently developed technique, RNA Expression and Protein sequencing (REAP-seq). This method enables us to obtain readout for whole transcriptome and proteins (>100) using oligo conjugated antibodies while retaining single cell resolution. Transcriptome and/or proteome analysis of bulk tumor often fails to detect signature of rare cell populations. Our study here highlights the use of single cell readouts for identifying novel oncology mechanisms, drug targets and drug combinations for more efficacious immunotherapies. Citation Format: Namit Kumar, Vanessa M. Peterson, Kelvin X. Zhang, Lixia Li, Philip W. Garrett-Engele, Raymond J. Moniz, Joel A. Klappenbach. Single cell RNA-sequencing of multiple myeloma clinical patients for identifying novel immuno-oncology drug targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-012.

After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.

Abstract The cell of origin of colon cancer is typically thought to be the resident somatic stem cells, which are immortal and escape the continual cellular turnover characteristic of the intestinal epithelium. However, recent studies have identified certain conditions in which differentiated cells can acquire stem-like properties and give rise to tumors. Defining the origins of tumors will inform cancer prevention efforts as well as cancer therapies, as cancers with distinct origins often respond differently to treatments. We report here a new condition in which tumors arise from the differentiated intestinal epithelium. Inactivation of the differentiation-promoting transcription factor SMAD4 in the intestinal epithelium was surprisingly well tolerated in the short term. However, after several months, adenomas developed with characteristics of activated WNT signaling. Simultaneous loss of SMAD4 and activation of the WNT pathway led to dedifferentiation and rapid adenoma formation in differentiated tissue. Transcriptional profiling revealed acquisition of stem cell characteristics, and colabeling indicated that cells expressing differentiated enterocyte markers entered the cell cycle and reexpressed stem cell genes upon simultaneous loss of SMAD4 and activation of the WNT pathway. These results indicate that SMAD4 functions to maintain differentiated enterocytes in the presence of oncogenic WNT signaling, thus preventing dedifferentiation and tumor formation in the differentiated intestinal epithelium. Significance: This work identifies a mechanism through which differentiated cells prevent tumor formation by suppressing oncogenic plasticity. Cancer Res; 78(17); 4878–90. ©2018 AACR.

We present a tool to measure gene and protein expression levels in single cells with DNA-labeled antibodies and droplet microfluidics. Using the RNA expression and protein sequencing assay (REAP-seq), we quantified proteins with 82 barcoded antibodies and > 20,000 genes in a single workflow. We used REAP-seq to assess the costimulatory effects of a CD27 agonist on human CD8(+) lymphocytes and to identify and characterize an unknown cell type.

BACKGROUND & AIMS: The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic contexts in mice. However, the function of LGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail.METHODS: We interrogated the function and expression of LGR family members using human pluripotent stem cell-derived tissues including definitive endoderm, mid/ hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5-GFP-IRES-CreERT2 mice.RESULTS: We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4 and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human-mouse species-specific differences at later time points of embryonic development.CONCLUSIONS: Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.