Molecular Reprogramming Research Articles

Activin A-Mediated Polarization of Cancer-Associated Fibroblasts and Macrophages Confers Resistance to Checkpoint Immunotherapy in Skin Cancer.

Cemiplimab is approved for the treatment of locally advanced basal cell carcinomas (BCC), although with mitigated results. We sought to interrogate the cellular and molecular transcriptional reprogramming underlying BCC resistance to immunotherapy. Here, we combined spatial and single-cell transcriptomics to deconvolute the spatial heterogeneity of the tumor microenvironment in regard with response to immunotherapy, in a cohort of both naïve and resistant BCCs. We identified subsets of intermingled cancer-associated fibroblasts (CAF) and macrophages contributing the most to CD8 T-cell exclusion and immunosuppression. Within this spatially resolved peritumoral immunosuppressive niche, CAFs and adjacent macrophages were found to display Activin A-mediated transcriptional reprogramming towards extracellular matrix remodeling, suggesting active participation to CD8 T-cell exclusion. In independent datasets of human skin cancers, Activin A-conditioned CAFs and macrophages were associated with resistance to immune checkpoint inhibitors (ICI). Altogether, our data identify the cellular and molecular plasticity of tumor microenvironment (TME) and the pivotal role of Activin A in polarizing the TME towards immune suppression and ICI resistance.

Transcriptional signatures of heroin intake and relapse throughout the brain reward circuitry in male mice.

Opioid use disorder (OUD) looms as one of the most severe medical crises facing society. More effective therapeutics will require a deeper understanding of molecular changes supporting drug-taking and relapse. Here, we develop a brain reward circuit-wide atlas of opioid-induced transcriptional regulation by combining RNA sequencing (RNA-seq) and heroin self-administration in male mice modeling multiple OUD-relevant conditions: acute heroin exposure, chronic heroin intake, context-induced drug-seeking following abstinence, and relapse. Bioinformatics analysis of this rich dataset identified numerous patterns of transcriptional regulation, with both region-specific and pan-circuit biological domains affected by heroin. Integration of RNA-seq data with OUD-relevant behavioral outcomes uncovered region-specific molecular changes and biological processes that predispose to OUD vulnerability. Comparisons with human OUD RNA-seq and genome-wide association study data revealed convergent molecular abnormalities and gene candidates with high therapeutic potential. These studies outline molecular reprogramming underlying OUD and provide a foundational resource for future investigations into mechanisms and treatment strategies.

Abstract IA016: Epigenetic crosstalk associated with prostate cancer lineage plasticity

Abstract Background: Prostate cancer arises as an androgen driven disease and therefore androgen receptor (AR) targeting therapies have been a major focus of prostate cancer treatment. Lineage plasticity, a process by which differentiated cells lose their identity and acquire alternative lineage programs, has recently been identified as an emerging mechanism of resistance to targeted therapies in prostate cancer. This plasticity can manifest as histologic transformation from an AR-positive prostate adenocarcinoma to neuroendocrine prostate cancer (NEPC). NEPC is clinically aggressive and prognosis is poor. Although NEPC tumors arise clonally from prostate adenocarcinoma and share genomic alterations, there is significant epigenetic deregulation during the transformation process. However, mechanistically, we still do not know how these epigenetic alterations arise and how best to leverage these alterations as a therapeutic opportunity. Methods: To model DNA methylation program associated with the transition from CRPC to NEPC, we performed reduced-representation bisulfite sequencing (RRBS) in a variety of clinically relevant models including a novel genetically engineered mouse model and patient-derived organoids/xenografts (PDO/X). We performed genetic (CRISPR/Cas9 technology) and pharmacologic inhibition of DNA methylation writers (DNMTs), erasures (TETs) and polycomb repressive complex 2 histone methyltransferase EZH2 to query their role in the establishment of the DNA methylation program. To assess EZH2 enzymatic activity, we performed H3K27me3 ChIP-seq in our murine and human models. Results: The transcriptome from adenocarcinoma or NEPC foci in our murine model closely resembled clinical CRPC or NEPC transcriptome, respectively. We also found increased expression of DNA methylation regulators (DNMT1/3B and TET1) in NEPC foci. A clinically relevant methylome signature based on murine model was found to segregate clinical samples into CRPC and NEPC subtypes which was also validated in androgen-deprived isogenic LNCaP cells as well as PDO/Xs. Interestingly, AR-target genes (e.g., FKBP5 and TMPRSS2) were found to be hypermethylated and downregulated while cell fate decision regulators (e.g., FOXA2 and SLIT2) and EZH2 targets were hypomethylated and upregulated in NEPC foci. While we found that genetic or pharmacologic inhibition of DNA methylation writers and erasures impacted the NEPC-associated DNA methylation program, inhibition of EZH2 resulted in significant alterations to the DNA methylation program. In turn, we also found that that DNMT inhibition led to equally significant alterations to EZH2 activity. Conclusion: Our data suggest that there is an interplay between EZH2 and DNA methylation that is associated with the molecular reprogramming associated with the progression from AR-positive adenocarcinoma towards NEPC as well as for the maintenance of the NEPC molecular program. More mechanistic studies are now needed to fully characterize this interplay and to determine if we can harness this synergy for therapeutic potential. Citation Format: David Rickman, Richa Singh, Varadha Balaji Venkadakrishnan, Yasutaka Yamada, Himisha Beltran. Epigenetic crosstalk associated with prostate cancer lineage plasticity [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr IA016.

Deciphering the single-cell transcriptome network in keloids with intra-lesional injection of triamcinolone acetonide combined with 5-fluorouracil.

Keloid is a highly aggressive fibrotic disease resulting from excessive extracellular matrix deposition after dermal injury. Intra-lesional injection of triamcinolone acetonide (TAC) in combination with 5-fluorouracil (5-FU) is a commonly used pharmacological regimen and long-term repeated injections can achieve sustained inhibition of keloid proliferation. However, the molecular mechanisms underlying the inhibitory effect on keloids remain insufficiently investigated. This study performed single-cell RNA sequencing analysis of keloids treated with TAC+5-FU injections, keloids, and skins to explore patterns of gene expression regulation and cellular reprogramming. The results revealed that TAC+5-FU interrupted the differentiation trajectory of fibroblasts toward pro-fibrotic subtypes and induced keloid atrophy possibly by inhibiting the FGF signaling pathway in intercellular communication. It also stimulated partial fibroblasts to develop the potential for self-replication and multidirectional differentiation, which may be a possible cellular source of keloid recurrence. T cell dynamics demonstrated elevated expression of secretory globulin family members, which may be possible immunotherapeutic targets. Schwann cell populations achieved functional changes by increasing the proportion of apoptotic or senescence-associated cell populations and reducing cell clusters that promote epidermal development and fibroblast proliferation. Our findings elucidated the molecular and cellular reprogramming of keloids by intra-lesional injection of TAC+5-FU, which will provide new insights to understand the mechanism of action and therapeutic targets.

Tri-modal single cell (TEA-seq) profiling reveals a novel, epigenetically distinct T cell subset in children and global molecular reprogramming across age

Abstract Most single cell analyses of human T cell heterogeneity use a single modality (i.e., RNA or ATAC or protein), with limited ability to deconvolute complex cellular and molecular alterations that can occur during human aging and disease. Here, we use a new trimodal single cell technology (TEA-seq) to elucidate the interplay between the surface proteome, transcriptome, and epigenome in more than 300,000 T cells from healthy children (11–13 yrs) and older adults (55–65 yrs). We uncover that age universally drives molecular reprogramming across all T cell subsets towards a more activated basal state. Naive CD4 T cells, considered relatively resistant to aging, exhibited far more pronounced transcriptional reprogramming than naive CD8 T cells, in which alterations are preferentially driven by compositional shifts in naive-like memory subsets. We further discover a novel naïve-like CD8aa T cell subset lost with age that displays an innate-like transcriptome, a KLF/SP-rich epigenome and high IL-21 receptor expression. These data reveal the complex molecular landscape of the human T cell compartment that may contribute to differential immune responses across age. Supported by grants from NIH (K01 AG068373 to CEG) and the Allen Institute

A Rejuvenation Signature in Skeletal Muscle That Is Mediated By Late-Life Exercise

The objectives of this study were to measure to what extent exercise stimulates partial molecular reprogramming in skeletal muscle. We hypothesized that exercise training, beyond its known functional improvements in aged tissues, could recapitulate some of the same effects that are seen with in vivo partial reprogramming by Yamanaka factors. Using transcriptome profiling from 1) a skeletal muscle-specific in vivo Oct3/4, Klf4, Sox2, and Myc (OKSM) reprogramming-factor expression murine model, 2) an in vivo inducible muscle-specific Myc induction murine model, 3) a translatable high-volume hypertrophic exercise training approach in aged mice, and 4) human exercise muscle biopsies, we collectively defined exercise-induced genes common to partial reprogramming. Late-life exercise training lowered murine DNA methylation age according to several contemporary muscle-specific clocks. A comparison of the murine soleus transcriptome after late-life exercise training to the soleus transcriptome after OKSM induction revealed an overlapping signature. Within this signature, downregulation of specific mitochondrial and muscle-enriched genes was conserved in skeletal muscle of long-term exercise-trained humans; among these was muscle-specific Abra/Stars. Myc is the OKSM factor most induced by exercise in muscle and was elevated following exercise training in aged mice. A pulse of MYC rewired the global soleus muscle methylome, and the transcriptome after a MYC pulse partially recapitulated OKSM induction. A common signature also emerged in the murine MYC-controlled and exercise adaptation transcriptomes, including lower muscle-specific Melusin and reactive oxygen species-associated Romo1. With Myc, OKSM, and exercise training in mice as well habitual exercise in humans, the complex I accessory subunit Ndufb11 was lower; low Ndufb11 is linked to longevity in rodents. Our data collectively suggest: 1) a biological age-mitigating effect on the epigenetic landscape by late-life exercise-training in murine skeletal muscle, 2) a common gene expression signature of partial reprogramming by OKSM and exercise training in muscle of humans and aged mice, and 3) that Myc is an exercise-responsive factor that contributes to a rewired molecular profile at the transcriptome and methylome levels. National Institutes of Health: Kevin A. Murach, AG063994; American Federation for Aging Research (AFAR): Kevin A. Murach, Junior Investigator Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 5916: Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance

Abstract Background: 41.9% of adult US population is obese and every 5-unit BMI increase is associated with 12 percent increase in breast cancer risk; it is an independent predictor of poor outcomes in breast cancer. Microbiome has emerged as a central regulator of obesity and we recently discovered that microbes can not only potentiate breast cancer growth and metastasis but also affect therapeutic response. We showed weight-loss by Diet/Exercise/Behavioral intervention (POWER-remote trial) exerts favorable effects. Here, we examine molecular underpinnings of poor therapy response in obese state and explore the gut microbiome to mitigate the same. Methods: We co-implanted lineage determined human White preAdipocytes (hWAT) with MCF7 breast cancer cells in the mammary glands of mice. Changes in tumor microenvironment and tumor cells were evaluated ex situ by RNA sequencing, flow cytometry, Raman spectroscopy, Quantitative phase microscopy, PCR, immunoblotting and functional assays. To inspect the gut microbiome, 16 obese breast cancer patients were subjected to weight loss intervention over 14 weeks. Gut microbiome was analyzed at baseline and post-intervention by microbial shotgun sequencing. Patients were divided into responders and non-responders based on % weight-loss. Results: We found that human breast cancer cells co-implanted with hWAT formed more aggressive tumors compared to controls. RNA sequencing revealed significant upregulation of multiple oncogenic pathways along with robust innate immune response. Tumor dissociated cells from hWAT-MCF7 tumors showed extensive molecular reprogramming rendering them more proliferative, motile and stem-like along with dramatic physiological changes like increased intercellular lipid accumulation. hWAT-MCF7 cells attained resistance to multiple drugs including Tamoxifen, Fulvestrant, Letrozole and Palbociclib. Simultaneous targeting of multiple signaling pathways using pharmacological inhibitors is infeasible in clinical setting hence we explored the obesity-microbiome axis. Obese breast cancer patients underwent POWER-remote intervention and their gut microbiome analysis showed significant differential expression of many microbes. Based on abundance and metabolite profile, F. prausnitzii was selected as the candidate of interest. On treatment with F. prausnitzii growth media, acquired multi-drug resistance in hWAT-MCF7 cells could be reversed suggesting that increase in gut population of the bacterium could be helpful in sensitizing patients unresponsive to therapy. Conclusion: Direct interaction between breast cancer cells and WAT rendered them resistant to multiple drugs. Metabolites produced by weight-loss-associated bacterium F. prausnitzii reversed adiposity-induced multi-drug resistance, hence, can potentially have therapeutic benefits in obese breast cancer patients. Citation Format: Sheetal Parida, Sumit Siddharth, Nethaji Muniraj, Zhenhui Liu, Ishan Barman, Michael Smith, Jenni Sheng, Vered Stearns, Janelle W. Coughlin, Dipali Sharma. Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5916.

Calcium signalling pathways in prostate cancer initiation and progression.

Cancer cells proliferate, differentiate and migrate by repurposing physiological signalling mechanisms. In particular, altered calcium signalling is emerging as one of the most widespread adaptations in cancer cells. Remodelling of calcium signalling promotes the development of several malignancies, including prostate cancer. Gene expression data from in vitro, in vivo and bioinformatics studies using patient samples and xenografts have shown considerable changes in the expression of various components of the calcium signalling toolkit during the development of prostate cancer. Moreover, preclinical and clinical evidence suggests that altered calcium signalling is a crucial component of the molecular re-programming that drives prostate cancer progression. Evidence points to calcium signalling re-modelling, commonly involving crosstalk between calcium and other cellular signalling pathways, underpinning the onset and temporal progression of this disease. Discrete alterations in calcium signalling have been implicated in hormone-sensitive, castration-resistant and aggressive variant forms of prostate cancer. Hence, modulation of calcium signals and downstream effector molecules is a plausible therapeutic strategy for both early and late stages of prostate cancer. Based on this premise, clinical trials have been undertaken to establish the feasibility of targeting calcium signalling specifically for prostate cancer.

A molecular signature defining exercise adaptation with ageing and in vivo partial reprogramming in skeletal muscle.

Exercise promotes functional improvements in aged tissues, but the extent to which it simulates partial molecular reprogramming is unknown. Using transcriptome profiling from (1) a skeletal muscle-specific in vivo Oct3/4, Klf4, Sox2 and Myc (OKSM) reprogramming-factor expression murine model; (2) an in vivo inducible muscle-specific Myc induction murine model; (3) a translatable high-volume hypertrophic exercise training approach in aged mice; and (4) human exercise muscle biopsies, we collectively defined exercise-induced genes that are common to partial reprogramming. Late-life exercise training lowered murine DNA methylation age according to several contemporary muscle-specific clocks. A comparison of the murine soleus transcriptome after late-life exercise training to the soleus transcriptome after OKSM induction revealed an overlapping signature that included higher JunB and Sun1. Also, within this signature, downregulation of specific mitochondrial and muscle-enriched genes was conserved in skeletal muscle of long-term exercise-trained humans; among these was muscle-specific Abra/Stars. Myc is the OKSM factor most induced by exercise in muscle and was elevated following exercise training in aged mice. A pulse of MYC rewired the global soleus muscle methylome, and the transcriptome after a MYC pulse partially recapitulated OKSM induction. A common signature also emerged in the murine MYC-controlled and exercise adaptation transcriptomes, including lower muscle-specific Melusin and reactive oxygen species-associated Romo1. With Myc, OKSM and exercise training in mice, as well habitual exercise in humans, the complex I accessory subunit Ndufb11 was lower; low Ndufb11 is linked to longevity in rodents. Collectively, exercise shares similarities with genetic in vivo partial reprogramming. KEY POINTS: Advances in the last decade related to cellular epigenetic reprogramming (e.g. DNA methylome remodelling) toward a pluripotent state via the Yamanaka transcription factors Oct3/4, Klf4, Sox2 and Myc (OKSM) provide a window into potential mechanisms for combatting the deleterious effects of cellular ageing. Using global gene expression analysis, we compared the effects of in vivo OKSM-mediated partial reprogramming in skeletal muscle fibres of mice to the effects of late-life murine exercise training in muscle. Myc is the Yamanaka factor most induced by exercise in skeletal muscle, and so we compared the MYC-controlled transcriptome in muscle to Yamanaka factor-mediated and exercise adaptation mRNA landscapes in mice and humans. A single pulse of MYC is sufficient to remodel the muscle methylome. We identify partial reprogramming-associated genes that are innately altered by exercise training and conserved in humans, and propose that MYC contributes to some of these responses.

Abstract PR002: Identification of metabolic adaptation mechanisms that drive anoikis suppression and metastasis in Ewing sarcoma

Abstract Metastasizing cancer cells must overcome anoikis (detachment-induced death) prior to colonization in distant organs. Ewing sarcoma (EwS) is a highly aggressive bone and soft tissue cancer that mainly occurs in children, adolescents, and young adults. EwS patients with metastatic disease have a 5-year survival rate of only 15-20%, which has not changed for decades. Our recent study identified that EwS cells are highly dependent on augmented cysteine metabolism and glutathione biogenesis in anoikis suppression and metastasis through the IL1RAP protein. Mechanistically, IL1RAP binds the cell surface system Xc- transporter to enhance exogenous cystine uptake, thereby replenishing cysteine and glutathione antioxidant pools. Moreover, under cystine depletion, IL1RAP induces cystathionine gamma lyase (CTH) to activate the transsulfuration pathway for de novo cysteine synthesis. Furthermore, we show that inhibitors of Glutaminase (GLS) and Glutamate cysteine ligase (GCL), two critical enzymes for glutamate and glutathione synthesis, synergistically induce massive anoikis and ferroptosis in EwS cells. However, as a single agent, GLSi or GCLi only had moderate effects on EwS survival. Our global proteomic analysis identified dramatic adaptive changes in response to GLSi and GCLi, which may facilitate EwS cell survival and metastasis upon blockade of these metabolic processes. To further pinpoint critical regulators of anoikis and metastasis, we found that anokis suppression is governed by EWS-FLI1, a driver oncogene in EwS, and depletion of EWS-FLI1 triggered anoikis and led to cell death of EwS tumor spheroids formed in 3D cultures, whereas only a mild cytostatic effect was induced in 2D cultures. Given the predominant role of oncogenic EWS-FLI1 in anoikis suppression, we performed proteomic analysis in EwS cells +/- EWS-FLI1 depletion or gene rescue cultured under either 2D or 3D conditions prior to anoikis onset. We found that the 2D-to-3D transition induced marked global proteomic changes, suggesting a molecular reprogramming in response to 3D-induced anoikis stress that might be crucial for anoikis suppression. Moreover, a significant subset of these adaptive proteomic changes was driven by EWS-FLI1. Among these proteins, we validated that CDH11 is highly expressed in EwS compared with >1000 other human cancer cell lines, and localized on the EwS cell surface, which might be targeted via immunotherapeutic strategies. Thus, these studies provide insights into adaptive mechanisms upon perturbation of glutathione metabolism and oncogenes that drive anoikis suppression and metastasis in EwS. Citation Format: Hai-Feng Zhang, Christopher S. Hughes, Alberto Delaidelli, Yue Zhou Huang, Taras Shyp, Xiaqiu Yang, Poul H. Sorensen. Identification of metabolic adaptation mechanisms that drive anoikis suppression and metastasis in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR002.

Integrated transcriptome and proteome analyses unravel a series of early defence responses in Sarcandra glabra against Colletotrichum gloeosporioides.

Anthracnose caused by Colletotrichum gloeosporioides critically threatens the growth and commercial cultivation of Sarcandra glabra . However, the defence responses and underlying mechanisms remain unclear. Herein, we aimed to investigate the molecular reprogramming in S. glabra leaves infected with C. gloeosporioides . Leaf tissues at 0, 24 and 48h post-inoculation (hpi) were analysed by combining RNA sequencing and Tandem Mass Tag-based liquid chromatography with tandem mass spectrometry. In total, 18 441 and 25 691 differentially expressed genes were identified at 24 and 48hpi compared to 0hpi (uninoculated control), respectively. In addition, 1240 and 1570 differentially abundant proteins were discovered at 24 and 48hpi compared to 0hpi, respectively. Correlation analysis revealed that transcription and translation levels were highly consistent regarding repeatability and expression. Analyses using databases KEGG and iPATH revealed tricitric acid cycle, glycolysis/gluconeogenesis and phenylpropanoid biosynthesis were induced, whereas photosynthesis and tryptophan were suppressed. Enzymatic activity assay results were consistent with the upregulation of defence-related enzymes including superoxide dismutases, catalases, peroxidases and chitinases. The transcriptome expression results were additionally validated by quantitative real-time polymerase chain reaction analyses. This study provides insights into the molecular reprogramming in S. glabra leaves during infection, which lay a foundation for investigating the mechanisms of host-Colletotrichum interactions and breeding disease-resistant plants.

Unraveling the Sweet Secrets of HCC: Glucometabolic Rewiring in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the primary form of liver cancer. It causes ∼ 800 000 deaths per year, which is expected to increase due to increasing rates of obesity and metabolic dysfunction associated steatotic liver disease (MASLD). Current therapies include immune checkpoint inhibitors, tyrosine kinase inhibitors, and monoclonal antibodies, but these therapies are not satisfactorily effective and often come with multiple side effects and recurrences. Metabolic reprogramming plays a significant role in HCC progression and is often conserved between tumor types. Thus, targeting rewired metabolic pathways could provide an attractive option for targeting tumor cells alone or in conjunction with existing treatments. Therefore, there is an urgent need to identify novel targets involved in cancer-mediated metabolic reprogramming in HCC. In this review, we provide an overview of molecular rewiring and metabolic reprogramming of glucose metabolism in HCC to understand better the concepts that might widen the therapeutic window against this deadly cancer.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

Exosomes from cisplatin-induced dormant cancer cells facilitate the formation of premetastatic niche in bone marrow through activating glycolysis of BMSCs.

Lung cancer is the leading cause of cancer-related deaths worldwide. Chemotherapy kills most cancer cells; however, residual cells enter a dormant state. The dormant cancer cells can be reactivated under specific circumstances. The "premetastatic niche" that is suitable for colonization of cancer cells is formed before the arrival of cancer cells. Tumor-derived exosomes are the main mediators of tumorigenesis. We are aiming to elucidate the roles of exosomes from cisplatin-induced dormant lung cancer cells in the formation of premetastatic niches in bone marrow. We performed differential proteomics in dormant A549 cell- and A549 cell-derived exosomes. Non-targeted metabolomics and RNA sequencing were performed to explore the molecular and metabolic reprogramming of bone marrow stromal cells (BMSCs). The growth and metastasis of A549 cells in vivo were monitored by bioluminescence imaging. We found that Insulin-like growth factor 2 (IGF-2) and Insulin-like growth factor binding protein 2 (IGFBP2) were upregulated in dormant A549 cell-derived exosomes. BMSCs that took up exosomes from dormant A549 cells showed enhanced glycolysis and promoted the growth and metastasis of A549 cells possibly through Insulin-like growth factor 1 receptor (IGF-1R)-induced metabolic reprogramming. Inhibition of the production of lactate and IGF-1R signaling can suppress the growth and metastasis of A549 cells from bone marrow. Overall, we demonstrated that BMSCs formed a premetastatic niche upon taking up exosomes from cisplatin-induced dormant lung cancer cells. BMSCs promoted lung cancer cell growth and metastasis through the reverse Warburg effect.

EPCO-39. ADAPTIVE CONVERGENCE OF METHYLOMES REVEALS EPIGENETIC DRIVERS AND BOOSTERS OF REPEATED RELAPSES IN PATIENT-MATCHED CHILDHOOD EPENDYMOMAS AND IDENTIFIES TARGETS FOR ANTI-RECURRENCE THERAPIES

Abstract Ependymoma (EPN) is the third most common brain tumor in children and frequently recurs. Here, we report an integrated longitudinal analysis of epigenetic, genetic and tumorigenic changes in 30 patient-matched repeated relapses obtained from 10 pediatric patients to understand the mechanism of recurrences. Genome-wide DNA methylation analysis revealed stable molecular subtypes and convergent epigenetic reprogramming during serial relapses of the 5 RELA and 5 PFA EPNs that paralleled with elevated patient-derived orthotopic xenograft (PDOX) (13/27) formation in the late relapses. Differentially methylated CpGs (DMCs) preexisted in the primary tumors and persisted in the relapses (driver DMCs) were detected, ranging from 51 hypo-methylated in RELA to 148 hyper-methylated DMCs in PFA tumors; while newly acquired DMCs sustained in all the relapses but was absent in the primary tumors (booster DMCs) ranged from 38- 323 hyper-methylated DMCs in RELA and PFA EPNs, respectively. Integrated analysis of these DMC associated DNA methylation regions (DMRs) and RNAseq in both patient and PDOX tumors identified a small fraction of the differentially expressed genes (4.6±4.4% in RELA and 4.5±1.1% in PFA) as regulated by driver DMRs (e.g., up-regulated CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and booster DMRs (including the sole upregulated PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). Most these genes were novel to EPN relapses. Seven DMCs in RELA and 22 in PFA tumors were also identified as potential relapse predictors. Finally, integrating DNA methylation with histone modification identified LSD1 as a relapse driver gene. Combined treatment of a novel inhibitor SYC-836 with radiation significantly prolonged survival times in two PDOX models of recurrent PFA. This high-resolution epigenetic and genetic roadmap of EPN relapse and our 13 new PDOX models should significantly facilitate biological and preclinical studies of pediatric EPN recurrences.

Epigenetic Alterations of Repeated Relapses in Patient-matched Childhood Ependymomas

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

Cellular Heterogeneity and Molecular Reprogramming of the Host Response during Influenza Acute Lung Injury.

An exuberant host response contributes to influenza A virus (IAV) (or influenza)-mediated lung injury. However, despite significant information on the host response to IAV, the cellular framework and molecular interactions that dictate the development of acute injury in IAV-infected lungs remain incompletely understood. We performed an unbiased single-cell RNA sequencing (scRNAseq) analysis to examine the cellular heterogeneity and regulation of host responses in the IAV model of acute lung injury. At the cellular level, IAV infection promoted the overwhelming recruitment of monocytes that exhibited the cell differentiation trajectory to monocyte-derived macrophages. Together, monocytes and monocyte-derived myeloid cells constituted over 50% of the total immune cells in IAV-infected lungs. In contrast, IAV infection resulted in a significant loss of nonhematopoietic cells. Molecularly, our data show the multidimensional cell-cell communication dynamics of interferon and chemokine signaling between immune and nonimmune cells and the cell-specific molecular pathways regulating the host responses during IAV-induced lung injury. Our data provide a foundation for further exploring the mechanistic association of the IAV host response with acute lung injury. IMPORTANCE A dysregulated host response develops acute lung injury during IAV infection. However, the pathological immune mechanism(s) associated with acute lung injury during IAV infection is yet to be elucidated. In this study, we performed scRNAseq to examine the dynamics of host responses during the peak of IAV-mediated lung injury. At the cellular level, our data reveal significant myelopoiesis predominated by monocytes and macrophages and the simultaneous disruption of the nonhematopoietic cell framework, crucial for regulating inflammation and barrier integrity in IAV-infected lungs. Molecularly, we observed a complex cellular network involving cell-cell communications and a number of unique regulons dictating the outcome of interferon and chemokine responses during peak lung injury. Our data present a unique atlas of cellular changes and the regulation of global and cell-specific host responses during IAV infection. We expect that this information will open new avenues to identify targets for therapeutic intervention against IAV lung injury.

Bacillus halotolerans KKD1 induces physiological, metabolic and molecular reprogramming in wheat under saline condition

Salt stress decreases plant growth and is a major threat to crop yields worldwide. The present study aimed to alleviate salt stress in plants by inoculation with halophilic plant growth-promoting rhizobacteria (PGPR) isolated from an extreme environment in the Qinghai–Tibetan Plateau. Wheat plants inoculated with Bacillus halotolerans KKD1 showed increased seedling morphological parameters and physiological indexes. The expression of wheat genes directly involved in plant growth was upregulated in the presence of KKD1, as shown by real-time quantitative PCR (RT-qPCR) analysis. The metabolism of phytohormones, such as 6-benzylaminopurine and gibberellic acid were also enhanced. Mining of the KKD1 genome corroborated its potential plant growth promotion (PGP) and biocontrol properties. Moreover, KKD1 was able to support plant growth under salt stress by inducing a stress response in wheat by modulating phytohormone levels, regulating lipid peroxidation, accumulating betaine, and excluding Na+. In addition, KKD1 positively affected the soil nitrogen content, soil phosphorus content and soil pH. Our findings indicated that KKD1 is a promising candidate for encouraging wheat plant growth under saline conditions.

Constitutive expression of JASMONATE RESISTANT 1 induces molecular changes that prime the plants to better withstand drought

In this study, we investigated Arabidopsis thaliana plants with altered levels of the enzyme JASMONATE RESISTANT 1 (JAR1), which converts jasmonic acid (JA) to jasmonoyl-l-isoleucine (JA-Ile). Analysis of a newly generated overexpression line (35S::JAR1) revealed that constitutively increased JA-Ile production in 35S::JAR1 alters plant development, resulting in stunted growth and delayed flowering. Under drought-stress conditions, 35S::JAR1 plants showed reduced wilting and recovered better from desiccation than the wild type. By contrast, jar1-11 plants with a strong reduction in JA-Ile content were hypersensitive to drought. RNA-sequencing analysis and hormonal profiling of plants under normal and drought conditions provided insights into the molecular reprogramming caused by the alteration in JA-Ile content. Especially 35S::JAR1 plants displayed changes in expression of developmental genes related to growth and flowering. Further transcriptional differences pertained to drought-related adaptive systems, including stomatal density and aperture, but also reactive oxygen species production and detoxification. Analysis of wild type and jar1-11 plants carrying the roGFP-Orp1 sensor support a role of JA-Ile in the alleviation of methyl viologen-induced H2 O2 production. Our data substantiate a role of JA-Ile in abiotic stress response and suggest that JAR1-mediated increase in JA-Ile content primes Arabidopsis towards improved drought stress tolerance.

"Cutting the Mustard" with Induced Pluripotent Stem Cells: An Overview and Applications in Healthcare Paradigm.

Treatment of numerous ailments has been made accessible by the advent of genetic engineering, where the self-renewal property has unfolded the mysteries of regeneration, i.e., stem cells. This is narrowed down to pluripotency, the cell property of differentiating into other adult cells. The generation of induced pluripotent stem cells (iPSCs) was a major breakthrough in 2006, which was generated by a cocktail of 4 Yamanaka Factors, following which significant advancements have been reported in medical science and therapeutics. The iPSCs are reprogrammed from somatic cells, and the fascinating results focused on developing authentic techniques for their generation via molecular reprogramming mechanisms, with a plethora of molecules, like NANOG, miRNAs, andDNA modifying agents, etc. The iPSCs have exhibited reliable results in assessing the etiology and molecular mechanisms of diseases, followed by the development of possible treatments and the elimination of risks of immune rejection. The authors formulate a comprehensive review to develop a clear understanding of iPSC generation, their advantages and limitations, with potential challenges associated with their medical utility. In addition, a wide compendium of applications of iPSCs in regenerative medicine and disease modeling has been discussed, alongside bioengineering technologies for iPSC reprogramming, expansion, isolation, and differentiation. The manuscript aims to provide a holistic picture of the booming advancement of iPSC therapy, to attract the attention of global researchers, to investigate this versatile approach in treatment of multiple disorders, subsequently overcoming the challenges, in order to effectively expand its therapeutic window.