Cell RNA Sequencing Research Articles

Comparison of B-Cell Lupus and Lymphoma Using a Novel Immune Imbalance Transcriptomics Algorithm Reveals Potential Therapeutic Targets.

Systemic lupus erythematosus (lupus) and B-cell lymphoma (lymphoma) co-occur at higher-than-expected rates and primarily depend on B cells for their pathology. These observations implicate shared inflammation-related B cell molecular mechanisms as a potential cause of co-occurrence. We consequently implemented a novel Immune Imbalance Transcriptomics (IIT) algorithm and applied IIT to lupus, lymphoma, and healthy B cell RNA-sequencing (RNA-seq) data to find shared and contrasting mechanisms that are potential therapeutic targets. We observed 7143 significantly dysregulated genes in both lupus and lymphoma. Of those genes, we found 5137 to have a significant immune imbalance, defined as a significant dysregulation by both diseases, as analyzed by IIT. Gene Ontology (GO) term and pathway enrichment of the IIT genes yielded immune-related "Neutrophil Degranulation" and "Adaptive Immune System", which validates that the IIT algorithm isolates biologically relevant genes in immunity and inflammation. We found that 344 IIT gene products are known targets for established and/or repurposed drugs. Among our results, we found 48 known and 296 novel lupus targets, along with 151 known and 193 novel lymphoma targets. Known disease drug targets in our IIT results further validate that IIT isolates genes with disease-relevant mechanisms. We anticipate the IIT algorithm, together with the shared and contrasting gene mechanisms uncovered here, will contribute to the development of immune-related therapeutic options for lupus and lymphoma patients.

Targeting Unique Ligand Binding Domain Structural Features Downregulates DKK1 in Y537S ESR1 Mutant Breast Cancer Cells.

Resistance to endocrine therapies remains a major clinical hurdle in breast cancer. Mutations to estrogen receptor alpha (ERα) arise after continued therapeutic pressure. Next generation selective estrogen receptor modulators and degraders/downregulators (SERMs and SERDs) show clinical efficacy, but responses are often non-durable. A tyrosine to serine point mutation at position 537 in the ERα ligand binding domain (LBD) is among the most common and most pathogenic alteration in this setting. It enables endocrine therapy resistance by superceding intrinsic structural-energetic gatekeepers of ER hormone-dependence, it enhances metastatic burden by enabling neomorphic ER-dependent transcriptional programs, and it resists SERM and SERD inhibiton by reducing their binding affinities and abilities to antagonize transcriptional coregulator binding. However, a subset of SERMs and SERDs can achieve efficacy by adopting poses that force the mutation to engage in a new interaction that favors the therapeutic receptor antagonist conformation. We previously described a chemically unconventional SERM, T6I-29, that demonstrates significant anti-proliferative activities in Y537S ERα breast cancer cells. Here, we use a comprehensive suite of structural-biochemical, in vitro, and in vivo approaches to better T6I-29's activities in breast cancer cells harboring Y537S ERα. RNA sequencing in cells treated with T6I-29 reveals a neomorphic downregulation of DKK1, a secreted glycoprotein known to play oncogenic roles in other cancers. Importantly, we find that DKK1 is significantly enriched in ER+ breast cancer plasma compared to healthy controls. This study shows how new SERMs and SERDs can identify new therapeutic pathways in endocrine-resistant ER+ breast cancers.

Single-cell analysis defines highly specific leukemia-induced neutrophils and links MMP8 expression to recruitment of tumor associated neutrophils during FGFR1 driven leukemogenesis

BackgroundLeukemias driven by activated, chimeric FGFR1 kinases typically progress to AML which have poor prognosis. Mouse models of this syndrome allow detailed analysis of cellular and molecular changes occurring during leukemogenesis. We have used these models to determine the effects of leukemia development on the immune cell composition in the leukemia microenvironment during leukemia development and progression.MethodsSingle cell RNA sequencing (scRNA-Seq) was used to characterize leukemia associated neutrophils and define gene expression changes in these cells during leukemia progression.ResultsscRNA-Seq revealed six distinct subgroups of neutrophils based on their specific differential gene expression. In response to leukemia development, there is a dramatic increase in only two of the neutrophil subgroups. These two subgroups show specific gene expression signatures consistent with neutrophil precursors which give rise to immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Analysis of gene expression in these precursor cells identified pathways that were specifically upregulated, the most pronounced of which involved matrix metalloproteinases Mmp8 and Mmp9, during leukemia progression. Pharmacological inhibition of MMPs using Ilomastat preferentially restricted in vitro migration of neutrophils from leukemic mice and led to a significantly improved survival in vivo, accompanied by impaired PMN-MDSC recruitment. As a result, levels of T-cells were proportionally increased. In clinically annotated TCGA databases, MMP8 was shown to act as an independent indicator for poor prognosis and correlated with higher neutrophil infiltration and poor pan-cancer prognosis.ConclusionWe have defined specific leukemia responsive neutrophil subgroups based on their unique gene expression profile, which appear to be the precursors of neutrophils specifically associated with leukemia progression. An important event during development of these neutrophils is upregulation MMP genes which facilitated mobilization of these precursors from the BM in response to cancer progression, suggesting a possible therapeutic approach to suppress the development of immune tolerance.

Switch of innate to adaptative immune responses in the brain of patients with Alzheimer’s disease correlates with tauopathy progression

Neuroinflammation is a key feature of Alzheimer’s disease (AD). In this work, analysis of single- cell RNA-sequencing (scRNA-seq) data obtained from the brain of patients with AD provides evidence supporting a switch from an innate to an adaptative immune response during tauopathy progression, with both disease-associated microglia (DAM) and CD8+ T cells becoming more frequent at advanced Braak stages.

The heterogeneity and drug resistance of malignant cells and intercellular communication of microenvironment in osteosarcoma: Based on single‐cell analysis

AbstractBackgroundOsteosarcoma (OS) is a prevalent bone cancer characterized by its aggressive nature, which often metastasizes to the lungs and contributes to a poor prognosis. Treatment progress has been hindered by drug resistance, distant metastases, and significant patient heterogeneity. However, recent advancements in technologies such as sequencing and multi‐omics have enabled more in‐depth investigations at the micro level.AimBy undertaking a critical analysis of related studies, the present work intends to provide an overview of the microenvironmental features of osteosarcoma discovered through single‐cell RNA sequencing (scRNA‐seq) analysis. The articles included in this review were from several medical and health databases.Results and discussionWe also discuss drug resistance traits, taking into account the heterogeneity of OS cells within the tumor microenvironment (TME). This includes inherent and acquired resistance in both pre‐ and post‐chemotherapy osteosarcoma, as well as the close association between cancer stem cells (CSCs) and drug resistance. Additionally, we examine the intercellular communication between OS cells and other cell types, as revealed by both traditional and single‐cell studies, and suggest several promising therapeutic targets.ConclusionSingle‐cell RNA sequencing (scRNA‐seq) provides a detailed view of the tumor microenvironment, revealing intercellular communication between osteosarcoma cells and other cells, and aiding in the identification of potential therapeutic targets.

Noncoding RNAs: Novel Targets for Opioid Tolerance.

As a global health problem, chronic pain is one of the leading causes of disability, and it imposes a huge economic and public health burden on families and society. Opioids represent the cornerstone of analgesic drugs. However, opioid tolerance caused by long-term application of opioids is a major factor leading to drug withdrawal, serious side effects caused by dose increases, and even the death of patients, placing an increasing burden on individuals, medicine, and society. Despite efforts to develop methods to prevent and treat opioid tolerance, no effective treatment has yet been found. Therefore, understanding the mechanism underlying opioid tolerance is crucial for finding new prevention and treatment strategies. Noncoding RNAs (ncRNAs) are important parts of mammalian gene transcriptomes, and there are thousands of unique noncoding RNA sequences in cells. With the rapid development of high-throughput genome technology, research on ncRNAs has become a hot topic in biomedical research. In recent years, studies have shown that ncRNAs mediate physiological and pathological processes, including chromatin remodeling, transcription, posttranscriptional modification and signal transduction, which are key regulators of physiological processes in developmental and disease environments and have become biomarkers and potential therapeutic targets for various diseases. An increasing number of studies have found that ncRNAs are closely related to the development of opioid tolerance. In this review, we have summarized the evidence that ncRNAs play an important role in opioid tolerance and that ncRNAs may be novel targets for opioid tolerance.

DMP1 POSITIVE ASTROCYTES MAINTAIN BLOOD-BRAIN BARRIER VIA TRANSFERRING MITOCHONDRIA TO VASCULAR ENDOTHELIAL CELLS

Though dentin matrix protein 1 (Dmp1) is known to play critical role in mediating bone mineralization, it has also been validated to be expressed in brain and helps maintain blood brain barrier (BBB). Our study aims to clarify the expression pattern of Dmp1 in mouse brain and explore whether intercellular mitochondrial transfer occurs between Dmp1 positive astrocytes (DPAs) and endothelial cells, and thus acting as a mechanism in maintaining BBB during aging.Single cell RNA sequencing (scRNAseq) of 1 month, 6 month, and 20 month old mice brain (n=1, respectively) was employed to identify Dmp1 positive cell types. Dmp1Cre-mGmT and Dmp1Cre-COX8a fluorescent mice were generated to visualize DPAs and investigate their mitochondrial activities. A 3D noncontact coculture system and mitochondrial transplantation were applied to study the role of mitochondrial transfer between astrocytes and bEnd.3 endothelial cells. Dmp1Cre-Mfn2f/f mice were generated by depleting the ER-mitochondria tethering protein Mfn2 in DPAs.Dmp1 was mainly expressed in astrocytes at different ages. GO analysis revealed that cell projection and adhesion of DPAs were upregulated. Confocal imaging on Dmp1Cre-mGmT mice indicated that DPAs are a cluster of astrocytes that closely adhere to blood vessels (n=3). Bioinformatics analysis revealed that mitochondrial activity of DPAs were compromised during aging. Enriched scRNAseq of fluorescent cells from Dmp1Cre-COX8a mice (n=2) and immunofluorescent imaging (n=3) validated the acquisition of extrinsic mitochondria in endothelial cells. 3D coculture of astrocytes and bEnd.3 and direct mitochondrial transplantation revealed the rescue effect of mitochondrial transfer on damaged bEnd.3. BBB was impaired after depleting Mfn2 in DPAs, expressing a similar phenotype with aging brain.Astrocytes that express Dmp1 play a significant role in maintaining BBB via transferring mitochondria to vascular endothelial cells. Compromised mitochondrial transfer between DPAs and endothelial cells might be the potential mechanism of impaired BBB during aging.

Urinary single-cell sequencing captures kidney injury and repair processes in human acute kidney injury

Acute kidney injury (AKI) is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells. Mechanisms underlying AKI remain incompletely understood, specific therapies are lacking and monitoring the course of AKI in clinical routine is confined to measuring urine output and plasma levels of filtration markers. Here we demonstrate feasibility and potential of a novel approach to assess the cellular and molecular dynamics of AKI by establishing a robust urine-to-single cell RNA sequencing (scRNAseq) pipeline for excreted kidney cells via flow cytometry sorting. We analyzed 42,608 single cell transcriptomes of 40 urine samples from 32 patients with AKI and compared our data with reference material from human AKI post-mortem biopsies and published mouse data. We demonstrate that tubular epithelial cells transcriptomes mirror kidney pathology and reflect distinct injury and repair processes, including oxidative stress, inflammation, and tissue rearrangement. We also describe an AKI-specific abundant urinary excretion of adaptive progenitor-like cells. Thus, single cell transcriptomics of kidney cells excreted in urine provides noninvasive, unprecedented insight into cellular processes underlying AKI, thereby opening novel opportunities for target identification, AKI sub-categorization, and monitoring of natural disease course and interventions.

Abstract P1020: Differential Contribution Of Bone Marrow Cells To The Heart In Steady-state And After Repetitive Ischemia

Multiple studies have implicated the important role of migration of bone marrow (BM)-derived cells in heart after ischemia. Various BM-derived cells have been demonstrated to exert beneficial or detrimental effects in ischemia. But the response of BM to repetitive ischemia (RI) and the difference in contribution of this heterogenous population to the heart after RI remain elusive. Here, we applied an occluder on the left anterior descending coronary arteries of rats and they went under RI over a period of 10-17 days. This RI model induced coronary collateral growth and increased myocardial blood flow in the ischemic region after 10-17 days. We performed single- cell RNA sequencing and analysis of 24103 bone marrow transcriptomes isolated from the heart and bone marrow of rats in steady-state and after RI. Unsupervised clustering of cardiac neutrophils revealed 28 major clusters and 16 different cell types. Compared to steady state heart, proportion of BM derived macrophage mainly CD163 positive population was increased in the heart after RI. The proportion of BM derived natural killer cells was decreased markedly in the RI group. There was a slightly higher percentage of BM derived endothelial cells and lower smooth muscle cell and fibroblast in the RI group. While neutrophil degranulation, leukocyte activation, leukocyte migration are the major biological pathways in both groups, RI group showed activation of regeneration. We report heterogeneity of BM cells migrated to the heart during RI and redefine the BM that respond to heart ischemia and participate in induction of angiogenesis after RI.

Abstract 5330: Nuclear protein export inhibitor selinexor chemotherapy combination for pancreatic cancer therapy

Abstract Background: The majority of pancreatic ductal adenocarcinoma (PDAC) patients experience disease progression after Gemcitabine-nab-Paclitaxel (GemPac) treatment indicating the need for more effective combination therapies for this highly recalcitrant disease. Earlier we showed that nuclear exporter protein exportin 1 (XPO1) is a valid therapeutic target in PDAC and selective inhibitor of nuclear export (SINE) selinexor (Sel), synergistically enhances the efficacy of GemPac in pancreatic cancer cells, spheroids, patient derived tumors and showed evaluable response in PDAC patients in a Phase I study. Here we investigated the mechanisms of this synergy utilizing PDAC cellular models in vitro and LSL-Kras G12D/+; Trp53 fl/+; Pdx1-Cre (KPC) mouse model. Methods: We used single-nuclei RNA sequencing in KPC tumors, and poly A RNA sequencing in PDAC cells post sel-Gem-Pac treatment. Prioritized sn-seq and RNA-seq identified molecules were validated in in vitro or in the PDAC patient samples through siRNA mediated silencing, quantitative gene expression, cytotoxicity and related assays. Results: In KPC model, selinexor-GemPac caused statistically significant enhancement in survival compared to controls (p less than 0.05). Molecular analysis of residual KPC tumors showed re-organization of tumor stromal architecture, suppression of proliferation and nuclear retention of tumor suppressors. Histopathological analysis for Ki67 suggests comparatively lower rate of proliferation and enhanced nuclear accumulation of FOXO3a in the treated mice. Loosening of stroma and lowering of collagen fiber is also evident in treated mice from Picrosirius and Masson’s trichrome staining. Additionally, the KPC derived primary tumor cells showed reduced colony formation upon combination treatment. Sn-seq showed reduction in tumor cell heterogeneity (less cell clusters based on gene expression), CDK8, CD44 and Camk1d stem cell and EMT related genes alongside significant alterations in mitochondrial signaling after combination treatment. In cellular models, GO and GSEA analyses of RNA-seq data predicted DNA templated regulation of transcription and DNA replication associated genes in Sel or Sel-GemPac mediated growth inhibition. The top downregulated genes including drug resistant related gene PFN1 and HSP90 identified in Sel or Sel-GemPac treated PDAC cells from RNA-seq data were validated using RNAi and confirmed their role in synergy. Conclusions: Taken together Selinexor in combination with Gemcitabine-nab-Paclitaxel enhanced the survival of KPC mice through inhibition of well-connected tumor promoting molecules resulting in broad penetration in PDAC supporting signaling. A phase II study involving selinexor-gemcitabine combination in patients with metastatic PDAC is ongoing. Citation Format: Md. Hafiz Uddin, Sahar Bannoura, Yiwei Li, Husain Y. Khan, Amro Aboukameel, Mohammed N. Al-Hallak, Rafic Beydoun, Steve Kim, Yosef Landesman, Ramzi M. Mohammad, Anthony Shields, Asfar S. Azmi. Nuclear protein export inhibitor selinexor chemotherapy combination for pancreatic cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5330.

Abstract 3646: Distinct functions of pancreatic stellate cells in PDAC stromal evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in the United States with a five-year relative survival rate of 10.8%. After surgery, radiation and chemotherapy are the next lines of therapy. However, these treatments have not significantly improved clinical outcomes due in part to its unique and characteristic desmoplasia, which limits treatment efficacy. Cancer-associated fibroblasts (CAFs) have been shown to contribute in PDAC’s stromal reaction in part viasecretion of extracellular matrix (ECM) components and growth factors, suggesting that CAFs are pro-tumorigenic. This understanding led to several CAF ablation preclinical studies; surprisingly, these studies demonstrated tumor-suppressive potential for CAFs, highlighting the functional heterogeneity among CAFs in PDAC. This emphasizes the need for greater developmental understanding of this influential cell population. Lineage tracing via our recently established Fabp4-Cre;Rosa26mTmG mouse model revealed that pancreatic stellate cells (PSCs), thought to be the main cellular origin of PDAC CAFs, contribute to a minor faction of CAFs yet appear to have non-redundant functions to alternatively-derived CAFs. Here we delve further into the specific functions of PSC-derived CAFs via single-cell RNA sequencing of pancreatic stellate cells in healthy and tumor pancreas with our Fabp4-Cre;Rosa26mTmG mouse model. To our surprise, initial scRNA-seq analysis illustrated that quiescent pancreatic stellate cells from healthy tissue appear to be more heterogeneous to PSC-derived CAFs, with quiescent pancreatic stellate cells clustering into three “super clusters” while PSC-derived CAFs clusters into one “super cluster”. Additionally, clusters specific to normal pancreas tissue revealed potential transcripts-of-interest that are anti-tumorigenic. Current efforts seek to analyze stromal evolution with respect to CAF cell of origin in pre-malignant lesions and throughout stepwise tumorigenesis to metastasis. Furthermore, we are investigating the functional significance of potentially anti-tumorigenic factors lost from the PSC compartment upon differentiation to a CAF phenotype during PDAC progression. These findings shed light on the increasingly appreciated heterogeneity of PDAC CAFs, whilst suggesting that PSC-derived CAFs may be specifically targeted as a singular cell population for therapeutic treatment. Importantly, these findings help gain a greater understanding of PDAC disease progression and development in order to better serve patient care. Citation Format: M. Kathrina Casapao Onate, Matthew Schleisman, Zheng Xia, Mara H. Sherman. Distinct functions of pancreatic stellate cells in PDAC stromal evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3646.

Abstract 5982: Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC)

Abstract Remote oligometastases are the predominant roots of the unsatisfactory 5-year survival rate in esophageal squamous cell carcinoma (ESCC) patients. The title of metastatic-initiating cells (MICs) has been designated to describe tumor subpopulation with heterogeneous genetic background and behaviors that potentially displays stemness features and favors metastatic lesion formation. However, preceding studies have overpassed the early surviving MICs due to limitations in bulk transcriptomic sequencing at macrometastatic stage. With the aim of denoting and characterizing MICs in ESCC, we established the pulmonary metastatic mice model via intravenous injection and followed by multiplex single-cell RNA sequencing (scRNA-Seq) of tumor cells collected from lung tissues along longitudinal end-points. The scRNA-Seq results uncover a 7-gene surface marker signature panel that could depict the early surviving MICs of ESCC, including CD44, TACSTD2, TM4SF1, S100A14, RHOD, CST6, and C19orf33. In silico gene ontology (GO) enrichment of the early surviving MICs subpopulation reveals numerous migration and adhesion-related biological processes (p<0.05). In addition to scRNA-Seq, immunohistochemistry (IHC) staining of tumor cells in mice lungs collected in sequential order evidenced the survival heterogeneity of tumor subpopulations. Likewise, flow cytometry fluorescence-activated cell sorting (FACS) of signature-enriched tumor cells was performed to resemble the target MICs. CD44Pos ESCC cells are found to show distinctively greater wound-healing (p<0.01), foci formation (p<0.05), migration (p<0.01) and invasion (p<0.01) ability than CD44Neg cells in vitro, and longer survival patterns (p<0.05) upon implantation in vivo. Multi-color immunofluorescence (mIF) staining of these gene signatures is underway in mice models and clinical samples for comprehensive validation. We anticipate this surface marker signature to accelerate development for clinical diagnosis together with molecular targeting in metastatic ESCC patients. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Hong Yu Zhou, Kwong Yiu Lam, Xin Yuan Guan. Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5982.

Abstract 1561: Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation

Abstract Long non-coding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides lacking protein-coding potential, which are involved in diverse pathophysiologic conditions including cancer progression and metastasis. Metastasis is initiated by a cellular process, epithelial-to-mesenchymal transition (EMT), and many lncRNAs can be affected by and participate in the EMT process. To find out lncRNAs regulated by the EMT signals, we performed an NGS-based RNA sequencing in murine lung cancer cells, and finally selected lnc-NR2F1 that is upregulated by ZEB1, an EMT-inducing transcription factor. lnc-NR2F1 expression levels showed a positive correlation with ZEB1 mRNA levels and EMT scores in 13 mouse lung cancer cells or in lung adenocarcinoma data from TCGA. The migration and invasion of lung cancer cells were decreased after lnc-NR2F1 knockdown and increased after lnc-NR2F1 overexpression. lnc-NR2F1 was transcriptionally up-regulated by NR2F1, a transcription factor transcribed from the antisense strand. Interestingly, NR2F1 was transcriptionally induced by ZEB1, and promoted the migration and invasion of lung cancer cells. Through the analysis of public ChIRP-seq data, we found that lnc-NR2F1 can modulate the expression of TWIST2, and add-back of TWIST2 restored the migration and invasion of lung cancer cells suppressed by lnc-NR2F1 knockdown. In conclusion, lnc-NR2F1 is induced by a ZEB1-NR2F1 axis and stimulates the migration and invasion of lung cancer cells via transcriptional regulation of TWIST2. Citation Format: Eun Ju Kim, Young-Ho Ahn. Lnc-NR2F1 upregulated by a ZEB1/NR2F1 axis promotes the migration and invasion of lung cancer cells via TWIST2 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1561.

Abstract 976: Interrogating the pre-metastastic gene signature to block brain metastases

Abstract BACKGROUND: The incidence of brain metastases (BM) is tenfold higher than that of primary brain tumors. BM predominantly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis, posing a large unmet clinical need to identify novel therapies against BM. The goal of this work is to uncover the molecular factors that drive the formation of BM and investigate whether we can slow down and ultimately block BM formation. METHODS: The Singh lab has generated a large in-house biobank of patient-derived BM cell lines that are established from BM patient tumor samples. We use these BM cell lines to generate murine orthotopic xenograft models of BM and interrogate the biological processes that lead to BM. These models have successfully recapitulated all the stages of their respective BM cascades and additionally captured a “pre-metastatic” population of BM cells that have just seeded the brains of mice before forming mature, clinically detectable tumors. Pre-metastatic cell populations are impossible to detect in human patients but represent a therapeutic window wherein metastasizing cells can be targeted and eradicated before establishing clinically detectable and difficult to treat brain tumors. RESULTS: RNA sequencing of pre-metastatic BM cells revealed a unique deregulated transcriptomic profile that is specific to pre-metastatic cells despite the tumor of origin. Subsequent Connectivity Map analysis revealed compounds that we biologically characterized in vitro for selective anti-BMIC phenotypes. This effort led to a lead compound that exhibits anti-BM activity in vitro, while remaining ineffective against normal brain cell controls. Preliminary in vivo work has shown that following both orthotopic and intracardiac injection of BM cells, treatment with this lead compound reduces the tumor burden compared to mice being treated by a vehicle control, while providing a significant survival advantage. Ongoing mechanistic investigations aim to delineate the protein target of this compound in the context of the observed selective anti-BM phenotype. CONCLUSION: Identification of novel small molecules that target premetastatic BM cells could slow or prevent the formation of BM and dramatically improve the prognosis of at-risk cancer patients. Citation Format: Agata Kieliszek, Blessing Bassey-Archibong, Chitra Venugopal, Sheila Singh. Interrogating the pre-metastastic gene signature to block brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 976.

Abstract 915: C/EBPβis required for mouse mammary tumorigenesis

Abstract Breast tumorigenesis relies on complex interactions between tumor cells and their surrounding environment, orchestrated by tightly regulated transcriptional networks. While much is known about the signaling pathways affecting breast cancer cell growth and apoptosis, the transcriptional programs driving these responses are less understood. C/EBPβ is a transcription factor essential for proper cellular proliferation, differentiation, tissue function, metabolism, and the immune response, and is deregulated in breast cancer. C/EBPβ and its protein isoform LIP have long been postulated to promote breast cancer progression, however, the precise mechanisms and gene regulatory networks induced by C/EBPβ are less understood. Analysis of available human patient gene expression data reveals that increased Cebpb mRNA is associated with poor overall survival amongst all breast cancer subtypes, and analysis of available liquid chromatography with tandem mass spectrometry data shows that increased C/EBPβ total protein is associated with poor overall survival in triple-negative breast cancer patients. To test the function of C/EBPβ in tumor initiation and progression, several genetic mouse models were generated. Genetic ablation of tumor C/EBPβ in mouse models of basal-like [C3(1)TAg] and luminal (MMTV-Neu) breast cancers results in a complete block in mammary tumorigenesis. Interestingly, C/EBPβ-null tumor cells upregulate genes involved in antigen presentation and interferon gamma response in vitro, while overexpression of the C/EBPβ-LIP induces chemokines involved in immune cell recruitment and immunosuppression, such as CCL5. In support, RNA sequencing of human breast cancer cells shows that deletion of Cebpb in BT-20 cells causes de-repression of immune genes. Specifically, genes associated with interferon α and γ signaling (JAK1, TNFA, CCRL16, CSF2, IL32) as well as genes involved in antigen processing and presentation (MHC class I and class II molecules) are upregulated in the absence of Cebpb. These data suggest that C/EBPβ may repress genes important for immune visibility. Ongoing studies are aimed at identifying gene regulatory programs induced by C/EBPβ and specifically by C/EBPβ-LIP. These studies will have critical implications for whether C/EBPβ-induced pathways can be targeted in combination with other drugs, such as immune checkpoint inhibitors, improving patient outcome. These studies are supported by NIH RO1CA212518 (H.L.M.). Citation Format: Caitlin M. Burke, Michelle D. Rojo, Jeffery M. Rosen, Tobias Ackermann, Christine Muller, Cornelis F. Calkhoven, Heather L. Machado. C/EBPβis required for mouse mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 915.

Abstract 3048: CSI-Microbes: Identifying cell-type specific intracellular microbes from single-cell RNA-seq data

Abstract Despite recent reports of microbes living within tumor cells, the identification of intracellular microbes remains an open and important challenge. Here we introduce CSI-Microbes (computational identification of Cell type Specific Intracellular Microbes), a computational approach for the discovery of cell-type specific intracellular microbial taxa from single-cell RNA sequencing of host cells. It is first validated on two gold-standard datasets of human immune cells exposed to Salmonella. Next, CSI-Microbes is tested on Merkel cell and colorectal carcinoma where it identifies both reported and previously unknown tumor-specific intracellular microbes. Finally, CSI-Microbes is applied to analyze the intracellular microbiome of thirteen lung tumors where it identifies four tumors with bacterial taxa enriched in tumor cells and two tumors with bacterial taxa enriched in stroma or immune cells. Notably, the infected tumor cells down-regulate pathways associated with anti-microbial response and antigen processing and presentation, testifying to the functional significance of bacterial presence. Citation Format: Welles Robinson, Fiorella Schischlik, E. Michael Gertz, Joo S. Lee, Kaiyuan Zhu, S. Cenk Sahinalp, Rob Patro, Mark D. Leiserson, Alejandro A. Schaffer, Eytan Ruppin. CSI-Microbes: Identifying cell-type specific intracellular microbes from single-cell RNA-seq data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3048.

Abstract LB128: Oncolytic HSV-P10 and effects on metabolism as potential therapy for treatment of glioblastoma

Abstract Glioblastoma is an aggressive tumor of the central nervous system associated with a very poor prognosis even with surgery and chemoradiation. We have previously developed an oHSV that encodes for PTENα (HSV-P10) and stimulates anti-tumor immunity. This virus is currently licensed by and is being developed by Mesoblast to evaluate safety and efficacy in patients. To understand the effect of PTENα on tumor cell responses to oHSV therapy we performed RNA sequencing of primary GBM cells infected with control or oHSV-P10. We identified a dysregulation of glycolysis in cells infected with oHSV-P10 relative to control oHSV. Since cancer cells rely heavily on glycolysis for energy production (Warburg effect) the impact of PTENα on metabolic switch was significant. RNA sequencing PCR and western blot analysis showed a significant reduction in cellular Hexokinase II expression and downregulation of HIF-1α in HSV-P10 infected cells. Consistent with the function of HIF1 and hexokinase in driving glycolysis we also observed a significant reduction in glucose uptake and lactate release: implying a reduction in glycolysis in cells after being treated with oHSV-P10. Thus, cellular energetics of HSV-P10 infected cells was directed away from glycolysis relative to HSVQ infected cells. Consistent with this we observed increased, oxidative phosphorylation, production of reactive oxidative species and ATP production. Many cancers, including Glioblastoma are known to utilize the glutamine as a substrate for energy production through its entry into the TCA cycle. Blocking this shows a significant decrease in cell viability and ATP production when combined with HSV-P10. These results indicate that combination of HSV-P10 with radiation has a significant therapeutic potential and should be tested in patients for safety and efficacy. Citation Format: Matthew P. Mullarkey, Upasana Sahu, Bangxing Hong, Balveen Kaur. Oncolytic HSV-P10 and effects on metabolism as potential therapy for treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB128.

Abstract LB020: PTEN-L expressing HSV induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice

Abstract GBM, a WHO classified Grade IV glioma, is one of the most lethal and heterogeous primary brain tumors with inevitable recurrence, limiting the median survival time less than 21 months. Current standard-of-care treatment including surgical resection followed by chemo- and radio-therapy remains palliative because of therapy resistance, majorly conferred by GBM stem cells (GSCs), leading to tumor recurrence. With no current effective treatments, novel approaches to overcome GSCs-mediated resistance to chemotherapy and irradiation are urgently required in order to achieve long-term success in GBM therapy. Oncolytic viral (OV) therapy represents a novel and promising biological therapy for solid tumor that preferentially targets tumor cells for lytic destruction, sparing the healthy cells and in the process activating host anti-tumor immune response. Among all OVs, oncolytic Herpes Simplex Virus (oHSV) is substantially ahead in the clinic, with an oHSV T-VEC approved by the FDA for metastatic melanoma treatment. Recently, G47∆, another HSV1 virus, has been granted conditional approval for the treatment of GBM in Japan. Further, several other oHSVs including G207 and HSV1716 are currently being tested for safety and efficacy against GBM. This has fueled great expectations towards OVs as a promising alternative to conventional therapies. Our group has previously shown that PTENα expression by an oHSV (HSV-P10) resulted in further improved long-term survivors in intracranial tumor-bearing mice compared to HSVQ treatment. Here we aim to dissect the molecular mechanisms associated with improved therapeutic efficacy of HSV-P10 against GBM, and if HSV-P10 can overcome GBM cell radioresistance. The RNA sequencing and GSEA analyses of primary human GBM cells infected with control HSVQ or HSV-P10 reveals that while HSVQ virus infection leads to an increase in genes regulating IL6-STAT3 pathway, pivotal in maintaining stemness properties, HSV-P10 infection causes a reduction in the genes regulating this pathway. As a consequence, HSV-P10 reduces CD133+/CD44+ stem cell fraction, induces DNA damage and sensitizes the GBM cells to irradiation. Our findings reveal a novel mechanism induced by HSV-P10 in combination with irradiation whereby HSV-P10 modulates IL6-STAT3 signaling downregulating Sox2, a core transcription factor in the maintenance of GSCs, with a simultaneous decrease in Nestin expression and enhanced GFAP expression promoting GSC differentiation. HSV-P10 infection in combination with irradiation reduces GSC tumor sphere formation in vitro and sensitizes GBMs to radiotherapy in an intracranial mouse xenograft model. Collectively, our findings provide a potential avenue to overcome GSC-mediated therapy resistance to improve the therapeutic efficacy for GBM patients. Citation Format: Upasana Sahu, Matthew P. Mullarkey, Bangxing Hong, Balveen Kaur. PTEN-L expressing HSV induces glioma stem cell differentiation and sensitizes glioblastoma to radiation in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB020.

Abstract 5467: NTX301 targets platinum resistant ovarian cancer

Abstract Background: The majority of patients with ovarian cancer (OC) experience tumor relapse and develop chemoresistant tumors, which are fatal. Epigenetic alterations, especially DNA methylation, result in silencing of tumor suppressor and differentiation-associated genes, confer chemoresistance and are linked to stemness. Although our and others’ previous studies demonstrated that hypomethylating agents (HMA) could re-sensitize OC cells to chemotherapy, translation to the clinic has been slow due the poor stability and bioavailability of existing HMAs. Novel agents are critically needed. Methods: NTX 301 is a novel, highly potent and orally bioavailable HMA, in early clinical development, provided by PinotBio. Here, we assessed its anti-tumor effects in OC models by using cell proliferation, stemness assays, and RNA sequencing and validation. Results: OC cells (SKOV3, IC50=5.089nM; OVCAR4, IC50=29.68nM; and OVCAR5 IC50=3.664nM) were highly sensitive to NTX301 (p<0.05) compared to immortalized fallopian tube epithelial cells (FT-190) (IC50=103.3nM). Treatment with NTX301 induced more significant downregulation of the DNA methyltransferases (DNMTs) 1-3 expression in SKOV3 and OVCAR5 cells compared with decitabine (p<0.05). Treatment with low dose NTX301 (100nM) reset the transcriptome of OC cells, inducing about 15,000 differentially expressed genes (DEGs) compared to DMSO (P<0.05). Gene Ontology Enrichment analysis classified upregulated DEGs enriched in biological processes related to cellular response to DNA damage stimulus, DNA repair, positive regulation of cell cycle process; down-regulated DEGs enriched in processes of regulation of alcohol, cholesterol, and fatty acid biosynthesis and regulation of cell migration. GO analysis indicated NTX301 down-regulated molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase activity, known features of cancer stem cells (CSCs). Indeed, treatment with low dose NTX301 (100nM, 4 days) reduced ALDH(+) cell population, inhibited their self-renewal ability, and repressed the stemness-related transcription factor, Sox2 (P<0.05). We next tested the effects NTX301 in cisplatin (CDDP) resistant OC cells, developed by repeated treatments with CDDP. Priming with NTX301 reduced the IC50 (~two-fold) to CDDP in OVCAR5_CDDP (5.43μM vs. 9.79μM) and OVCAR4_CDDP (2.19μM vs. 9.29μM) resistant cells (p<0.05). NTX301 with CDDP induced phospho-γH2AX and cleaved caspase 3. To discover the mechanisms by which NTX301 restored chemosensitivity, we measured its effects on Schlafen-11 (SLFN11), a DNA damage response regulator silenced through methylation. SLFN11 expression was repressed in CDDP resistant compared to parental cells (P<0.05) and treatment with NTX301 induced its re-expression (P<0.05). Conclusions: Our data indicate that NTX301 is a potent HMA which targets ovarian CSCs and re-sensitizes OC cells to chemotherapy, supporting its further development in OC. Citation Format: Yinu Wang, Xiaolei Situ, Horacio Cardenas, Daniela Matei. NTX301 targets platinum resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5467.

Abstract LB558: Single cell profiling of PBMCs reveals correlates of clinical response to glofitamab

Abstract Glofitamab is a novel CD20-targeted bispecific T cell-engaging antibody that has demonstrated significant clinical efficacy in aggressive forms of Non-Hodgkin Lymphoma (aNHL). Despite significant clinical efficacy in aNHL (Hutchings et al. JCO 2021), some patients only achieve a partial response or progress upon treatment. To investigate the cellular and molecular factors that correlate with clinical activity of glofitamab, we applied CyTOF, single cell RNA and TCR sequencing to study PBMCs collected at baseline and at cycle 2 of treatment from patients with aNHL. Our cohort included 9 patients with complete response (CR) and 9 patients with progressive disease (PD) status (response assessed at cycle 3) recruited during dose escalation of glofitamab in a Ph1 clinical trial (NP30179). We identified a higher proportion of CD4 T cells and a lower proportion of monocytes in CR versus PD patients. Complementary to changes in cell proportions, we explored the covariation of genes across single cells to isolate co-regulated processes and identified several gene expression programs associated with the response status, of which some were shared between two or more cell types while others were cell type-specific. Focusing on the T cell compartment, we further identified subtypes of CD8 and CD4 T cells enriched in either CR or PD patients, and used topic modeling, a computational approach that excels in settings of continuous phenotypes, to contrast the transcriptional state of T cells in CR patients compared to PD patients at baseline and on treatment. Interestingly, we found CR patients to exhibit a less exhausted CD8 T cell population at baseline, confirming our previous observations using tumor bulk RNA sequencing data. Moreover, we observed that hyper-expanded clonotypes were generally enriched in the CD8 T cell compartment, and that the clonal cells had a stronger cytotoxic phenotype compared to non-clonal T cells. This observation was further corroborated when looking at TCR diversity. Indeed, we observed that distinct T cell populations exhibited differences in TCR diversity, with exhausted CD8 T cells showing decreased clonal diversity. Finally, to better understand the changes in CD8 T cells, we performed trajectory inference and observed that the position of individual cells along the pseudotime varied largely according to timepoint and response status. This analysis further allowed us to identify the expression dynamic of numerous markers of T cell exhaustion, as well as transcription factors associated with progenitor and self-renewing state. Notwithstanding the limited sample size and patient heterogeneity, our analysis provides a deep characterization of the cellular and molecular features associated with response to glofitamab using peripheral biomarkers. As glofitamab is being evaluated in a number of clinical trials either as a single agent or in combination, our dataset and findings can help broaden the understanding of its mode of action, and might be relevant for patient enrichment and monitoring response. Citation Format: Sina Nassiri, Lucas Habegger, Petra Gerber, Vinko Tosevski, Tamara Hüsser, Emilio Yanguez, Sylvia Herter, Martin Weisser, Koorosh Korfi, Pablo Umana, Emily Piccione, Ann-Marie Bröske, Marina Bacac. Single cell profiling of PBMCs reveals correlates of clinical response to glofitamab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB558.