Seq Analysis Research Articles

TFAP2 paralogs facilitate chromatin access for MITF at pigmentation and cell proliferation genes.

In developing melanocytes and in melanoma cells, multiple paralogs of the Activating-enhancer-binding Protein 2 family of transcription factors (TFAP2) contribute to expression of genes encoding pigmentation regulators, but their interaction with Microphthalmia transcription factor (MITF), a master regulator of these cells, is unclear. Supporting the model that TFAP2 facilitates MITF’s ability to activate expression of pigmentation genes, single-cell seq analysis of zebrafish embryos revealed that pigmentation genes are only expressed in the subset of mitfa-expressing cells that also express tfap2 paralogs. To test this model in SK-MEL-28 melanoma cells we deleted the two TFAP2 paralogs with highest expression, TFAP2A and TFAP2C, creating TFAP2 knockout (TFAP2-KO) cells. We then assessed gene expression, chromatin accessibility, binding of TFAP2A and of MITF, and the chromatin marks H3K27Ac and H3K27Me3 which are characteristic of active enhancers and silenced chromatin, respectively. Integrated analyses of these datasets indicate TFAP2 paralogs directly activate enhancers near genes enriched for roles in pigmentation and proliferation, and directly repress enhancers near genes enriched for roles in cell adhesion. Consistently, compared to WT cells, TFAP2-KO cells proliferate less and adhere to one another more. TFAP2 paralogs and MITF co-operatively activate a subset of enhancers, with the former necessary for MITF binding and chromatin accessibility. By contrast, TFAP2 paralogs and MITF do not appear to co-operatively inhibit enhancers. These studies reveal a mechanism by which TFAP2 profoundly influences the set of genes activated by MITF, and thereby the phenotype of pigment cells and melanoma cells.

MiR778 mediates gene expression, histone modification, and DNA methylation during cyst nematode parasitism.

Despite the known critical regulatory functions of microRNAs, histone modifications, and DNA methylation in reprograming plant epigenomes in response to pathogen infection, the molecular mechanisms underlying the tight coordination of these components remain poorly understood. Here, we show how Arabidopsis (Arabidopsis thaliana) miR778 coordinately modulates the root transcriptome, histone methylation, and DNA methylation via post-transcriptional regulation of the H3K9 methyltransferases SU(var)3-9 homolog 5 (SUVH5) and SUVH6 upon infection by the beet cyst nematode Heterodera schachtii. miR778 post-transcriptionally silences SUVH5 and SUVH6 upon nematode infection. Manipulation of the expression of miR778 and its two target genes significantly altered plant susceptibility to H. schachtii. RNA-seq analysis revealed a key role of SUVH5 and SUVH6 in reprograming the transcriptome of Arabidopsis roots upon H. schachtii infection. In addition, chromatin immunoprecipitation (ChIP)-seq analysis established SUVH5 and SUVH6 as the main enzymes mediating H3K9me2 deposition in Arabidopsis roots in response to nematode infection. ChIP-seq analysis also showed that these methyltransferases possess distinct DNA binding preferences in that they are targeting transposable elements under noninfected conditions and protein-coding genes in infected plants. Further analyses indicated that H3K9me2 deposition directed by SUVH5 and SUVH6 contributes to gene expression changes both in roots and in nematode feeding sites and preferentially associates with CG DNA methylation. Together, our results uncovered multi-layered epigenetic regulatory mechanisms coordinated by miR778 during Arabidopsis-H. schachtii interactions.

Disease Associated Microglia Is Identified As A Major Contributor To Neuroinflammation Associated To Chronic Hypoxia Induced‐Pulmonary Hypertension

IntroductionPulmonary hypertension (PH) is a progressive disease that leads to right heart failure and death. Increased sympathetic activity in PH patients is associated with a worse prognosis due to a series of well‐established downstream sequelae. However, the mechanism behind chronic elevation in sympathetic activity in PH is poorly understood. Our fundamental hypothesis is that neuroinflammation in the hypothalamic paraventricular nucleus (PVN) is essential for elevated sympathetic activity in PH. This hypothesis is supported by our published data demonstrating: 1) increased number of microglia in the PVN is observed in multiple animal models of PH; 2) Microglia inhibition attenuates sympathetic activity and PH cardiopulmonary effects. However, microglia are, a heterogeneous with respect to gene expression and function. Indicating that characterization of the specific microglia subtypes involved in PH pathophysiology would be the key to identify possible targets to improve PH therapy.Methods3‐months‐old SD rats were subjected to monocrotaline induced PH model. After 28 days, CD45+CD11b+ cells were isolated from the PVN followed by single‐cell RNA Sequencing (scRNA Seq) analysis. The disease associated microglia (DAM), which highly express Trem2, displayed the most significant phenotypic changes in PH. 3‐months‐old WT mice (n=12/group) were exposed to chronic hypoxia induced‐PH (CH‐PH) (10%O2) or Normoxia for 4 weeks in a ventilated chamber. CD45+CD11b+ cells were isolated from the PVN to evaluate TREM2 expression by flow cytometry in the same population. Then, TREM2 global knock‐out mice (TREM2‐KO) and WT (n=10/group) were exposed to CH‐PH model and pulmonary hemodynamics were evaluated using a Millar pressure catheter in a terminal procedure.ResultsWe identified 10 clusters as microglia/macrophage. Following evaluation of several cluster markers used to identify microglia subtypes, we found the cluster with the most significant differences in gene profile and biological processes by comparing control and PH samples. This cluster was identified as the disease‐associated microglia (DAM), characterized by high expression of TREM2. Data was also confirmed in chronic hypoxia induced PH (CH‐PH) model by flow cytometry. CH induced a two‐fold increase of TREM2+microglia (CD11+, CD45+, TMEM119+) expression compared to normoxia controls. Fascinatingly, TREM2 deficient mice demonstrated a significant attenuation in the pulmonary pressure, represented by its surrogate the right ventricular systolic pressure (RVSP), and remarkably, reduction in overall lung inflammation induced by hypoxia.ConclusionsWhile TREM2 contribution has been extensively studied in neurodegenerative diseases and cancer therapy, these experiments are the first to demonstrate the potential of Trem2 targeted therapy in context of PH. Moreover, we believe that data generated from these studies may lead to translational studies evaluating the potential of TREM2 as a therapeutic target for PH.

Abstract 486: Role Of Matrix Gla Protein In Vascular Regulation In Brown Adipose Tissue

Obesity is a world-wide problem; an estimated 38% of the world population will be overweight and 20% will be obese by 2030. Brown adipose tissue (BAT) is a thermogenic tissue that plays an important role in energy expenditure and glucose uptake. It is also highly vascularized but the regulation of vascular formation in BAT is poorly understood. The bone morphogenetic proteins (BMPs) are essential in both brown adipogenesis and angiogenesis. BMP7 controls progenitor commitment to pre-adipocytes in BAT, and BMP4 is a known angiogenic factor that is essential for white and brown adipogenic differentiation. Matrix Gla protein (MGP) acts as an inhibitor of BMP4/7 and is highly expressed in pre-adipocytes. We hypothesize that modulation of MGP affects BAT vascularization and function. Our results show that mice with global Mgp gene deletion (Mgp-/-) mice have poorly functional BAT, with abnormal mitochondria and decreased expression of thermogenic lineage markers as determined by RNA Seq analysis and qPCR. In addition, the vasculature showed significant increases in the number of brown adipose endothelial cells (ECs) and expression of markers for both EC lineage and progenitor cells. Furthermore, targeted Mgp deletion in ECs or adipose cells as directed by Cdh5- or Adiponectin-Cre transgenes in Mgp-floxed mice also affected the vascular outgrowth in BAT. Together the results suggest that MGP is an essential regulator of vascular outgrowth in BAT, likely through its ability to inhibit BMPs. A better understanding of this kind of regulation may be helpful in strategies to limit metabolic dysfunction in obesity and diabetes.

Adaptation to High‐Salt Diet Requires H,K‐ATPase Type 2 Dependent NaCl Secretion

IntroductionThe kidney controls the extracellular volume and the blood pressure because of its ability to excrete efficiently Na+ and Cl‐ ions. These last decades in Western countries the consumption of salt has dramatically increase and the question arises as to how the kidney adapts to this chronic situation. Up to now, the mechanisms described to excrete a large load of salt consist in the inhibition of Na+ reabsorption all along the nephron. We have recently identified a novel pathway that mediates the secretion of Na+ in the collecting duct (Morla, L. et al. 2016). This system involves A‐type intercalated cells (AIC), NKCC1 and the H,K‐ATPase type 2 (HKA2), which is known to be able to transport Na+. Here, we investigated the role of HKA2 in NaCl renal secretion, as well as the physiological relevance of this secretory pathway in vivo, in response to salt load.Material and MethodsC57BL6J mice wild type or knockout for the Atp12a gene (HKA2‐KO) were used and placed under a control (NS) or high‐Na+ (HS) diets for 3 days. Outer medullary collecting ducts (OMCD) of WT mice under NS or HS were isolated for RNAseq and RT‐PCR analysis. Type A intercalated cells were isolated by FACS (following labelling with c‐kit antibodies) from WT mice under NS or HS for RT‐PCR analysis. The mice were also placed in metabolic cages for measurements of physiological parameters (food and water intakes, urine excretion, plasma parameters). Blood pressure was assessed by tail‐cuffed measurement.ResultsThe RNA seq analysis of OMCD revealed that, as expected, genes involved in Na+ reabsorption (ENaCa, SGK) are downregulated. However, we also observed that HS diet also leads to an increased expression of the Atp12a gene that encodes for the catalytic subunit of the HKA2 both in OMCD and ICA. To understand the role of the HKA2, we placed the HKA2‐KO mice under high‐salt diet and observed that those mice exhibit a salt loosing phenotype with low blood pressure that is due to a stronger down‐regulation of NKCC2 than in WT in the same context. Therefore, the absence of HKA2 is over‐compensated by the inhibition of a reabsorption pathway.ConclusionAltogether, these results demonstrate that the secretion of Na+ in OMCD is stimulated in response to high salt intake.

Anti‐Tumor Effect of Embryonic Stem Cell Derived Exosomes in Triple Negative Breast Cancer: Potential Role of TCF7‐E‐Cadherin and VEGF

BackgroundTriple Negative Breast Cancer (TNBC) is an aggressive form of cancer and accounts for 10‐20 % of all breast cancer types. TNBC patients are widely treated with chemotherapeutic drugs such as doxorubicin (Dox), which is a primary standard of care. Dox treatment results in unwanted side effects including cardiac dysfunction and skeletal muscle weakness. We previously demonstrated the anti‐tumor effects and cardioprotective effects of embryonic stem cell derived exosomes (Es‐Exos) in the xenograft model of TNBC. The purpose of this study was to identify the biomolecules present in Es‐Exos, which may be linked to the anti‐tumor effects in the xenograft model.Methods and ResultsOrthotopic xenograft model of TNBC was generated by injecting human MDA‐MB‐231‐Tom‐Luc cell line in mammary intraductal T4 region of nude mice. Non‐tumor bearing female mice were used as control (NT‐Con;n=6) as applicable. The animals (n=10/group) were randomized to receive Saline (Con); Dox (5 mg/kg/week); Es‐Exos (50 ug/animal/twice a week; Dox+Es‐Exos; Es‐Exos alone and MEF‐Exos‐(mouse embryonic fibroblast cell derived exosomes 50 ug/animal/twice a week) for up to 4 weeks. Skeletal muscle grip strength declined both in Con and upon treatment with Dox in tumor bearing mice as compared to NT‐Con, which was restored upon treatment with Es‐Exos. Cardiac function measured by echocardiography showed decline in ejection fraction (EF) in saline treated tumor bearing Con and in Dox group, when compared to NT‐Con. Treatment with Dox+Es‐Exos and Es‐Exos improved ejection fraction compared to Con and Dox cohort. Treatment with Es‐Exos significantly reduced the tumor size compared to Dox and saline treated Con groups (Con Vs Dox and Dox+Es‐Exos; p‐value <0.05). Picro Sirius red staining showed increased deposition of collagen in saline treated Con, which was reduced in Dox+Es‐Exos and Es‐Exos groups. RNA seq analysis of Es‐Exos identified several anti‐proliferative genes including a 6‐fold expression of Transcription Factor 7 (TCF7) and 6.5‐fold higher expression of E‐Cadherin (CDH1). Immunohistochemistry analysis of tumor tissue showed abundant expression of VEGF in untreated Con, which was reduced by combined treatment with Es‐Exos and Dox.ConclusionEmbryonic stem cells derived exosomes contain anti‐proliferative genes which may possibly lead to their anti‐tumor property in the TNBC tumor model.

Genome-Wide Analysis of Invertase Gene Family, and Expression Profiling under Abiotic Stress Conditions in Potato.

Simple SummaryInvertase genes are among the important genes responsible for carbon metabolism in plants, significantly contributing to plant development and stress responses. In this study, for the first time, we performed genome-wide analysis for Invertase gene family in potato, identified and conducted expression profiling in different tissues by RNA seq analysis and validated it by Q-PCR. We also performed invertase family genes expression profiling under drought, salt and heat stress to elucidate their involvement in stress responses. Findings of this study will be helpful for future functional and genetic studies not only in potato but also in other plants.The potato is one of the most important and valuable crops in terms of consumption worldwide. However, abiotic stressors are the critical delimiters for the growth and productivity of potato. Invertase genes play key roles in carbon metabolism, plant development, and responses to stress stimuli. Therefore, a comprehensive genome-wide identification, characterization and expression analysis of invertase genes was performed in the potato. The current study identified 19 invertase genes, randomly distributed throughout the potato genome. To further elucidate their evolutionary, functional and structural relationship within family and with other plant species, we performed sequence and phylogenetic analysis, which segregated invertase genes into two main groups based on their sequence homology. A total of 11 genes are included in acidic invertases and 8 genes are in neutral or alkaline invertases, elucidating their functional divergence. Tissue specific expression analyses (RNA sequencing and qRT-PCR) of different plant tissues showed differential expression pattern. Invertase genes have higher expression in flower, leaf, root and shoot tissues, while under abiotic stress conditions, the expression of the invertase gene is significantly upregulated. Results of this study revealed that vacuolar and cell wall destined invertases are mainly the functional member genes of the invertase family. This study provides comprehensive data and knowledge about StINV genes in Solanum tuberosum for future genetic and epigenetic studies.

CeO2 nanoparticles improved cucumber salt tolerance is associated with its induced early stimulation on antioxidant system

Salinity is a global issue limiting efficient agricultural production. Nano-enabled plant salt tolerance is a hot topic. However, the role of nanoparticles induced possible early stimulation on antioxidant system in its improved plant salt tolerance is still largely unknown. Here, poly (acrylic) acid coated nanoceria (cerium oxide nanoparticles) (PNC, 7.8 nm, −31 mV) with potent ROS (reactive oxygen species) scavenging ability are used. Compared with control, no significant difference of H2O2 and O2•─ content, MDA (malondialdehyde) content, relative electric conductivity, and Fv/Fm was found in leaves and/or roots of cucumber before onset of salinity stress, regardless of leaf or root application of PNC. While, before onset of salinity stress, compared with control, the activities of SOD (superoxide dismutase, up to 1.8 folds change), POD (peroxidase, up to 2.5 folds change) and CAT (catalase, up to 2.3 folds change), and the content of GSH (glutathione, up to 3.0 folds change) and ASA (ascorbic acid, up to 2.4 folds change) in leaves and roots of cucumber with PNC leaf spray or root application were significantly increased. RNA seq analysis further confirmed that PNC foliar spray upregulates more genes in leaves over roots than the root application. These results showed that foliar sprayed PNC have stronger early stimulation effect on antioxidant system than the root applied one and leaf are more sensitive to PNC stimulation than root. After salt stress, cucumber plants with foliar sprayed PNC showed better improvement in salt tolerance than the root applied one. Also, plants with foliar sprayed PNC showed significant higher whole plant cerium content than the root applied one after salt stress. In summary, we showed that foliar spray of nanoceria is more optimal than root application in terms of improving cucumber salt tolerance, and this improvement is associated with better stimulation on antioxidant system in plants.

Identification of the toxic 6mer seed consensus for human cancer cells

6mer seed toxicity is a novel cell death mechanism that kills cancer cells by triggering death induced by survival gene elimination (DISE). It is based on si- or shRNAs with a specific G-rich nucleotide composition in position 2–7 of their guide strand. An arrayed screen of 4096 6mer seeds on two human and two mouse cell lines identified G-rich 6mers as the most toxic seeds. We have now tested two additional cell lines, one human and one mouse, identifying the GGGGGC consensus as the most toxic average 6mer seed for human cancer cells while slightly less significant for mouse cancer cells. RNA Seq and bioinformatics analyses suggested that an siRNA containing the GGGGGC seed (siGGGGGC) is toxic to cancer cells by targeting GCCCCC seed matches located predominantly in the 3′ UTR of a set of genes critical for cell survival. We have identified several genes targeted by this seed and demonstrate direct and specific targeting of GCCCCC seed matches, which is attenuated upon mutation of the GCCCCC seed matches in these 3′ UTRs. Our data show that siGGGGGC kills cancer cells through its miRNA-like activity and points at artificial miRNAs, si- or shRNAs containing this seed as a potential new cancer therapeutics.

Profilin is involved in G1 to S phase progression and mitotic spindle orientation during Leishmania donovani cell division cycle.

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

Abstract P3-06-05: Human endogenous retrovirus-K (HERV-K) is aberrantly expressed in triple negative breast cancer (TNBC) and associated with increased distant metastasis: Impact of HERV-K knockdown on gene expression patterns and invasive potential of mesenchymal TNBC

Abstract Background: Human endogenous retrovirus K (HERV-K) belongs to a family of endogenous retroviruses that are present in our genome with similarities to present day exogenous retroviruses. HERV-K, like other endogenous retroviruses, is transmitted vertically in a Mendelian fashion through the human genome. This virus can express several proteins but our knowledge of HERV-K expression in human cancers is mainly limited to the envelope (Env) protein. Elevated HERV-K env protein expression has been shown in breast cancer both in in vitro and in vivo studies. Previous work from this laboratory has shown increased HERV-K expression in blood is predictive of a prostate cancer diagnosis. Aims & Experimental Approach: This project aimed to decipher whether individual HERV-K proteins (Env and Gag) display differential association with histological type, molecular subtype and patient outcomes in an Irish cohort of triple negative breast cancer patients (n=177). Additionally we aimed to assess the impact of HERV-K targeting shRNA mediated knockdown on TNBC characteristics including cell migration, invasion and proliferative capacity in 2D and 3D cell culture models. RNA seq and bioinformatics analysis was performed to identify key molecular processes regulated by HERV-K in TNBC. Results: HERV-K Env expression showed a trend towards an association with poor survival in TNBC (Log rank test, p=0.105), while HERV-K Gag was not found to be associated with survival. HERV-K Env positive metastatic patients had an increased involvement of the bone and lungs compared to HERV-K Env negative patients, while HERV-K gag positive patients showed reduced liver metastasis involvement. Knockdown of HERV-K in TNBC cell lines MDA-MB-231 and MDA-MB-468 reduced their proliferative, migratory and invasive potential. Preliminary screens using a cancer drug target array shows significantly reduced expression of HDAC4, a regulator of cell growth and the anti-apoptotic protein. GSEA analysis of RNAseq data from MDA-MB-231 HERV-K shRNA knockdown demonstrated enrichment for genes associated with both the Charafe downregulated in Luminal A versus Mesenchymal gene signature (p= 5.14x10-133), and the Charafe downregulated in Luminal A versus Basal gene signature (p= 7.03x10-110), indicating that HERV-K may play an important role in mesenchymal and basal phenotype TNBC. Additionally pathway analysis points toward a role in the regulation of many different pathways and cellular process, including focal adhesion (p= 9.77x10-7), cellular senescence (p= 7.63x10-7) and viral signalling pathways (p= 6.86x10-14). Discussion: Reduced rates of migration, invasion and proliferation in HERV-K knockdowns points towards the essential role of HERV-K in tumorigenesis and metastasis. HERV-K knockdown also modulated key gene expression signatures traditionally associated with the basal and mesenchymal phenotypes in breast cancer. Taken together, our findings indicate that HERV-K may be a useful molecular target for the treatment of TNBC. Citation Format: Sharon A Glynn, Dibyangana Bhattacharyya, Shauna Lambe, Emma Kerr, Simon McDade, Faizan H Khan, Eoin Dervan, Feng Wang-Johanning, Sean Hynes, Grace Callagy. Human endogenous retrovirus-K (HERV-K) is aberrantly expressed in triple negative breast cancer (TNBC) and associated with increased distant metastasis: Impact of HERV-K knockdown on gene expression patterns and invasive potential of mesenchymal TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-05.

Abstract P5-13-35: Mechanisms of CDK4/6 inhibitor resistance in hormone receptor positive metastatic breast cancer: Single institution retrospective analysis

Abstract Background: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are the standard of care for hormone receptor positive (HR+) metastatic breast cancer (MBC). Mechanisms of intrinsic and acquired resistances have been proposed but are poorly understood in the real-world setting. The current study aims to study the association of genomic biomarkers and CDK 4/6i resistance mechanisms utilizing genomic data acquired through routine clinical practice. Methods: Tumor genomic data from patients with HR+ MBC who received FDA-approved CDK4/6i (palbociclib, ribociclib, and abemaciclib) were obtained by chart review. Progression free survival (PFS) was determined from the first day of treatment until date of disease progression. Patients were divided by number of lines of treatment (first line vs. ≥ 2 lines of therapy) and subcategorized into early progressors (< 6 months PFS), intermediate progressors (6-24 months PFS for first line and 6-9 months PFS for ≥ 2 lines of therapy), and late progressors (>24 months PFS for first line and > 9 months PFS for ≥ 2 lines of therapy). Genomic alterations from next-generation sequencing (NGS) were analyzed with PFS. PFS stratified by first vs. >2 lines of treatment were performed using a Cox proportional hazards model with hazard ratios and 95% confidence intervals reported. Results: Among 795 patients with HR+ MBC treated with CDK 4/6i between 2015 and 2020 including 673 patients from a main campus and 122 patients from community sites, a total of 131 patients had genomic data available for the analysis. The obtaining rate of genomic data was 18% (122 out of 673 patients) from the main campus and 7% (9 out of 122 patients) from community sites. Seventy-one patients were treated with CDK4/6i as the first line of MBC, and 60 were treated as second or sequential lines. 35 patients had early progression, 40 patients had intermediate progression, 44 patients had late progression, and 12 patients were indeterminate. The distribution of genomic alterations was not significantly different between each group. The genomic alterations that were associated with impaired PFS were ZNF703(hazard ratio, HR 2.1, N=13), ERBB2 (HR 1.9, N=12), MDM2 (HR 3.3, N=6), PALB2 (HR 3.8, N=5), ARFRP1 (HR 3.1, N=4), FRS1 (HR 2.9, N=4), IRS2 (HR 6.9, N=2), and JAK2 (HR 6, N=2). RNA seq analysis is currently underway. Conclusion: Tumor genomic tests are not routinely performed for patients with HR+ MBC, particularly less performed from community practices. In this single institution retrospective analysis, we identified genomic alterations, such as ZNF703, ERBB2, PALB2, and MDM2, as potential resistance mechanisms of CDK4/6i. Tumor genomics through standard clinical practice are essential to understand resistance mechanisms to CDK4/6i. Citation Format: Jin Sun Lee, Susan Yost, Yujie Cui, Paul Frankel, Sierra Min Li, Yate-Ching Yuan, Denise Morse, Judy Wong-Toh, Linda Bosserman, Joanne Mortimer, Yuan Yuan. Mechanisms of CDK4/6 inhibitor resistance in hormone receptor positive metastatic breast cancer: Single institution retrospective analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-35.

MAX deficiency impairs human endometrial decidualization through down-regulating OSR2 in women with recurrent spontaneous abortion

Human uterine stromal cell undergoes decidualization for pregnancy establishment and maintenance, which involved extensive proliferation and differentiation. Increasing studies have suggested that recurrent spontaneous abortion (RSA) may result from defective endometrial stromal decidualization. However, the critical molecular mechanisms underlying impaired decidualization during RSA are still elusive. By using our recently published single-cell RNA sequencing (scRNA-seq) atlas, we found that MYC-associated factor X (MAX) was significantly downregulated in the stromal cells derived from decidual tissues of women with RSA, followed by verification with immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (qRT-PCR). MAX knockdown significantly impairs human endometrial stromal cells (HESCs) proliferation as determined by MTS assay and Ki67 immunostaining, and decidualization determined by F-actin, and decidualization markers. RNA-seq together with chromatin immunoprecipitation sequencing (ChIP-seq) and cleavage under targets and release using nuclease sequencing (CUT&RUN-seq) analysis were applied to explore the molecular mechanisms of MAX in regulation of decidualization, followed by dual-luciferase reporter assay to verify that MAX targets to (odd-skipped related transcription factor 2) OSR2 directly. Reduced expression of OSR2 was also confirmed in decidual tissues in women with RSA by IHC and qRT-PCR. OSR2 knockdown also significantly impairs HESCs decidualization. OSR2-overexpression could at least partly rescue the downregulated insulin-like growth factor binding protein 1 (IGFBP1) expression level in response to MAX knockdown. Collectively, MAX deficiency observed in RSA stromal cells not only attenuates HESCs proliferation but also impairs HESCs decidualization by downregulating OSR2 expression at transcriptional level directly.

Integrated Analysis of mRNA-Seq and MiRNA-Seq Reveals the Molecular Mechanism of the Intestinal Immune Response in Marsupenaeus japonicus Under Decapod Iridescent Virus 1 Infection.

The intestine is not only an important digestive organ but also an important immune organ for shrimp; it plays a key role in maintaining homeostasis. Decapod iridescent virus 1 (DIV1) is a new type of shrimp-lethal virus that has received extensive attention in recent years. To date, most studies of the shrimp intestinal immune response under viral infections have relied on single omics analyses; there is a lack of systematic multi-omics research. In the current study, intestinal mRNA-seq and microRNA (miRNA)-seq analyses of Marsupenaeus japonicus under DIV1 infection were performed. A total of 1,976 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs (DEMs) were identified. Among them, 21 DEMs were negatively correlated with 194 DEGs from a total of 223 correlations. Functional annotation analysis revealed that M. japonicus can regulate glycosaminoglycan biosynthesis (chondroitin sulfate, dermatan sulfate, and keratan sulfate), vitamin metabolism (retinol metabolism and ascorbate and aldarate metabolism), immune pathway activation (Toll and IMD signaling pathways, Wnt signaling pathway, IL-17 signaling pathway, and Hippo signaling pathway), immunity enzyme activity promotion (triose-phosphate isomerase), antimicrobial peptide (AMP) expression, reactive oxygen species (ROS) production, and cell apoptosis through miRNAs to participate in the host’s antiviral immune response, while DIV1 can influence Warburg effect-related pathways (pyruvate metabolism, glycolysis/gluconeogenesis, and citrate cycle), glycosphingolipid biosynthesis-related pathways (glycosphingolipid biosynthesis—globo and isoglobo series and glycosphingolipid biosynthesis—lacto and neolacto series), and the tight junction and adhesion junction of the intestinal mucosal epithelium through the host’s miRNAs and mRNA to promote its own invasion and replication. These results indicate that intestinal miRNAs play important roles in the shrimp immune response against DIV1 infection. This study provides a basis for further study of the shrimp intestinal antiviral immune response and for the formulation of effective new strategies for the prevention and treatment of DIV1 infection.

Abstract PO-137: Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race

Abstract African American (AA) men exhibit 2-3 times higher mortality from prostate cancer compared with Caucasian American (CA) men. Factors contributing to the disparity include societal-, neighborhood- and institutional-level determinants of health. In addition, a number of studies have reported individual-level ancestry-related biological differences, including in mutations, copy number variation, aggregate gene expression and response to treatment between AA and CA prostate cancer patients. Previously, by comparing the transcriptome between 20 AA and 15 CA prostate cancer patients, we identified a large number of race-related Alternative RNA Splicing (ARS) events. Among these, we further demonstrated that an exon skipping event involving exon 20 within PIK3CD increased tumor growth rate, metastatic potential and drug resistance in prostate cancer. To expand our previous findings, we collected non-neoplastic and tumor-paired tissue from 37 AA and 40 CA prostate cancer patients with different Gleason score categories: 14 high grade (4 AA and 10 CA), 22 low grade (10 AA and 12 CA), and 41 intermediate grade (23 AA and 18 CA). DNA and RNA were isolated for ancestral genotyping and RNA seq analysis, respectively. To achieve RNA sequencing depth adequate for ARS analysis, we performed RNA seq of 150 bp paired-end and an average of 5 × 106 reads per sample. The read alignment was done using Star 2 TwoPass pipeline. The rMATS pipeline was used for ARS annotation and quantification. We identified 105,403 ARS events, including 60,657 exon skipping, 17,439 alternative acceptor, 12,737 alternative donor, 9,555 retained intron and 5,015 mutually exclusive exon. Using the Wilcoxon rank-sum test, we compared the Percent Spliced In (PSI) between AA and CA of the same Gleason score category and identified ARS events exhibiting ΔPSI > 15% and p-value < 0.05. Specifically, we identified 536 race-related ARS events in high grade, 492 race-related ARS events in low grade, and 447 race-related ARS events in intermediate grade. Gene Ontology analysis demonstrated that the genes undergoing race-related ARS events function in cellular processes relevant to cancer biology, including metabolic processes in low grade, NF-kappaB signaling in intermediate grade and cell motility in high grade. Specific examples of these genes include ERG and PARP2 in high grade, KLK2 and DNMT1 in intermediate grade, and AURKA and SEMA3A in low grade. These findings support a potential role for the ARS process in diversifying gene expression, potentially contributing to prostate cancer aggressiveness in AA patients. The race-related ARS events identified in our work represent potential biomarkers and/or therapeutic targets for precision oncology in the context of prostate cancer. Further analysis of the function of prioritized race-related ARS events and their association with local ancestry is ongoing. Funding: DoD Prostate Cancer Research Program Health Disparity Research Award. NIH Basic Research in Cancer Health Disparities R01 Award. Prostate Cancer Foundation Movember Challenge Award. Citation Format: Muthana Al Abo, Wen-Chi Foo, Daniel J. George, Steven R. Patierno, Jennifer A. Freedman. Comparative transcriptomic analysis of prostate cancer from African American and Caucasian American men by Gleason score and race [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-137.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments†.

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (<P10) of the cell lines revealing there were no genetic drift over the passages. Karyotyping also revealed no chromosomal anomalies. Creation of these cell lines can standardize experimental approaches, eliminate subject to subject variabilities, and benefit the reproductive biological studies on pregnancies by using these cells.

Yersinia remodels epigenetic histone modifications in human macrophages.

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61% of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response.

Abstract 9857: Epicardial Hdac3 Promotes Myocardial Growth Through a Novel MicroRNA Pathway

Growth cues from epicardium-derived cells (EPDCs) such as Insulin growth factors (IGFs) and Fibroblast Growth Factors (FGFs) are required for compact myocardial growth, although it is unclear whether/how their expression is regulated by chromatin modifications during this process. To understand the potential function of Histone deacetylase 3 (Hdac3) in the developing epicardium, we conditionally inactivated Hdac3 in the EPDCs. Strikingly, deletion of Hdac3 in the epicardium led to ventricular myocardial wall hypoplasia along with the reduction of EPDCs. To identify disrupted growth signals sending from epicardium to myocardium, we deleted Hdac3 in cultured mouse epicardial cells (MECs) by CRISPR/CAS9 and performed RNA Seq analyses. We found that IGF2 and FGF9 were significantly down regulated in Hdac3 knockout (KO) MECs, which can be rescued by re-expression of intact Hdac3 but not by catalytically dead Hdac3. Importantly, the proliferation of cultured primary embryonic cardiomyocytes was significantly decreased when treated with Hdac3 KO MEC supernatant as compared to those treated with control MEC supernatant, and this proliferation decrease was rescued by supplementation of IGF2 or FGF9. Next, we sought to understand how Hdac3 regulates the expression of IGF2 or FGF9. Interestingly, we found that miR-322/503 was significantly upregulated in Hdac3 KO MECs and Hdac3 epicardial KO hearts. Overexpression of miR-322/503 repressed the expression of IGF2 and FGF9, while knockdown of miR-322/503 restored the expression of IGF2 and FGF9 in Hdac3 KO MECs. Altogether, our findings suggest that Hdac3 in the developing epicardium promotes compact myocardial growth by stimulating IGF2 expression through repressing miR-322/503. Our findings shed light on the potential novel mechanisms of congenital heart defects, and new strategies for myocardial regeneration.

Abstract 14414: Embryonic Stem Cell Derived Exosomes Enhance Anti-Tumor Efficacy of Doxorubicin and Improve Cardiac Function in Triple-Negative Breast Cancer Xenograft Mice

Background: Doxorubicin (Dox) is a potent chemotherapeutic drug widely used for the treatment of triple negative breast cancer (TNBC). However, Dox treatment results in inadvertent cardiotoxic effects which often require supplemental therapy. We previously showed that treatment with embryonic stem cells derived exosomes (ES-Exos) enhanced chemosensitivity of Dox in breast cancer cells. Here we tested the hypothesis whether adjuvant therapy with ES-Exos in mice with orthotopic TNBC xenograft can enhance the anti-tumor effect of Dox and improve cardiac function. Methods and Results: Athymic female nude mice (6-wk old) were injected with human MDA-MB-231-Luc cell line in mammary intraductal T4 region to generate orthotopic xenografts of TNBC. After 2 weeks of tumor growth, mice (n=10/group) were treated for 4 weeks with: Saline (Con); Dox (5 mg/kg/week); ES-Exos (50 μg, twice a week); Dox+ES-Exos; and MEF (50 μg/twice a week; non-stem cell exosomes derived from mouse fibroblasts). ES-Exos were labelled with ExoGlow-Vivo near-IR dye to track the localization of exosomes in the tumor. Treatment with Dox+ES-Exos and Es-Exos showed improved survival as compared to Dox (2 deaths) and Con (3 deaths) (Fig.A) . Tumor size was significantly reduced with Dox+ES-Exos and ES-Exos treatments as compared to Con and Dox treatment (Fig.B) . Cardiac function measured by echocardiography showed improved ejection fraction in Dox+ES-Exos and ES-Exos as compared to Con group (Fig.C) . Cell proliferation rate assessed by Ki-67 score with immunofluorescence showed significantly higher rate in Con as compared to Dox and Dox+ES-Exos groups. RNA seq analysis identified upregulation of unique antitumorigenic regulators such as Semaphorin 5 (Sema5) and FOXO15 in ES-Exos compared to MEF, which may contribute to the therapeutic effect. Conclusion: Treatment with ES-Exos may be a promising novel strategy to improve chemosensitivity with attenuation of its cardiotoxic effect in TNBC.

Arbuscular Mycorrhizal Fungus Alters Root System Architecture in Camellia sinensis L. as Revealed by RNA-Seq Analysis.

Arbuscular mycorrhizal fungus (AMF), forming symbiosis with most terrestrial plants, strongly modulates root system architecture (RSA), which is the main characteristic of root in soil, to improve plant growth and development. So far, the studies of AMF on tea plant seedlings are few and the relevant molecular mechanism is not deciphered. In this study, the 6-month-old cutting seedlings of tea plant cultivar “Wancha No.4” were inoculated with an AMF isolate, Rhizophagus intraradices BGC JX04B and harvested after 6 months of growth. The indexes of RSA and sugar contents in root were determined. The transcriptome data in root tips of mycorrhizal and non-mycorrhizal cutting seedlings were obtained by RNA-sequence (Seq) analysis. The results showed that AMF significantly decreased plant growth, but increased the sucrose content in root and the higher classes of lateral root (LR) formation (third and fourth LR). We identified 2047 differentially expressed genes (DEGs) based on the transcriptome data, and DEGs involved in metabolisms of phosphorus (42 DEGs), sugar (39), lipid (67), and plant hormones (39) were excavated out. Variation partitioning analysis showed all these four categories modulated the RSA. In phosphorus (P) metabolism, the phosphate transport and release (DEGs related to purple acid phosphatase) were promoted by AMF inoculation, while DEGs of sugar transport protein in sugar metabolism were downregulated. Lipid metabolism might not be responsible for root branching but for AMF propagation. With respect to phytohormones, DEGs of auxin (13), ethylene (14), and abscisic acid (5) were extensively affected by AMF inoculation, especially for auxin and ethylene. The further partial least squares structural equation modeling analysis indicated that pathways of P metabolism and auxin, as well as the direct way of AMF inoculation, were of the most important in AMF promoting root branching, while ethylene performed a negative role. Overall, our data revealed the alterations of genome-wide gene expression in tea plant roots after inoculation with AMF and provided a molecular basis for the regulatory mechanism of RSA (mainly root branching) changes induced by AMF.