Global Differential Gene Expression Research Articles

Transcriptomics and starch biosynthesis analysis in leaves and developing seeds of mung bean provide a basis for genetic engineering of starch composition and seed quality.

Mung bean starch is distinguished by its exceptional high amylose content and regulation of starch biosynthesis in leaves and storage tissues, such as seeds, share considerable similarities. Genetic engineering of starch composition and content, requires detailed knowledge of starch biosynthetic gene expression and enzymatic regulation. In this study we applied detailed transcriptomic analyses to unravel the global differential gene expression patterns in mung bean leaves and in seeds during various stages of development. The objective was to identify candidate genes and regulatory mechanisms that may enable generation of desirable seed qualities through the use of genetic engineering. Notable differences in gene expression, in particular low expression of the Protein Targeting to Starch (PTST), starch synthase (SS) 3, and starch branching enzyme1 (SBE1) encoding genes in developing seeds as compared to leaves were evident. These differences were related to starch molecular structures and granule morphologies. Specifically, the starch molecular size distribution at different stages of seed development correlated with the starch biosynthesis gene expression of the SBE1, SS1, granule-bound starch synthases (GBSS) and isoamylase 1 (ISA1) encoding genes. Furthermore, putative hormonal and redox controlled regulation were observed, which may be explained by abscisic acid (ABA) and indole-3-acetic acid (IAA) induced signal transduction, and redox regulation of ferredoxins and thioredoxins, respectively. The morphology of starch granules in leaves and developing seeds were clearly distinguishable and could be correlated to differential expression of SS1. Here, we present a first comprehensive transcriptomic dataset of developing mung bean seeds, and combined these findings may enable generation of genetic engineering strategies of for example starch biosynthetic genes for increasing starch levels in seeds and constitute a valuable toolkit for improving mung bean seed quality.

Abstract 6842: The effect of intra-tumoral Candida albicans on host gene expression

Abstract The gut and tumor microbiome have been shown to affect tumorigenesis, tumor immune infiltration, and response to traditional cancer therapeutics such as radiation therapy. Further, specific microbe-cancer relationships have been established. One such microbe is Candida albicans, which has been identified within several gastrointestinal cancers and is associated with an increased risk of metastases and decreased overall survival. By completing RNA-sequencing on murine rectal tumors colonized with C. albicans, we can better understand whether intratumoral changes in gene expression exist based on colonization of specific microbes. This study utilizes gut-to-tumor translocation and whole transcriptome analysis for a discovery-based approach to identify the effects of C. albicans on the host tumor. We established a rectal tumor model with C57BL/6 mice using MC38 rectal cancer cells subcutaneously injected on the right flank, local radiation to the tumor (standard-of-care for rectal cancer), and gavage with either C. albicans (Ca) or PBS. Fungal presence within tumors was validated by staining with calcofluor white and subsequently, tumors were sent for bulk RNA-sequencing to determine differences in gene expression using DeSeq2 and a gene set enrichment analysis (GSEA). In unsupervised analysis of gene expression differences between PBS-gavaged and Ca-gavaged with bulk RNA sequencing, tumors gavaged with C. albicans possessed unique gene expression and pathway analysis when compared to PBS-gavaged mice. Global differential gene expression showed 1,947 genes upregulated in Ca gavage and 593 genes upregulated in the PBS gavage (p < 0.05). C. albicans colonized tumors showed decreased expression in gene pathways relating to immune activation (interferon alpha response, IL-6 and STAT3 signaling), p53 expression, DNA repair, oxidative phosphorylation, NOTCH signaling, and apoptosis. We subsequently used a network-based approach to identify the most important genes driving the differences in the tumors, calculating the degree, closeness, and betweenness centralities. Several microbes have been regarded as potential contributors to decreased treatment response and survival in rectal cancer. In the case of tumors colonized with C. albicans, bulk RNA-sequencing indicates down regulation of gene pathways related to cell proliferation, immune activation, and DNA maintenance. Down regulation of gene pathways related to cell cycle control and arrest suggests that intratumoral colonization of C. albicans may causally affect tumorigenesis. Decreases in immune signaling in tumors suggest a potentially causal role of C. albicans immune suppression. These results highlight initial steps to understand how intratumoral C. albicans may be affecting rectal cancer progression and treatment response and serve as a technique to allow better insight to individual microbial impact on tumors. Citation Format: Alexander Loncar, Dennis Grencewicz, Rebecca Hoyd, Aaditya Pallerla, Nyelia Williams, Martin Benej, Yogita Mehra, Shiva Jahanbakhshi, Matthew Anderson, Nicholas Denko, Daniel Spakowicz. The effect of intra-tumoral Candida albicans on host gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6842.

MYB alternative promoter activity is increased in adenoid cystic carcinoma metastases and is associated with a specific gene expression signature

ObjectiveAdenoid cystic carcinoma (ACC) is a head and neck cancer with a poor long-term prognosis that shows frequent local recurrences and distant metastases. The tumors are characterized by MYB oncogene activation and are notoriously unresponsive to systemic therapies. The biological underpinnings behind therapy resistance of disseminated ACC are largely unknown. Here, we have studied the molecular and clinical significance of MYB alternative promoter (TSS2) usage in ACC metastases. Materials and methodsMYB TSS2 activity was investigated in primary tumors and metastases from 26 ACC patients using RNA-sequencing and quantitative real-time PCR analysis. Differences in global gene expression between MYB TSS2 high and low cases were studied, and pathway analyses were performed. ResultsMYB TSS2 activity was significantly higher in ACC metastases than in primary tumors (median activity 15.1 vs 3.0, P = 0.0003). MYB TSS2 high ACC metastases showed a specific gene expression signature, including increased expression of multi-drug resistance genes and canonical MYB target genes, and suppression of the p53 and NOTCH pathways. ConclusionsCollectively, our findings indicate that elevated MYB TSS2 activity is associated with metastases, potential drug resistance, and augmented MYB-driven gene expression in ACC. Our study advocates the need for new therapies that specifically target MYB and drug resistance mechanisms in disseminated ACC.

Gene expression bias between the subgenomes of allopolyploid hybrids is an emergent property of the kinetics of expression.

Hybridization coupled to polyploidy, or allopolyploidy, has dramatically shaped the evolution of flowering plants, teleost fishes, and other lineages. Studies of recently formed allopolyploid plants have shown that the two subgenomes that merged to form that new allopolyploid do not generally express their genes equally. Instead, one of the two subgenomes expresses its paralogs more highly on average. Meanwhile, older allopolyploidy events tend to show biases in duplicate losses, with one of the two subgenomes retaining more genes than the other. Since reduced expression is a pathway to duplicate loss, understanding the origins of expression biases may help explain the origins of biased losses. Because we expect gene expression levels to experience stabilizing selection, our conceptual frameworks for how allopolyploid organisms form tend to assume that the new allopolyploid will show balanced expression between its subgenomes. It is then necessary to invoke phenomena such as differences in the suppression of repetitive elements to explain the observed expression imbalances. Here we show that, even for phenotypically identical diploid progenitors, the inherent kinetics of gene expression give rise to biases between the expression levels of the progenitor genes in the hybrid. Some of these biases are expected to be gene-specific and not give rise to global differences in progenitor gene expression. However, particularly in the case of allopolyploids formed from progenitors with different genome sizes, global expression biases favoring one subgenome are expected immediately on formation. Hence, expression biases are arguably the expectation upon allopolyploid formation rather than a phenomenon needing explanation. In the future, a deeper understanding of the kinetics of allopolyploidy may allow us to better understand both biases in duplicate losses and hybrid vigor.

Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae.

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50g/kg) inclusion diet, or a high-frass (300g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1β type a, IL-1β type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Pooled microarray expression analysis of failing left ventricles reveals extensive cellular-level dysregulation independent of age and sex

Existing cardiovascular studies tend to suffer from small sample sizes and unaddressed confounders. Re-profiling of 9 microarray datasets revealed significant global gene expression differences between 358 failing and 191 non-failing left ventricles independent of age and sex (p = 5.1e-10). Covariate-adjusted mixed-effect regression revealed 17 % (945/5553) genes with >1.5-fold changes. The extracellular matrix and integral membrane ontologies were significantly enriched and depleted in failing ventricles, respectively. Furthermore, MTSS1 implicated in cardiovascular dysfunction showed the greatest change in ischemic compared to dilated cardiomyopathy (Bonferroni p < 0.05 for all genes and ontologies). Transcriptomic meta-profiling here provided deeper insight into heart failure at the cellular level.

Cancer predisposition signaling in Beckwith-Wiedemann Syndrome drives Wilms tumor development.

Wilms tumor (WT) exhibits structural and epigenetic changes at chromosome 11p15, which also cause Beckwith-Wiedemann Syndrome (BWS). Children diagnosed with BWS have increased risk for WT. The aim of this study is to identify the molecular signaling signatures in BWS driving these tumors. We performed whole exome sequencing, methylation array analysis, and gene expression analysis on BWS-WT samples. Our data were compared to publicly available nonBWS data. We categorized WT from BWS and nonBWS patients by assessment of 11p15 methylation status and defined 5 groups- control kidney, BWS-nontumor kidney, BWS-WT, normal-11p15 nonBWS-WT, altered-11p15 nonBWS-WT. BWS-WT samples showed single nucleotide variants in BCORL1, ASXL1, ATM and AXL but absence of recurrent gene mutations associated with sporadic WT. We defined a narrow methylation range stratifying nonBWS-WT samples. BWS-WT and altered-11p15 nonBWS-WT showed enrichment of common and unique molecular signatures based on global differential methylation and gene expression analysis. CTNNB1 overexpression and broad range of interactions were seen in the BWS-WT interactome study. While WT predisposition in BWS is well-established, as are 11p15 alterations in nonBWS-WT, this study focused on stratifying tumor genomics by 11p15 status. Further investigation of our findings may identify novel therapeutic targets in WT oncogenesis.

Divergent responses in desiccation experiments in two ecophysiologically different Zygnematophyceae.

Water scarcity can be considered a major stressor on land, with desiccation being its most extreme form. Land plants have found two different solutions to this challenge: avoidance and tolerance. The closest algal relatives to land plants, the Zygnematophyceae, use the latter, and how this is realized is of great interest for our understanding of the conquest of land. Here, we worked with two representatives of the Zygnematophyceae, Zygnema circumcarinatum SAG 698-1b and Mesotaenium endlicherianum SAG 12.97, who differ in habitats and drought resilience. We challenged both algal species with severe desiccation in a laboratory setup until photosynthesis ceased, followed by a recovery period. We assessed their morphological, photophysiological, and transcriptomic responses. Our data pinpoint global differential gene expression patterns that speak of conserved responses, from calcium-mediated signaling to the adjustment of plastid biology, cell envelopes, and amino acid pathways, between Zygnematophyceae and land plants despite their strong ecophysiological divergence. The main difference between the two species appears to rest in a readjustment of the photobiology of Zygnema, while Mesotaenium experiences stress beyond a tipping point.

Abstract 033: Innate Lymphoid Cells Mediate Hypertension In T Cell Deficient Hosts

T cells have been shown to contribute to hypertension, however, hypertension occurs in settings of T cell deficiency, such as human immunodeficiency virus infection and following chemotherapy. We studied two colonies of RAG-1 -/- mice with disparate responses to angiotensin II (Ang II) infusion. Ang II increased both systolic and diastolic pressure in RAG-1 -/- mice from Jackson labs (RAG-1 -/-US ) but had no effect in a colony of RAG-1 -/- mice housed at our Pozzilli facility (RAG-1 -/-POZ ) for the past 14 years (SBPs 169 ± 2 vs 114 ± 3 mmHg, p &lt; 0.003). While both lacked T and B cells, the RAG-1 -/-US mice exhibited a striking expansion of natural killer and both type 1 and type 3 innate lymphoid cells, while the RAG-1 -/-POZ mice did not. In RAG-1 -/-US mice, NK cells and ILC1s produced IFNγ, while ILC3s produced IL-17A. Immunoclearing of IL-17A, but not IFNγ prevented the hypertension in RAG-1 -/-US mice. There were differences in global gene expression and DNA methylation status in NK1.1 + /NKp46 + cells between these two colonies and notably β2-AdR and IL-10 expression was significantly lower in RAG-1 -/-US mice. Knockdown of the β2-AdR using intra-splenic infusion of siRNA in RAG-1 -/-POZ mice led to a hypertensive response resembling that observed in wild type and RAG-1 -/-US mice. We also found striking differences in DNA methylation patterns in innate lymphoid cells of RAG-1 -/-US vs RAG-1 -/-POZ mice and treatment with the DNA methyltransferase inhibitor 5-azacytidine prevented hypertension in RAG-1 -/-US mice. Single cell sequencing showed that 5-azacytidine also reduced splenic populations of NK and ILC3 cells and reduced the presence of the transcription factor RORγt, which defines ILC3s. Ang II infusion caused only a transient elevation of blood pressure in NSG/MHCI/II -/- mice that lack T cells and innate lymphoid cells but led to sustained hypertension following adoptive transfer of either T cells or NK1.1 + /NKp46 + cells to these animals. We conclude that DNA methylation likely modulates expansion of NK and innate lymphoid cells. Either T cells or innate lymphoid cells can contribute to hypertension, and the latter can compensate by serving as a source of cytokines like IL-17A when T cells are deficient.

Methotrexate Provokes Disparate Folate Metabolism Gene Expression and Alternative Splicing in Ex Vivo Monocytes and GM-CSF- and M-CSF-Polarized Macrophages.

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six-eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six-ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy.

Transcriptome analysis of the ink sac and brain tissues from Sepiella inermis: A resource for discovering genes related to the inking of cephalopods.

The common Chinese cuttlefish (Sepiella inermis) is an important cephalopod with nutritional and commercial value. Intensive inking stimulated by swilling seawater in transfer containers threatens the survival of cephalopods during transportation. However, the molecular basis for the inking behavior of S. inermis remains unclear. In the present study, transcriptome analysis was performed on ink sac and brain tissues from S. inermis under two different conditions, i.e. the control group (with individuals immersed in static seawater) and the experimental group (with individuals immersed in swilling seawater) to determine the global gene expression differences. The individuals from the experimental group ejected ink in response to the swilling of seawater. 330,699 unigenes were obtained from twelve transcriptome libraries via the Illumina Hiseq X platform, and the differentially expressed genes in the ink sac and brain tissues were identified respectively. Multiple upregulated genes in the ink sac were involved in cation transporter activity. Besides, an autocrine/paracrine factor wnt10b like and two important transcription factors (homeobox 1 and Hes-1-b-like) were also significantly upregulated in the ink sac. Moreover, a neuronal nitric oxide synthase (nNOS) was significantly downregulated in the brain. The findings from this study provide an important transcriptomic resource for discovering critical genes related to inking behavior of S. inermis, providing a basis for developing potential methods for protecting S. inermis from intensive inking.

Identifying mechanisms of acquired immune escape from sequential, paired biopsies.

2519 Background: Resistance to immune checkpoint blockade (ICB) can manifest as disease progression either at the initiation of treatment (primary resistance) or after some initial response (acquired resistance). To better understand the mechanisms underlying acquired resistance, the imCORE Network is conducting a study (NCT03333655) to examine changes in tumor biology from pre-treatment to disease progression across cancer types. Methods: Eligible patients included those experiencing clinical benefit on ICB (defined as objective response or stable disease longer than 6 months) and had evaluable tissue samples from both pre-treatment and within 30 days of progression. Samples were subjected to whole exome sequencing (WES), RNA sequencing (RNA-Seq), and immunohistochemistry (IHC). Whole blood samples or adjacent normal tissue were used as a reference for tumor variant calling. Results: As of December 3rd, 2021, 24 enrolled patients have complete sample pairs. Of those, melanoma, bladder, and lung were most common (n = 7, 6 and 5 pairs, respectively), but our cohort also included patients with breast cancer, squamous head &amp; neck, and renal cell carcinoma. IHC data unexpectedly showed a modest, but consistent increase in tumor infiltrating CD8+ T cells at progression. In addition, IHC evidence of a decrease in MHC-I proteins (HLA-A and B2M) suggest that in 5 out of 24 cases, key proteins needed for antigen presentation are lost. Global differential gene expression analysis showed that immune gene expression was significantly increased at progression, including numerous chemokines and significant enrichment in gene sets responsible for antigen presentation machinery. Protein-altering mutations at progression appeared in B2M in one patient, and CXCL9 in another. However, in most cases it is unclear what genetic alterations are responsible. We do not observe evidence of consistent IFNγ and Jak/stat signaling loss or antigen presentation loss. Conclusions: While ICB resistance is thought to be associated with a lack of immune response within the TME, we found that acquired resistance is usually associated with either maintenance or an increase in immune infiltration. Multiple alterations that are unique to individual patients also continue to emerge. Our data shows ICB resistance is multifactorial and associated with dynamic changes to markers amid immune activation and inhibition. Clinical trial information: NCT03333655.

Distinct early life stage gene expression effects of hybridization among European and North American farmed and wild Atlantic salmon populations.

Due to multigeneration domestication selection, farmed and wild Atlantic salmon diverge genetically, which raises concerns about potential genetic interactions among escaped farmed and wild populations and disruption of local adaptation through introgression. When farmed strains of distant geographic origin are used, it is unknown whether the genetic consequences posed by escaped farmed fish will be greater than if more locally derived strains are used. Quantifying gene transcript expression differences among divergent farmed, wild and F1 hybrids under controlled conditions is one of the ways to explore the consequences of hybridization. We compared the transcriptomes of fry at the end of yolk sac absorption of a European (EO) farmed ("StofnFiskur", Norwegian strain), a North American (NA) farmed (Saint John River, NB strain), a Newfoundland (NF) wild population with EO ancestry, and related F1 hybrids using 44K microarrays. Our findings indicate that the wild population showed greater transcriptome differences from the EO farmed strain than that of the NA farmed strain. We also found the largest differences in global gene expression between the two farmed strains. We detected the fewest differentially expressed transcripts between F1 hybrids and domesticated/wild maternal strains. We also found that the differentially expressed genes between cross types over-represented GO terms associated with metabolism, development, growth, immune response, and redox homeostasis processes. These findings suggest that the interbreeding of escaped EO/NA farmed and NF wild population would alter gene transcription, and the consequences of hybridization would be greater from escaped EO farmed than NA farmed salmon, resulting in potential effects on the wild populations.

Submergence of the filamentous Zygnematophyceae Mougeotia induces differential gene expression patterns associated with core metabolism and photosynthesis

The streptophyte algal class Zygnematophyceae is the closest algal sister lineage to land plants. In nature, Zygnematophyceae can grow in both terrestrial and freshwater habitats and how they do this is an important unanswered question. Here, we studied what happens to the zygnematophyceaen alga Mougeotia sp., which usually occurs in permanent and temporary freshwater bodies, when it is shifted to liquid growth conditions after growth on a solid substrate. Using global differential gene expression profiling, we identified changes in the core metabolism of the organism interlinked with photosynthesis; the latter went hand in hand with measurable impact on the photophysiology as assessed via pulse amplitude modulation (PAM) fluorometry. Our data reveal a pronounced change in the overall physiology of the alga after submergence and pinpoint candidate genes that play a role. These results provide insight into the importance of photophysiological readjustment when filamentous Zygnematophyceae transition between terrestrial and aquatic habitats.

Effects of Training Status and Exercise Mode on Global Gene Expression in Skeletal Muscle.

The aim of this study was to investigate differences in skeletal muscle gene expression of highly trained endurance and strength athletes in comparison to untrained individuals at rest and in response to either an acute bout of endurance or strength exercise. Endurance (ET, n = 8, VO2max 67 ± 9 mL/kg/min) and strength athletes (ST, n = 8, 5.8 ± 3.0 training years) as well as untrained controls (E-UT and S-UT, each n = 8) performed an acute endurance or strength exercise test. One day before testing (Pre), 30 min (30′Post) and 3 h (180′Post) afterwards, a skeletal muscle biopsy was obtained from the m. vastus lateralis. Skeletal muscle mRNA was isolated and analyzed by Affymetrix-microarray technology. Pathway analyses were performed to evaluate the effects of training status (trained vs. untrained) and exercise mode-specific (ET vs. ST) transcriptional responses. Differences in global skeletal muscle gene expression between trained and untrained were smaller compared to differences in exercise mode. Maximum differences between ET and ST were found between Pre and 180′Post. Pathway analyses showed increased expression of exercise-related genes, such as nuclear transcription factors (NR4A family), metabolism and vascularization (PGC1-α and VEGF-A), and muscle growth/structure (myostatin, IRS1/2 and HIF1-α. The most upregulated genes in response to acute endurance or strength exercise were the NR4A genes (NR4A1, NR4A2, NR4A3). The mode of acute exercise had a significant effect on transcriptional regulation Pre vs. 180′Post. In contrast, the effect of training status on human skeletal muscle gene expression profiles was negligible compared to strength or endurance specialization. The highest variability in gene expression, especially for the NR4A-family, was observed in trained individuals at 180′Post. Assessment of these receptors might be suitable to obtain a deeper understanding of skeletal muscle adaptive processes to develop optimized training strategies.

High-Dimensional Analysis Identifies Mechanisms of Gilteritinib Resistance in FLT3-Mutated AML

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. DisclosuresBlachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq.

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end‐stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co‐expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA‐seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine‐derived renal progenitor cells and human kidney‐derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single‐cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co‐expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.

Abstract 27: Unravelling Cell-specific Interactions At The Preeclamptic Maternal-foetal Interface From Early To Late Pregnancy

Preeclamptic syndrome arrises in the fetal part of the placenta (villi). In this study, we analyse placental development by single nuclei RNA-sequencing in early and term pregnancy and draw conclusions about pathological processes in preeclampsia (PE) that originate early in gestation. We profiled the transcriptome of 101,067 nuclei obtained from a total of 12 pregnancies, spanning early, term and PE doners. Using unsupervised computational approaches, we identified 12 and 16 different cell types and states in decidua and villi, respectively. Our comprehensively identified catalogue of cell types and states aligns well with the previous single cell studies. We identified different subpopulations of syncytiotrophoblast and GATA3+/GREM2+ trophoblast stem cells (TSC) in villi. Through gestation, gene expression in cell populations from the matrisome or vascular environments show dynamic expression reflecting vascular development associated with spiral artery remodelling and concordant decidual stroma reorganisation. Global differential gene expression analysis shows that trophoblast cell types are most dysregulated in PE. Cell-cell communication analysis revealed important dysregulation between villi and decidual cell types. The secretory signalling characteristic of this trophoblastic disease may be used for early biomarker screening. Overall, this study paves the way to a deeper understanding of the early pathophysiology of PE. Figure 1: Villi (v) and decidua (d) cell clusters from early, late control and preeclampsia (PE) villi and decidua visualised as a UMAP. Datasets were integrated separately for each tissue and merged for embedding.

Abstract 1590: An experimental tumor model resistant to anti-PD-1 is sensitive to local mRNA immunotherapy

Abstract Immune checkpoint blockade elicits durable anti-cancer responses in the clinic, however a large proportion of patients do not benefit from treatment. In an effort to better understand acquired resistance to checkpoint blockade, we generated a mouse tumor model exhibiting in vivo resistance to anti-PD-1 antibody treatment. MC38 tumors acquired resistance to PD-1 blockade following serial in vivo passaging. Lack of sensitivity to PD-1 blockade was not attributed to dysregulation of PD-L1 or β2M expression, as both were expressed at similar levels in parental and resistant cells. Similarly, IFNγ signaling and antigen processing and presentation pathways were functional in both parental and resistant cell lines. RNA-sequencing revealed substantial differences in global gene expression, with tumors resistant to anti-PD-1 displaying a marked reduction in expression of immune-related genes relative to parental MC38 tumors. Indeed, resistant tumors exhibited reduced immune infiltration across multiple cell types, including T and NK cells. Pathway analysis revealed activation of TGFβ and Notch signaling in anti-PD-1 resistant tumors, and activation of these pathways was associated with poorer survival in human cancer patients. While pharmacological inhibition of TGFβ and Notch in combination with PD-1 blockade decelerated tumor growth, local administration of a mixture of mRNAs encoding immune-stimulatory cytokines (IL12sc, GM-CSF, IL15s and IFNα) potently induced regression of resistant tumors and resulted in complete tumor remission. Overall, this study describes a novel anti-PD-1 resistant mouse tumor model and underscores the role of two well-defined signaling pathways in response to immune checkpoint blockade. Furthermore, our data highlights the potential of intratumoral mRNA therapy in overcoming acquired resistance to PD-1 blockade. Citation Format: Marie Bernardo, Tatiana Tolstykh, Yu-an Zhang, Dinesh S. Bangari, Hui Cao, Kerstin A. Heyl, Joon Sang Lee, Natalia Malkova, Jack Pollard, Hui Qu, Fangxian Sun, Dmitri Wiederschain, Timothy R. Wagenaar. An experimental tumor model resistant to anti-PD-1 is sensitive to local mRNA immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1590.

Contrasting Stage Dependent Requirements for the Histone Methyltransferase EZH2 during the Evolution of Acute Myeloid Leukemia

Epigenetic regulators function in a context-dependent manner and are commonly mutated in tumors. Activating mutations and overexpression of the histone-methyltransferase, enhancer of zeste, homolog 2 (EZH2), occur in lymphoma and other malignancies, whilst loss-of-function mutations are predicted in myeloid malignancies. We study this apparent contradiction, examining the importance of cellular context and disease stage for Ezh2 loss, genetically deleting Ezh2 during the induction or maintenance of a single malignancy, Acute Myeloid Leukemia (AML).We initially focused on the role of Ezh2 in AML maintenance, using conditional Ezh2fl/fl mice. Following in vitro retroviral immortalization of haematopoietic stem and progenitor cells (HSPCs) from these with AML-fusion oncogenes: MLL-AF9 or AML1-ETO9a (reflecting two separate models of AML clinically associated with good and poor prognosis, markedly differing in mechanism of leukemic transformation and representing AML heterogeneity), these were then transduced with p-babe-Cre-puro vector (or empty vector) to delete Ezh2 . Strikingly, Ezh2 loss completely abolished cellular growth and colony formation, whilst empty vector transduced cells maintained their clonogenic output. To interrogate Ezh2 as a target in AML maintenance in vivo, we generated secondary MLL-AF9 or AML1-ETO9a leukemias through transplantation of bulk leukemia derived from Ezh2fl/fl ; Mx1-Cre primary leukemic mice. Following engraftment, deletion of Ezh2 was achieved by pIpC administration. Loss of Ezh2 in vivo markedly impaired both secondary MLL-AF9 and AML1-ETO9a leukemia progression. These data suggest that Ezh2 is an oncogene across multiple disparate subtypes of established AML and propose it as a possible therapeutic target in this context .Addressing this hypothesis, we examined the role of pharmacological inhibition of EZH2 in AML, utilizing GSK343, an S-Adenosyl Methionine competitive EZH2 inhibitor. Through colony formation and functional assays, we demonstrated in vitro sensitivities of MLL-AF9 transformed murine BM HSPCs, primary murine Ezh2+/+ MLL-AF9 tumours , the AML1-ETO fusion driven human cell line Kasumi-1 and primary leukemic cells isolated from patients with various AML genotypes to GSK343 at similar IC50 (~10µM) across all cells. In contrast, minimal effects of GSK343 were seen on normal primary CD34+ HSCs clonogenic function. Together these data provide evidence of therapeutic potential, validating EZH2 as a target in AML.To characterise the role of Ezh2 during AML induction in vivo, we utilized Ezh2fl/fl; Mx1-Cre mice to generate Ezh2-/- mice and compared them to Ezh2+/+ controls. HSPCs from these were retrovirally transduced with MLL-AF9 or AML1-ETO9a and transplanted into lethally irradiated wild-type recipients, and leukemias allowed to develop. Remarkably in the context of disease induction, and in stark contrast to the maintenance experiments, Ezh2 functioned as a tumor suppressor gene, with its loss significantly accelerating leukemia generation across both subtypes of AML (compared to control).To interrogate the molecular mechanisms of these findings we performed integrated genomic analysis with differential global gene expression analysis via RNA-sequencing (in Ezh2+/+ vs Ezh2-/- non-transformed HSPCs, Ezh2+/+ vs Ezh2-/- AML1-ETO9a and Ezh2+/+ vs Ezh2-/- MLL-AF9 leukemias) and ChIP-sequencing of the histone modifications H3K27me3 and H3K4me3. These revealed that during disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a number of genes including the transcription factor Plag1 (with known oncogenic function in CBFB-MYH11 murine leukemogenesis). Of note, overexpression of Plag1 phenocopied Ezh2 loss i n vivo to significantly accelerate MLL-AF9 leukemia induction.In addition, we demonstrated virtual mutual exclusivity of genes de-repressed in established MLL-AF9 leukemias following pharmacological Ezh2 inhibition compared to those de-repressed in the induction of Ezh2-/- MLL-AF9 leukemias. This de-repression of entirely separate gene programmes explains the starkly contrasting phenotypic outcomes of targeting Ezh2 in AML induction and maintenance, and further provides reassurance that EZH2 inhibition in established AML is a safe strategy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.