Cell RNA Sequencing Research Articles

Abstract B43: Division of labor between YAP and TAZ in lung cancer

Abstract Paralogs originating from a gene duplication event can diverge and accumulate changes that contribute to a division of labor. Therefore, we wished to explore differential activities of YAP and TAZ. Traditionally, research has tended to consider YAP and TAZ as functionally redundant transcriptional cofactors, with similar biologic impact. However, there is growing evidence that each of them possesses distinct attributes. Hence, it is of great interest to find out whether YAP and TAZ have nonredundant involvement in cancer. Lung cancer is the leading cause of cancer-related deaths worldwide. Despite significant progress, the 5-year survival is still lower than 15%. Therefore, there is an urgent need for novel lung cancer biomarkers. Recent evidence suggests that YAP/TAZ play important roles in lung tumorigenesis. We explored differences in the transcriptional programs regulated by YAP and TAZ in lung adenocarcinoma cells, and the physiologic implications of such differences. Towards this aim, we performed RNA-sequencing in lung cancer cells following knockdown of either YAP or TAZ. Our analysis revealed a broad spectrum of expression patterns, including genes that are regulated preferentially by either YAP, TAZ, or both. Interestingly, a group of genes related to extracellular matrix remodeling was substantially regulated by TAZ but not YAP, while cell cycle- and proliferation-related genes were affected mainly by YAP but not TAZ. These differences in gene regulation correlated with functional differences between YAP and TAZ. Knockdown of TAZ strongly affected cell migration, whereas depletion of YAP had only mild effects on migration. Conversely, YAP silencing resulted in reduced proliferation compared to TAZ silencing. Importantly, YAP- and TAZ-dependent transcriptomes were correlated to lung cancer patient survival. In summary, our findings suggest that YAP and TAZ have distinct but complementary roles, which are associated with different cancer features. Hopefully, the identification of different activities and downstream effects of YAP and TAZ may enable more refined stratification of lung cancers and provide clues for the development of more effective targeted therapies. Citation Format: Michal Shreberk-Shaked, Bareket Dassa, Silvia Di Agostino, Yarden Nuriel, Noa Wigoda, Ron Rotkopf, Giovanni Blandino, Yael Aylon, Moshe Oren. Division of labor between YAP and TAZ in lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B43.

Abstract A19: Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling

Abstract Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G-protein coupled receptors signaling. Data from The Cancer Genome Atlas demonstrated remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate of tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin. These are generated, respectively, through sphingosine kinase, autotaxin, and tissue factor, all of which are elevated in glioblastoma tumors or cell lines. Signaling to RhoA leads to activation of the transcriptional co-activator YAP (Yes-associated protein 1). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al., Oncogene 2018) using human glioblastoma cell lines and GBM stem cells (GSCs) derived from patient-derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC-23 PDX cells significantly attenuated in vitro self-renewal capability assessed by limiting dilution, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild-type cells. Our current work examines the effects of Gα12 since it transduces effects of multiple GPCRs upstream to RhoA that could be activated in the tumor environment. Targeting Gα12 using shRNA in two GBM cancer stem cell lines, GSC-23 and HK-281, decreased Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC-23 stemness as determined by limiting dilution assay and reduced mRNA expression levels of canonical stem cells genes (CCND1, NANONG, OCT4, SOX2, and NESTIN by approximately 50%). We are currently examining tumor growth of GSC-23 control and Gα12 knockdown cells as orthotopic xenografts. We are also examining the role of specific YAP-regulated target genes identified by RNA-sequencing of S1P-treated control and YAP knockout glioblastoma cells. In particular, one of the genes highly dependent on YAP is F3, which encodes the protein for tissue factor (TF) which is involved not only in coagulation but also in generation of thrombin, a ligand for PAR1, a GPCR that is upregulated in GBM and regulates RhoA signaling. Expression of TF was also selectively decreased in tumors from PDX cells lacking YAP, indicating that this transcriptional pathway is regulated in preclinical GBM models. Thus, Gα12/RhoA-mediated YAP signaling pathway appears to play a pivotal role in GBM tumor growth and progression. Citation Format: Olga M. Chaim, Jinhui Ma, Jacqueline Lara, Frank Furnari, Shigeki Myamoto, Joan H. Brown. Regulation of glioblastoma tumor growth and stem cell properties through Gα12 and tissue factor, upstream and downstream players in YAP signaling [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A19.

Abstract 548: Cellular Contributions to Neonatal Cardiac Regeneration

While the neonatal mouse heart possesses a remarkable ability to regenerate up to a week after birth, the adult mammalian heart is susceptible to irreversible scar formation that impedes cardiac function. Such a scar is formed by the precocious deposition of extracellular matrix (ECM) by resident cardiac fibroblasts (CFs). Unlike their adult counterparts, neonatal CFs in the regenerative widow may have a unique phenotype that contributes to cardiac regeneration and scar resolution. Indeed, the neonatal cardiac ECM secreted by CFs is reported to stimulate regeneration, yet the underlying mechanisms of CF-mediated cardiac repair in the neonate has not been examined. Here, we present a strategy to establish the role of tissue resident CFs in mouse neonatal cardiac regeneration through selective cell depletion and RNA sequencing. Through the initial analysis of published RNA sequencing data, we identified an enrichment of pro-regenerative molecules such as amphiregulin (Areg) during the neonatal regenerative window. Areg, an epidermal growth factor ligand, has been shown to paradoxically stimulate both cardiac repair and pathological fibrosis after adult cardiac injury. To assess its impact on the neonatal cardiac injury response, we developed an adeno-associated virus serotype 9 with the complete coding sequence of mouse Areg (AAV9:Areg) controlled by the cardiomyocyte-specific cardiac troponin T promoter. In vivo , AAV9:Areg treated mice accumulate BrdU+ (proliferative) non-myocytes adjacent to Areg-expressing cardiomyocytes. Ongoing studies are aimed at evaluating the contribution of CFs to neonatal cardiac repair, including whether Areg-dependent cellular changes impact CFs in the neonatal regenerative window in a manner distinct from that in the adult injury response.

Abstract B23: EWS-FLI1 partners with EWSR1 to regulate transcription in Ewing sarcoma

Abstract Transcription factors are important regulators in normal cell proliferation and cancer. Ewing sarcoma is a pediatric bone cancer driven by a translocation between two transcription factor genes, EWSR1 and FLI1, leading to the expression of an aberrant transcription factor, EWS-FLI1. While FLI1 is not expressed, constitutive expression of EWSR1 is retained in Ewing sarcoma cells. However, very little is known about the effect of wild-type EWSR1 on EWS-FLI1 transcriptional activity. EWSR1 and EWS-FLI1 coimmunoprecipitate in a complex together in Ewing sarcoma cells and HEK293T cells. RNA-sequencing in Ewing sarcoma cells revealed a pool of genes coregulated by EWS-FLI1 and EWSR1. Furthermore, loss of EWSR1 in Ewing sarcoma cells inhibits anchorage-independent growth. Taken together, this suggests EWSR1 may play a key role in the transcriptional activity of EWS-FLI1 in Ewing sarcoma biology. Citation Format: Nasiha Ahmed, Jacob Schwartz. EWS-FLI1 partners with EWSR1 to regulate transcription in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B23.

An Integrated Genomic and Transcriptomic Analysis Reveals Candidates of Susceptibility Genes for Crohn\u2019s Disease in Japanese Populations

Expression quantitative trait locus (eQTL) analyses have enabled us to predict the function of disease susceptibility SNPs. However, eQTL for the effector memory T cells (TEM) located in the lamina propria mononuclear cells (LPMCs), which play an important role in Crohn’s disease (CD), are not yet available. Thus, we conducted RNA sequencing and eQTL analyses of TEM cells located in the LPMCs from IBD patients (n = 20). Genome-wide association study (GWAS) was performed using genotyping data of 713 Japanese CD patients and 2,063 controls. We compared the results of GWAS and eQTL of TEM, and also performed a transcriptome-wide association study using eQTL from Genotype Tissue Expression project. By eQTL analyses of TEM, correlations of possible candidates were confirmed in 22,632 pairs and 2,463 genes. Among these candidates, 19 SNPs which showed significant correlation with tenascin-XA (TNXA) expression were significantly associated with CD in GWAS. By TWAS, TNFSF15 (FDR = 1.35e-13) in whole blood, ERV3-1 (FDR = 2.18e-2) in lymphocytes, and ZNF713 (FDR = 3.04e-2) in the sigmoid colon was significantly associated with CD. By conducting integration analyses using GWAS and eQTL data, we confirmed multiple gene transcripts are involved in the development of CD.

2113-P: Discrepancy between Single-Cell RNA Sequencing and Protein Expression Assessments of GLP-1Rr Heterogeneity in Beta Cells

The glucagon-like peptide 1 receptor (GLP-1R) is insulinotropic in β-cells and the target of multiple classes of diabetes drugs. People with T2D secrete normal amounts of GLP-1, but are less responsive to its actions; the mechanisms for this are unknown. Studies have shown that metabolic stress decreases GLP-1R in islets, however, it is unknown if this occurs in all, or just a subset, of β-cells. We hypothesized that GLP-1R expression is heterogeneous in β-cells, such that not all β-cells express the GLP-1R, and that metabolic stress would decrease the number of GLP-1R+ β-cells. To test this, we first performed single-cell RNA sequencing (scRNAseq) in mouse and human islet cells and found that the majority of β-cells were GLP-1R negative, supporting significant heterogeneity. To validate the scRNAseq results, we generated a GLP-1R reporter mouse by crossing GLP-1R-Cre with mTmG mice. Using FACS to separate and qPCR to characterize positive (GFP+) from negative (tdTomato+) dispersed islet cells, we found GLP-1R+ cells were enriched for Ins2 and Sst, while GLP-1R- cells were enriched for Gcg, suggesting GLP-1R promoter activity is localized to β- and d-cells. In addition, β-cell markers were not present in the tdTomato+ population, suggesting very few GLP-1R negative β-cells. Next, we stained dispersed islets from the reporter mice with a validated GLP-1R antibody and found >90% congruence with GFP+ cells and no staining in tdTomato+ cells. In dispersed islets from wild type mice, ∼90% of β-cells were GLP-1R+; very few of the α- or d-cells were GLP-1R+. Metabolic stress induced through chronic high-fat feeding or using multiparous models did not alter the number of GLP-1R+ β-cells. In conclusion, assessment of GLP-1R expression and GLP-1R protein revealed the majority of β-cells are GLP-1R+, a finding that conflicts with the interpretation of scRNAseq data. These findings suggest caution in interpreting the results of scRNAseq for low abundant transcripts such as the GLP-1R. Disclosure S.M. Gray: None. B.M. Chazotte: None. E.C. Ross: None. B. Svendsen: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.

Abstract A065: The Cancer Research Education Program (CREP): Training the next generation of cancer health disparities researchers through the Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD)

Abstract The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Louisiana State University Health Sciences Center (LSUHSC) and Moffitt Cancer Center (MCC) to advance translational research on cancer health disparities and to establish a Cancer Research Education Program (CREP). The CREP addresses a national priority to develop an educational training pipeline for one-on-one mentoring of undergraduates and medical students by a diverse group of LSUHSC and MCC faculty with unique expertise to conduct cancer health disparities research and outreach in underserved communities. The CREP supports 8-week internships providing: (1) hands-on summer research experiences; (2) a curriculum focusing on biobanking, precision medicine, and cancer health disparities; and (3) community outreach experiences in underserved communities. The curriculum includes web-based training modules, immersion experiences (e.g., biobank tour), professional development workshops, and learning activities (e.g., book and journal clubs). Data from the students' pre/post summative (impact/outcome) evaluations determine the acceptability and impact of these research and educational activities, students' knowledge, career aspirations, goal attainment, and their satisfaction based on nationally normed scales. Baseline and post-training data will be analyzed in August, at training completion, to assess program impact. Long-term yearly follow-up data will focus on the impact of CREP on student career trajectories. These data will help modify the CREP for years 2-4 of the SPIRIT-CHD. Seventy-five percent of the student participants in the first cohort were female. Students self-identified as Black/African American non-Hispanic (62.5%), White Hispanic (25%), and Asian non-Hispanic (12.5%). Student projects included genomic, immunologic, and cellular wet-lab research (analyzing proliferation of renal cell carcinoma, RNA sequencing and bioinformatics of Luminal B breast carcinomas, expression differences of polyamine enzymes in prostate cancer) and clinical studies (detection, prevention, and treatment of anal cancer in HIV-positive populations). Dry-lab projects focused on the analysis of Behavioral Risk Factor Surveillance System to assess policy-related trends in colorectal cancer screenings, studying the effectiveness of barbers as lay health educators for skin cancer prevention, and smoking cessation among Hispanics. CREP students also participated in cancer education outreach events to explain their projects to the communities at an elevated risk for certain types of cancer. This program has applicability to undergraduate and medical students nationwide on best practices for efficacious training in cancer health disparities, precision medicine, and biobanking. Ultimately, these efforts will enhance the diversity of the cancer research workforce, while contributing to the reduction of cancer health disparities. Citation Format: Fern Tsien, Paula Gregory, Gwendolyn Quinn, Vani N. Simmons, Z'Kera Sims, Megan E. Sutter, Ayesha Umrigar, Arnold H. Zea, Cathy Meade, Clement K. Gwede. The Cancer Research Education Program (CREP): Training the next generation of cancer health disparities researchers through the Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A065.

Hematopoietic maladaptation facilitates septic screening

Single-cell RNA sequencing of peripheral blood cells from patients with bacterial infections reveals a monocyte subset that can distinguish those at risk of sepsis.

Pharmacologic and genetic approaches define human pancreatic β cell mitogenic targets of DYRK1A inhibitors.

Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human β cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human β cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater β cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human β cell proliferation, and we provide clarity for future efforts in structure-based drug design for human β cell regenerative drugs.

Single cell and classic RNA sequencing – analysis and options for data integration

Single cell and classic RNA sequencing – analysis and options for data integration

Integrated Genomics and Functional Validation Identifies a Global Kinase Gene Signature Impacting Genome Stability in Myeloma

Identification of mechanisms underlying genomic instability is necessary to understand disease progression, including development of drug resistance. Our previous data demonstrates that dysregulation of DNA repair and maintenance/modification activities (including homologous recombination (HR), apurinic/apyrimidinic nuclease and APOBEC) significantly contribute to genomic instability in multiple myeloma (MM). However, how these and other pathways involved in genomic instability are dysregulated, remains to be explored. Since kinases play a critical role in the regulation of the maintenance of genomic integrity, we have performed a genome-wide kinome profiling to identify those involved in genomic instability in cancer. First, we analyzed genomic database for ten human cancers (including MM) from TCGA with both tumor cell gene expression and SNP/CGH array-based copy number information for each patient.We assessed genomic instability in each patient based on the total number of amplification and deletion events. We next interrogated all 550 kinases expressed in humans and identified those whose expression correlated with copy number alteration (based on FDR ≤ 0.05) in all tumor types. We identified six kinases whose elevated expression correlated with increased genomic instability defined by genomic amplification/deletion events in all ten cancers, including MM. To demonstrate functional relevance of these kinases, we conducted a CRISPR-based loss of function screen (using 3 guides per gene) in MM cells and evaluated the impact of each gene-knockout on micronuclei, a marker of ongoing genomic rearrangements and instability. For all six kinases, at least one guide resulted in ≥ 65% inhibition of micronuclei formation. Moreover, for five out of the six kinases, at least two guides showed ≥ 60% inhibition of micronuclei. All together, these data establishes a strong relevance of these kinases with genomic instability in MM. PDZ Binding Kinase (PBK) was among top kinases impacting genome stability in this data set with 2 out of 3 guides causing > 88% and 3rdguide causing 35% inhibition of micronuclei formation. We further report that inhibition of PBK, by knockdown or small molecule, inhibits DNA breaks, RAD51 recombinase expression and homologous recombination in MM cells. We further investigated molecular mechanisms involved in PBK-mediated genomic instability in MM. Expression profiling using RNA sequencing of MM cells treated with a specific PBK inhibitor showed that top ten pathways downregulated by treatment were mostly DNA repair/recombination followed by replication and G2/M checkpoint. Interestingly, we identified a notable overlap between PBK-regulated genes with FOXM1 target genes. FOXM1 is a major transcriptional regulator of genes involved in DNA repair, G2/M regulation and chromosomal stability. We, therefore, investigated PBK/FOXM1 interaction and show that PBK interacts with FOXM1 in MM cells. Moreover, the inhibition of PBK, by knockdown or small molecule, inhibits phosphorylation of FOXM1 as well as downregulates FOXM1-regulated HR and cell cycle genes RAD51, EXO1 and CDC25A. These results suggest that PBK-dependent phosphorylation of FOXM1 activity controls transcriptional networks involved in genomic instability in MM. Ongoing work is investigating role of PBK and other kinases in progression of MGUS/SMM to active MM and their impact on ongoing genomic changes with influence on multiple DNA repair pathways including HR. In conclusion, we describe a kinase panel that may have significant role in maintaining genome stability, and their perturbation may allow to improve genome stability in MM. Disclosures Munshi: Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Takeda: Consultancy.

Single-Cell RNA Sequencing of Healthy Human Marrow Hematopoietic Cells

Hematopoiesis, especially the early events of blood cell formation, has been mainly studied in bulk populations of cells and using progenitor colony formation assays; the familiar hierarchy of cell lineage differentiation and maturation, and associated regulatory factors have been inferred from these methods. However, these techniques often require extensive manipulation of cells, the exposure of cells to unphysiological conditions, aggregation of heterogeneous populations, and prior assumptions concerning cell function and gene expression. New single cell methodology avoids many of these potential experimental deficiencies. Here we have applied single-cell RNA-sequencing(scRNA-seq)to fresh human bone marrow CD34+cells: we profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from four healthy donors by deep sequencing of individual cell transcriptomes. An average of 4560 protein-coding genes were detected per cell. Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations (Fig 1A), based on expression of lineage-specific genes. Lin-CD34+CD38+cells emerged as locally clustered cell populations (Clusters 2-6, including MEP, GMP, ETP and ProB), while Lin-CD34+CD38-cells formed a single cluster (HSC/MLP). Reconstruction of differentiation trajectories by transcription in single cells revealed four committed lineages derived from stem cell compartment. The earliest fate split separates MEPs from MLPs, which partition further into lymphoid, and granulocyte-monocyte progenitors (Fig 1B). The overall pattern differs from the classical hematopoietic model describing a single binary split between myeloid and lymphoid differentiation immediately downstream of multipotent cells. However, our data align well to recently published scRNA-seq data showing sequential commitment of stem cells to the lymphoid, erythroid/megakaryocytic, and finally myeloid lineages (Setty M, Nat Biotechnol2019; Pellin D, Nat Commun2019). We further examined trends in gene expression in each of the branches and found dynamic expression changes underlying cell fate during early lineage differentiation (Fig 1C). As confirmation, PCA plot of published single-cell assay for transposase-accessible chromatin (scATAC-seq) shows similar differentiation pattern. After projecting scATAC-seq data to our transcriptomic clusters' specific genes, MEP-dependent and myeloid/lymphoid-dependent genes were located on opposing sides of the PC1 with same direction (Fig 1D), indicating transcriptome and epigenome work on differentiation in concerted effort. scRNA-seq provides opportunities for discovery and characterization at the molecular levels of early HSC differentiation and developmental intermediates, retrospectively, without the need to isolate purified populations. However, information inferred from scRNA-seq may be obscured due to missing reads and limited cell numbers. More cells would provide greater detail and higher resolution mapping.Given the low frequency of megakaryocyte progenitors within the CD34+cells as well as the neglected Lin-CD34-BM compartment, we could not fully resolve the separation and maturation of all lineages. Nonetheless, we found good coverage of cell types and a similar HSPC Atlas as other published studies (Velten L, Nat Cell Biol2017; Pellin D, Nat Commun2019)despite our limited numbers of starting cells. Our data accurately reflect the pattern of normal hematopoiesis, which may help to revise and refine characterization of hematopoiesis and provide a general reference framework to investigate the complexities of blood cell production at single-cell resolution - especially when cell numbers are limited, as from patient samples and in marrow failure syndromes. Fig. 1scRNA-seq of human hematopoietic stem and progenitor cells. (A) Unsupervised hierarchical clustering of gene expression data for all cells. C1, HSC/MLP; C2, MEP; C3, GMP; C4, ProB; C5-C6, ETP. (B)Visualization of the HSPC continuum. Each ball represents one cell.(C) Large-scale shifts in gene expression during development of hematopoietic cells.Bars on top indicate locations of individual cells, colored by stages of development, along this developmental trajectory. (D) Projections of five transcriptomic gene modules onto PCA of scATAC-seq data (Buenrostro JD,Cell 2018). Each dot represents a transcriptional factor. Figure 1 Disclosures No relevant conflicts of interest to declare.

Reduced Antigen Presentation May Contribute to Immunomodulatory Drug Resistance in Multiple Myeloma

Introduction: Maintenance therapy with the immunomodulatory drug (IMiD) lenalidomide improves progression-free survival (PFS) and overall survival among patients with multiple myeloma (MM). It has been suggested that IMiDs enhance immune-mediated anti-tumor responses through increased activation and proliferation of T cells and natural killer (NK) cells as well as inhibition of regulatory T cells. Despite the effectiveness of this therapy, nearly all patients relapse for reasons that remain uncharacterized. We sought to elucidate mechanisms of resistance to lenalidomide maintenance through investigation of mutations in cell-free DNA (cfDNA) and identification of changes in the innate and adaptive immune system by single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs). While MM is a cancer of plasma cells that primarily reside in the bone marrow, we focused on the peripheral blood which offers a comprehensive view of multiple sites of disease including extramedullary locations commonly found at the time of relapse. Methods: Twenty-four patients with MM and no minimal residual disease by 10-color flow cytometry and IGHVnext-generation sequencing (Invivoscribe) after induction therapy with or without an autologous stem cell transplant were selected for analysis. All patients were enrolled in NCT02538198 at Memorial Sloan Kettering Cancer Center. Peripheral blood was collected serially including timepoints prior to lenalidomide maintenance and after disease progression. Plasma and PBMCs were isolated by Ficoll-Paque density gradient centrifugation. cfDNA was extracted from plasma and targeted next-generation sequencing using a 70-gene panel of recurrently mutated genes in myeloma was performed using the QIAseq Targeted DNA kit (QIAGEN). DNA sequencing was also performed using the same gene panel on granulocytes to exclude germline variants. On PBMC samples, 5' gene expression and T and B cell receptor single-cell sequencing were performed using the Chromium Single Cell V(D)J kit (10X Genomics). Analysis of single-cell sequencing was performed on the top 100 principal components and visualized by Uniform Manifold Approximation and Projection (UMAP). A regression-based classifier was trained to automatically assign clustered cells into 26 immune cell phenotypes using lists of genes reported to be uniquely expressed by each cell type. Results: cfDNA sequencing was performed on 12 patients characterized as durable responders (PFS > 57 months) and 12 early progressors (median PFS 25 months). Prior to maintenance therapy, no significant difference was observed in the number of cfDNA mutations between these groups. The number of non-synonymous somatic mutations in cfDNA tended to increase over time and was significantly higher among patients taking lenalidomide for more than 2 years compared to patients at the time of relapsed disease (mean number of mutations was 7 in responders versus 3 in progressors).ZNF292was among the genes most significantly mutated in responders compared to progressors. ZNF292encodes a zinc finger protein involved in RNA binding and has previously been identified as a potential source of tumor-associated antigens in patients with monoclonal gammopathy of undetermined significance. To identify changes in immune cell phenotype and function that associate with disease progression, we performed single-cell RNA sequencing on PBMCs from the 12 progressors before maintenance and at the time of relapse. The median number of cells sequenced per PBMC sample was 3,132. Over 90% of cells could automatically be classified into a specific immune cell type by their gene expression. We observed the frequency of NK cells and plasmablasts were significantly increased at the time progression on lenalidomide while monocytes, dendritic cells, and basophils were decreased. Conclusion: Mechanisms of resistance among patients receiving treatment with IMiDs is not well known. Using targeted sequencing of cfDNA, we identify patients responding to maintenance tended to have a higher mutational load in cfDNA including genes previously reported to encode tumor-associated antigens. Additionally, we show that a decline in antigen-presenting dendritic cells is more common at the time of disease progression. Taken together, these results suggest reduced neoantigen production and a decline in antigen presentation may contribute to IMiD resistance. Disclosures Landgren: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Other: IDMC; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Green:Celgene: Consultancy; Cellectar Biosciences: Research Funding; Seattle Genetics: Research Funding; Juno Therapeutics: Consultancy, Patents & Royalties, Research Funding; GSK: Consultancy.

Abstract LB-325: Developmental hierarchy in Langerhans cell histiocytosis

Abstract Langerhans Cell Histiocytosis (LCH) is a neoplastic disease characterized by the accumulation of CD1a+CD207+ cells of unknown origin. Constitutive activation of the ERK signaling pathway is a common feature of LCH, but the pathogenesis of this disease remains unclear. Here we present a comprehensive analysis of the composition of LCH lesions that provides new insight into the development and molecular mechanisms of this disease. We used a multilayered approach employing immunohistochemistry, flow cytometry, single- cell RNA-sequencing, and ATAC- sequencing of LCH biopsies obtained from different patients. Based on the single- cell RNA expression data, we identified different LCH cell populations within LCH lesions that display distinct pathway signatures, including a stem-cell-like, proliferative state. Computational analysis discovered a hierarchy between these cells that suggest a directed developmental program in each tumor. Finally, functional chromatin mapping inferred distinct regulatory signatures discriminating the different cell states. Taken together, our study shows at single-cell resolution that there is considerable intra-tumoral heterogeneity in LCH and provides detailed insight into the molecular framework underlying tumor development. Our transcriptomic and epigenomic analyses have generated a reference map of subpopulations of LCH cells with distinct functional and phenotypic properties. We propose that the lesions are comprised of different LCH cell subsets that arise in an intrinsic developmental process. This work can serve as a template for the analysis of tumorigenesis beyond LCH. Citation Format: Florian Halbritter, Matthias Farlik, Christoph Bock, Caroline Hutter. Developmental hierarchy in Langerhans cell histiocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-325.

Abstract 4332: Targeting MYC overexpressing leukemia with cardiac glycoside proscillaridin through downregulation of histone acetyltransferases

Abstract Targeting MYC oncogene remains a major therapeutic goal in anticancer therapies. Here, we demonstrate that proscillaridin, a cardiac glycoside approved for heart failure treatment, causing Na+/K+ pump inhibition, targets efficiently MYC overexpressing cancer cells. At clinically relevant doses, proscillaridin induced rapid downregulation of MYC protein level, and produced growth inhibition preferentially against MYC overexpressing leukemic cell lines including lymphoid and myeloid stem cell populations. Whole transcriptome analysis with RNA sequencing of acute lymphoblastic leukemia cells showed a downregulation of gene sets involved in MYC pathways and cell replication, and an upregulation of genes involved in hematopoietic differentiation induced by proscillaridin treatment. Gene expression changes were associated with an epigenetic remodeling of chromatin active marks. Proscillaridin induced a significant loss of lysine acetylation in histone H3 (at lysine 9, 14, 18 and 27). In addition, mass spectrometry analysis revealed a loss of lysine acetylation in non-histone proteins such as MYC itself, MYC target proteins, and a series of histone acetylation regulators. Global loss of acetylation correlated with the rapid downregulation of histone acetyltransferase proteins (such as CBP, P300, TIP60 and GCN5) involved in histone and MYC acetylation. Overall, these results strongly support the repurposing of proscillaridin in MYC overexpressing leukemia and suggest a novel strategy to target MYC by inducing the downregulation of histone acetyltransferases involved in its stability. Citation Format: Elodie Marie Da Costa, Gregory Armaos, Gabrielle McInnes, Annie Beaudry, Gael Moquin-Beaudry, Virginie Bertrand-Lehouillier, Maxime Caron, Pascal St-Onge, Jeffrey R. Jonhson, Nevan Krogan, Yuka Sai, Michale Downey, Moutih Rafei, Meaghan Boileau, Kolja Eppert, Ema Florez-Diaz, Andre Haman, Trang Hoang, Daniel Sinnett, Christian Beausejour, Serge McGraw, Noel J. Raynal. Targeting MYC overexpressing leukemia with cardiac glycoside proscillaridin through downregulation of histone acetyltransferases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4332.

Abstract 4044: The immune phenotype in serial biopsies from metastatic TNBC undergoing chemo-immunotherapy

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis when metastatic. Immunotherapy, especially in combination with taxane chemotherapy has recently been shown to improve outcomes with PD-L1 as predictive biomarker for survival. To understand the mechanisms of response/resistance to chemo-immunotherapy for TNBC, we obtained serial core biopsies of TNBC patients (n=30) undergoing nab-paclitaxel and pembrolizumab treatment in a prospective trial at three time points, including baseline (before treatment), after 1 cycle (nab-paclitaxel treatment without immunotherapy), and after 2 cycles (nab-paclitaxel plus pembrolizumab treatment). fluorescence-activated cell sorter (FACS). We performed 10x genomics single-cell RNA sequencing (scRNA-seq) of immune cells isolated from these fresh, serial tumor specimens from two patients, a patient with subsequent RECIST response and a patient with rapid disease progression. Results were further validated by FACS and immunohistochemistry analyses. ScRNA-seq showed significant baseline heterogeneity as well as perturbations of the immune cell infiltrates with chemotherapy and chemo-immunotherapy. In the responder patient there was a significant expansion of T cells, including both CD4+ and CD8+ T cells. Before the pembrolizumab treatment, there are 1.84% of CD4+ T cells and 6.45% of CD8+ cells among total tumor infiltrating leukocytes (TILs). After the treatment, the percentage of CD4+ and CD8+ cells increased to 33.32% and 23.42% among TILs, respectively, as determined by FACS analysis. Cytotoxic T lymphocytes (CTLs) showed enrichments for transcripts of tissue-resident memory T cells (Trm). In contrast, tumors from a patient with rapid disease progression showed persistence of a myeloid compartment that suppress anti-tumor immunity. Although T cells were initially present, they mainly consisted of a naïve T cell phenotype and did not expand even after treatment. No Trm cells were detected within TILs by scRNAseq from this patient after pembrolizumab treatment. When comparing baseline samples from these two patients, we found there is a distinct tumor cell population in the non-responder with differentially expressed genes related to oxygen transport and proliferation. The association between this population and the response to chemo-immunotherapy remains to be explored. Our study provides proof of concept that single cell analyses of serial biopsies are a feasible tool to understand or even predict responses to chemo-immunotherapy for TNBC patients. Citation Format: Jiehui Deng, Suhagi Shah, Aatish Thennavan, Michelle Krogsgaard, Charles M. Perou, Kwok-Kin Wong, Sylvia Adams. The immune phenotype in serial biopsies from metastatic TNBC undergoing chemo-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4044.

Abstract 4995: Mechanism of action of anti-PD-L1 antibody in a PD-L1-negative and immune desert-like tumor model

Abstract Purpose: Intratumoral PD-L1 plays an important role in preventing T cell-mediated cancer cell killing. Anti-PD-1/PD-L1 antibodies generally show higher clinical benefit specifically in patients with high intratumoral PD-L1 expression. However, anti-PD-L1 antibody, atezolizumab, provide clinical benefit even in the subgroup of cancer patients where PD-L1 is low or undetectable on tumor cells and tumor-infiltrating immune cells (TC0/IC0). In this study, we investigated the mechanism of action by which PD-L1-negative tumors responded to an anti-PD-L1 therapy by using a syngeneic mouse tumor model. Experimental Design: We tested the antitumor effect of an anti-mouse PD-L1 monoclonal antibody (mAb) in vivo in murine models. Flow cytometry, tumor-stimulated IFNγ release assay, T cell repertoire analysis, RNA sequencing, protein immunoassays, and immunohistochemistry were performed on isolated tumors or on tumor-draining lymph nodes (dLN). Results: The anti-PD-L1 mAb showed significant antitumor activity in 6 of the 17 tumor models tested. Model FM3A, one of these 6 sensitive models, showed little intratumoral PD-L1 expression and was classified as an immune desert-like tumor model, but PD-L1 was found to be highly expressed on CD103+CD11c+ cells in dLN in this model. Some of the CD44+CD62L−CD8α+ effector T cells in dLN expressed PD-L1 receptors, i.e. they were PD-1- and/or B7-1-positive cells. Using lymphocytes from dLN, we found that tumor cell-stimulated IFNγ production was significantly increased even in the control group, and it was further significantly increased in the anti-PD-L1 mAb group compared to that in the control group. We found that anti-PD-L1 mAb treatment firstly enhanced T cell priming and increased CXCR3+ activated T cells in dLN. FM3A tumors expressed CXCR3 ligands regardless of anti-PD-L1 mAb treatment. CXCR3 blockade significantly prevented activated CD8α+ T cells from increasing in tumors as a result of anti-PD-L1 mAb treatment but not in dLN and significantly attenuated tumor growth inhibition of anti-PD-L1 mAb. Conclusions: We showed that an anti-PD-L1 mAb exerted antitumor activity in a TC0/IC0-like and immune desert-like tumor model through the blocking of PD-L1 in dLN. Tumor antigen presentation and T cell priming had already occurred in dLN at baseline prior to anti-PD-L1 mAb treatment in the FM3A model but PD-L1 on antigen-presenting cells prevented further T cell priming. We suggested that PD-L1 blockade in dLN appeared to trigger re-activation of T cells leading to antitumor activity. The results also suggested that the antitumor activity of anti-PD-L1 Ab requires both the enhancement of T cell priming by PD-L1 blockade as well as the trafficking of T cells to the tumor in response to the tumor via CXCR3 and its ligands system. Citation Format: Toshiki Iwai, Masamichi Sugimoto, Osamu Kondo. Mechanism of action of anti-PD-L1 antibody in a PD-L1-negative and immune desert-like tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4995.

Abstract 580: Detailed analysis of immune responses induced against patient-specific neoantigens using the ex-vivo induction protocol, NEO-STIM™

Abstract Background: Neoantigens are tumor-specific antigens that have been shown to be critical in the anti-tumor immune response. These antigens are not subject to central immune tolerance and are therefore potentially more immunogenic than tumor-associated antigens. The goal of our studies is to assess the immunogenic potential of high-quality neoantigen targets, as defined through our bioinformatic engine RECON®, as well as perform detailed characterization of the induced T cell responses towards these neoantigen targets. Methods: Patient-specific neoantigens were predicted using our bioinformatic engine RECON. The predicted high quality neoantigens were utilized in our proprietary ex-vivo stimulation protocol NEO-STIM to assess immunogenicity. NEO-STIM is used to prime, activate and expand memory and de novo T cell responses from both the CD4+ as well as the CD8+ compartment. In-depth analysis was performed to characterize the specificity, functionality (cytokine production and cytolytic capacity) and diversity of the induced T cell responses. Results: Here we present the successful induction of memory and de novo CD8+ T cell responses in PBMCs isolated by leukapheresis from three patients using NEO-STIM (2 with melanoma & 1 with NSCLC). We then deeply characterize the induced CD8+ T cell responses of one of the melanoma patients and show that these responses are functional, specific and have cytolytic capacity. Furthermore, we use paired single cell RNA and TCR sequencing to assess the diversity and gene expression profile of these responses. Conclusions: NEO-STIM is a novel platform to understand the immunogenic potential of high-quality neoantigen-targets in detail. Moreover, this platform can be utilized to generate T cell products for adoptive cell therapy for patients with a variety of solid and liquid tumors. Citation Format: Divya Lenkala, Jessica Kohler, Brian McCarthy, Michael Nelson, Rachel DeBarge, Yvonne Ware, Yuting Huang, Janani Sridar, Yusuf Nasrullah, Dewi Hartjano, Asaf Poran, Sejuti Sengupta, Joost H. van den Berg, Matt Goldstein, Richard B. Gaynor, Marit M. van Buuren. Detailed analysis of immune responses induced against patient-specific neoantigens using the ex-vivo induction protocol, NEO-STIM™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 580.

Abstract 2611: Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression

Abstract EVI1 (ecotropic viral integration site 1) is a transcription factor essential for the development and progression of acute myeloid leukemia (AML). Since high EVI1 expression, seen in approximately 10% of AML patients (EVI1high AML), is associated with poor clinical outcome, development of a novel therapeutic strategy is required. In this study, we investigated key molecules that are induced by aberrant EVI1 expression and modulate the metabolomics of leukemia cells. We conducted whole transcriptome analysis by RNA-sequencing in murine leukemia cells induced by retroviral transduction with EVI1 followed by transplantation into irradiated mice. We identified 45 genes with more than 10-fold expression in EVI1-transduced bone marrow cells compared to those with the mock vector at both pre-leukemic phase and leukemia phase. Intriguingly, the fructose bisphosphatase 1 gene (Fbp1), which encodes a key enzyme of gluconeogenesis was highly upregulated in EVI1-induced AML cells. We confirmed that FBP1 mRNA expression is quickly upregulated by ectopic expression of EVI1 in murine LSK (Linneg, c-Kitpos, Sca-1pos) cells. Moreover, we observed an enrichment of EVI1 in the promoter and enhancer region of Fbp1 by chromatin immunoprecipitation followed by qPCR analysis in murine hematopoietic cells, suggesting that Fbp1 expression is directly regulated by EVI1. Although high FBP1 expression suppresses glycolytic metabolism and negatively affects cellular proliferation in various types of solid tumor, its role in AML has not been investigated. In contrast to the results previously reported in other types of cancers, pharmacologic inhibition or short hairpin RNA (shRNA)-mediated knockdown of FBP1 significantly decreased colony-forming cell (CFC) capacity in EVI1-transduced murine LSK cells and significantly delayed progression of leukemia in the mice secondarily transplanted with EVI1-induced AML cells in vivo. These results suggest that aberrant expression of FBP1 is important for progression of EVI1high AML. While high FBP1 enzymatic activity negatively affects the glycolytic pathway, it in turn enhances the pentose phosphate pathway (PPP) through elevating the fructose-6-phosphate level. Activated PPP results in enhanced nucleotide synthesis essential for rapidly dividing cells. Importantly, individual knockdown of main PPP enzymes (G6pd, Pgd, Rpia) by shRNA transduction significantly decreased the CFC capacity of EVI1 transduced murine LSK cells, further confirming an important role of PPP in driving proliferation of EVI1high leukemia cells. Collectively, these results indicate that the activated PPP through transcriptional upregulation of FBP1 is crucial for progression of EVI1-driven leukemia. Since inhibition of FBP-1 did not compromise normal hematopoiesis, targeting the enzyme can be a promising therapeutic approach for the EVI1high AML. Citation Format: Hideaki Mizuno, Yuki Kagoya, Akira Chiba, Junji Koya, Yosuke Masamoto, Mineo Kurokawa. Activated pentose phosphate pathway mediated by FBP1 upregulation supports progression of acute myeloid leukemia with high EVI1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2611.

1711-P: Pancreas Single-Cell Patch-Seq Links Physiologic Dysfunction in Diabetes to Transcriptomic Phenotypes

Pancreatic islet-cells regulate glucose levels in the body through insulin and glucagon secretion; dysfunction of these cells leads to severe disease such as diabetes. Several single-cell transcriptome studies have shown heterogeneous gene expression in major islet cell-types such as beta and alpha cells. However, it has remained an open challenge to reconcile the heterogeneity in transcriptomic signatures with physiological observations of functional heterogeneity. Here we combined electrophysiological profiling and single-cell RNA sequencing of the same cells (pancreas patch-seq) to link transcriptomic phenotypes of islet cells to their physiologic properties. We collected 1,369 pancreas patch-seq cells from fresh and cryopreserved tissue and assessed function-gene expression networks in nondiabetic, type 1 diabetic (T1D) and type 2 diabetic (T2D) humans. We discovered that ‘hub’ genes and pathways drive functional heterogeneity in nondiabetic beta-cells and show that specific transcriptional programs correlate with physiologic dysfunction early in T2D. We have also found evidence that alpha-cell transcriptional plasticity acts as a driver of dysfunction in T1D. Disclosure J. Camunas Soler: None. X. Dai: None. Y. Hang: None. S. Kim: None. P. MacDonald: None. Funding Chan Zuckerberg Biohub; California Institute for Regenerative Medicine; National Institutes of Health