Identification Of Cells Research Articles

HAMSAB diet ameliorates dysfunctional signaling in pancreatic islets in autoimmune diabetes

An altered gut microbiota is associated with type 1 diabetes (T1D), affecting the production of short-chain fatty acids (SCFA) and glucose homeostasis. We previously demonstrated that enhancing serum acetate and butyrate using a dietary supplement (HAMSAB) improved glycemia in non-obese diabetic (NOD) mice and patients with established T1D. The effects of SCFA on immune-infiltrated islet cells remain to be clarified. Here, we performed single-cell RNA sequencing on islet cells from NOD mice fed an HAMSAB or control diet. HAMSAB induced a regulatory gene expression profile in pancreas-infiltrated immune cells. Moreover, HAMSAB maintained the expression of β-cell functional genes and decreased cellular stress. HAMSAB-fed mice showed preserved pancreatic endocrine cell identity, evaluated by decreased numbers of poly-hormonal cells. Finally, SCFA increased insulin levels in human β-like cells and improved transplantation outcome in NOD/SCID mice. Our findings support the use of metabolite-based diet as attractive approach to improve glucose control in T1D.

LncRNA MIR200CHG inhibits EMT in gastric cancer by stabilizing miR-200c from target-directed miRNA degradation

Gastric cancer (GC) is a heterogeneous disease, threatening millions of lives worldwide, yet the functional roles of long non-coding RNAs (lncRNAs) in different GC subtypes remain poorly characterized. Microsatellite stable (MSS)/epithelial-mesenchymal transition (EMT) GC is the most aggressive subtype associated with a poor prognosis. Here, we apply integrated network analysis to uncover lncRNA heterogeneity between GC subtypes, and identify MIR200CHG as a master regulator mediating EMT specifically in MSS/EMT GC. The expression of MIR200CHG is silenced in MSS/EMT GC by promoter hypermethylation, associated with poor prognosis. MIR200CHG reverses the mesenchymal identity of GC cells in vitro and inhibits metastasis in vivo. Mechanistically, MIR200CHG not only facilitates the biogenesis of its intronic miRNAs miR-200c and miR-141, but also protects miR-200c from target-directed miRNA degradation (TDMD) through direct binding to miR-200c. Our studies reveal a landscape of a subtype-specific lncRNA regulatory network, providing clinically relevant biological insights towards MSS/EMT GC.

A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates β-Cell Identity and Function.

As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of beta cell identity and function. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.

3D Enhancer-promoter networks provide predictive features for gene expression and coregulation in early embryonic lineages.

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages: the trophectoderm, the epiblast and the primitive endoderm. Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements through which transcriptional regulators enact these fates remain understudied. Here, we characterize, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observe extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although distinct groups of genes are irresponsive to topological changes. In each lineage, a high degree of connectivity, or 'hubness', positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a predictive model for transcriptional regulation (3D-HiChAT) that outperforms models using only 1D promoter or proximal variables to predict levels and cell-type specificity of gene expression. Using 3D-HiChAT, we identify, in silico, candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments, we validate several enhancers that control gene expression in their respective lineages. Our study identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to comprehensively understand lineage-specific transcriptional behaviors.

DEK oncoprotein participates in heterochromatin replication via SUMO-dependent nuclear bodies.

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis and stemness, but a characteristic oncogenic mechanism has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress, and delineates an avenue of further study for unraveling the contribution of DEK to cancer development.

Abstract B025: A multi-omic, spatial approach using patient prostatectomies to identify actionable drug targets for combatting prostate cancer health disparities

Abstract Prostate cancer (PC) is the most commonly diagnosed cancer among African American men (AA) and continues to be the largest difference in absolute incidence and cancer-specific deaths between AA and Caucasian men (CA). AA have a 70% higher incidence of PC than CA and over a 2 times higher mortality rate. Among men undergoing prostate biopsy In an equal access setting (i.e. the VA), AA are ~50% more likely to have PC and ~80% more likely to have high-grade PC than CA. The frequency of PC-associated genetic alterations and differences in oncogene expression in PCs is altered from CA and AA. While social economic status and environmental factors may play a role in PC health disparities, there are inherent genetic/molecular differences that account for the differences in PC outcomes between CA and AA. We sought to test our hypothesis that there is a subset of actionable gene products that are essential for enhanced PC progression in AA. A focused and clinically informed CRISPR knockout screen was conducted to identify and functionally validate race-specific actionable genes that contribute to the increased risks of fatal PC in AA. The CRISPR screen was designed leveraging a radical prostatectomy transcriptomic dataset from the Durham VA Medical Center that was linked with long-term clinical outcome data. This unique dataset consists of ~600-profiled radical prostatectomy samples of which over 50% were from AA patients. A Cox hazard analysis revealed differentially expressed genes (DEGs) which are positively associated with an increased risk of metastatic disease unique to AA PC patients. To identify drug-targetable genes, these DEGs were filtered against the drug-gene interaction database (DGIdb). This approach yielded 89 genes positively associated with an increased risk of metastatic outcome in AA and 236 genes in CA only. The CRISPR KO screen resulted in 19 genes significantly depleted genes. Of these 19 hits, 11 genes were genes associated with metastasis in AA, while 8 hits were CA genes, with 5 of 11 AA hits having documented small molecule inhibitors. To further prioritize our hits, we have conducted a spatial transcriptomic analysis of radical prostatectomy tissues from AA and CA PCs. By standard H and E staining these tumors look quite similar. But spatial analysis of AA compared to CA tissues revealed differences in immune cells and cells associated with the tumor microenvironment. This integrative multi-omic approach reveals new cell identities that may contribute to the aggressive pathogenicity of PC in AA compared to CA. CRISPR models and small molecule inhibitors of our top candidates will be used to investigate the druggability of these DEGs. This study lays the groundwork for potential therapeutics targeted toward PC in AA men. Citation Format: Felipe Segato Dezem, Aiza Akhtar, Arjumand Wanj, Sunyoung You, Hannah Chasteen, So Young Kim, Jasmine Plummer, Everardo Macias. A multi-omic, spatial approach using patient prostatectomies to identify actionable drug targets for combatting prostate cancer health disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B025.

Abstract IA047: A multiomic approach towards identifying actionable targets to combat prostate cancer health disparities

Abstract Prostate cancer (PC) is the most commonly diagnosed cancer among African American men (AA) and continues to be the largest difference in absolute incidence and cancer specific deaths between AA and Caucasian men (CA). AA have a 70% higher incidence of PC than CA and over a 2 times higher mortality rate. Among men undergoing prostate biopsy In an equal access setting (i.e. the VA), AA are ~50% more likely to have PC and ~80% more likely to have high-grade PC than CA. The frequency of PC associated genetic alterations and differences in oncogene expression in PCs is altered from CA and AA. While social economic status and environmental factors may play a role in PC health disparities, there are inherent genetic/molecular differences that account for the differences in PC outcomes between CA and AA. We sought to test our hypothesis that there is subset of actionable gene products that are essential for enhanced PC progression in AA. A focused and clinically informed CRISPR knockout screen was conducted to identify and functionally validate race specific actionable genes that contribute to the increased risks of fatal PC in AA. The CRISPR screen was designed leveraging a radical prostatectomy trancscriptomic dataset from the Durham VA Medical Center that was linked with long-term clinical outcome data. This unique dataset consists of ~600-profiled radical prostatectomy samples of which over 50% were from AA patients. A cox hazard analysis revealed differentially expressed genes (DEGs) which positively associated with increased risk of metastatic disease unique to AA PC patients. To identify drug targetable genes, these DEGs were filtered against the drug gene interaction database (DGIdb).This approach yielded 89 genes positively associated with increased risk of metastatic outcome in AA and 236 genes in CA only. The CRISPR KO screen resulted in 19 genes significantly depleted genes. Of these 19 hits, 11 genes were genes associated with metastasis in AA, while 8 hits were CA genes, with 5 of 11 AA hits have documented small molecule inhibitors. To further prioritize our hits, we have conducted spatial transcriptomic analysis of radical prostatectomy tissues from AA and CA PCs. By standard H and E staining these tumors looking quite similar. But spatial analysis of AA compared to CA tissues revealed differences in immune cells and cells associated with tumor microenvironment. These integative multiomic approach reveals new cell identities that may contribute to the aggressive pathogenicity of PC in AA compared to CA. CRISPR models and small molecule inhibitors of our top candidates will be used in investigate druggability of these DEGs. This study lays the groundwork for potential therapeutics targeted towards PC in AA men. Citation Format: Felipe Dezem, Aiza Akhtar, Sungyong You, So Young Kim, Jasmine Plummer, Everardo Macias. A multiomic approach towards identifying actionable targets to combat prostate cancer health disparities [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr IA047.

Regulatory architecture of cell identity genes and housekeeping genes.

Gene regulation and chromosome architecture are intimately linked. Genes with prominent roles in cell identity are often regulated by clusters of enhancer elements. By contrast, a recent study shows housekeeping genes are often regulated through clustering of promoters. We discuss here new regulatory insights for these two types of genes.

A reversible epigenetic memory of inflammatory injury controls lineage plasticity and tumor initiation in the mouse pancreas

Inflammation is essential to the disruption of tissue homeostasis and can destabilize the identity of lineage-committed epithelial cells. Here, we employ lineage-traced mouse models, single-cell transcriptomic and chromatin analyses, and CUT&TAG to identify an epigenetic memory of inflammatory injury in the pancreatic acinar cell compartment. Despite resolution of pancreatitis, our data show that acinar cells fail to return to their molecular baseline, with retention of elevated chromatin accessibility and H3K4me1 at metaplasia genes, such that memory represents an incomplete cell fate decision. Invivo, we find this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second insult but increased tumorigenesis with an oncogenic Kras mutation. The lowered threshold for oncogenic transformation, in turn, can be restored by blockade of MAPK signaling. Together, we define the chromatin dynamics, molecular encoding, and recall of a prolonged epigenetic memory of inflammatory injury that impacts future responses but remains reversible.

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Abstract A041: FHD-286, a potent and selective inhibitor of BRG1 and BRM, shifts metastatic uveal melanoma tumor towards a less immunosuppressive state in patient samples

Abstract BRG1/BRM are key components of the SWI/SNF complex that are critical in regulation of transcriptional programs that control cell fate and identity. Tumor cells aberrantly hijack and upregulated BRG1 levels, hijacking “stemness features”. Furthermore, BRG1 has been shown to impact immune cell polarization, driving programs associated with T cell exhaustion and an immunosuppressive cell state. Our group previously demonstrate that the combination of FHD-286 and anti-PD-1 antibody is synergistic in syngeneic mouse models from various lineages, and substantially shifted with tumor microenvironment (TME) towards a more tumor killing state (Ichikawa, K. 2022, SITC). FHD-286 was evaluated in a phase 1 dose escalation (FHD-286-001, NCT04879017), in metastatic uveal melanoma (mUM) a tumor with low response rate to standard immune checkpoint therapy as well as high levels of immunosuppressive cell infiltration. Thus, we endeavored to determine if there was evidence of biological changes in peripheral blood and TME shifts in samples taken from our phase 1 dose escalation in patients with metastatic uveal melanoma. Subjects were on a daily dosing regimen ranging from 2.5 to 10 mg or an intermittent regimen of 1-week on/1-week-off ranging from 10 to 22.5 mg in 73 patients. RNA sequencing was performed in 46 patient blood samples collected in Paxgene tubes during dose escalation (2.5 mg- 15mg, C1D1, C1D15, C3D1 and other intermediate timepoints) and analyzed for transcriptional changes associated with shifts in immune polarization and activation. Tumor biopsies were collected at screening and either Cycle 3 or end of treatment. Biomarker changes in the tumor were quantified using multiplex IF (mIF) panel (Neogenomics) to assess co-localization and spatial orientation of 20 different immune related markers in 16 patients, 13 of which had samples from screening and on-treatment. Further changes were assessed using RNA sequencing of paired biopsies in 9 patients. Analysis of peripheral blood identified changes in expression of genes associated with a reduction in immunosuppressive signatures, particularly a dose dependent reduction of FOXP3 from baseline, as well as an increase in transcripts associated with activation of neutrophils. The mIF panel highlighted a reduction in immunosuppressive cells of multiple lineages including in on treatment samples, with 12/13 patients showing a reduction of one or more of the following: FOXP3+ Tregs (8/13), PD-L1+ Macrophages (8/13), an increase in the M2 Mf to tumor cells distance (8/13), markers of T cell exhaustion (PD1) on both CD4 (7/13) and CD8 T cells (8/13), or an increase in CD8/Treg ratio (5/13). Taken together these results suggest FHD-286 may reduce the immunosuppressive “blockade”, priming mUM patients to response in combination with an immune checkpoint inhibitor. Citation Format: Liv H Johannessen, Jessica Wan, Kim Horrigan, Mike Collins, GiNell Elliott, Kana Ichikawa, Ammar Adam, Sam Agresta, Jessica Piel, Martin Hentemann. FHD-286, a potent and selective inhibitor of BRG1 and BRM, shifts metastatic uveal melanoma tumor towards a less immunosuppressive state in patient samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A041.

Age‐equivalent induced neurons from patients reveal neuronal fate loss and metabolic transformation underlying neurodegeneration in sporadic Alzheimer’s disease

AbstractBackgroundOld age is the dominating risk factor for Alzheimer’s Disease (AD), which exclusively affects people at older ages. Sporadic AD represents the overwhelming majority of all cases, but most research on AD has been performed on genetic causes and their directly related pathways, also because we were in lack of models that can reflect complex human genetics and age in a neuronal context.MethodPatient‐specific iPSC models represent an attractive solution, but iPSC reprogramming results in cellular rejuvenation and thus yields phenotypically young neurons mirroring fetal development. By contrast, direct conversion of old patient fibroblasts into induced neurons (iNs) preserves endogenous signatures of aging and produces neurons that resemble the adult human brain on an epigenome‐ and transcriptome‐wide scale. To control for the involvement of aging in AD, we generated age‐equivalent fibroblast‐derived iNs and rejuvenated iPSC‐derived neurons from a cohort of AD patients and controls.ResultPatient‐derived AD iNs reflect a hypo‐mature neuronal identity characterized by markers of stress, cell cycle, glycolytic reprogramming, and de‐differentiation, which share similarities with malignant cancer transformation and age‐dependent epigenetic erosion. We identified an isoform switch of pyruvate kinase M (PKM) to the PKM2 isoform as a trigger of a Warburg effect‐like metabolic switch, and further as a key transcriptional regulator leading to cell fate instability. Metabolomics, glucose tracing, and acetylome analysis confirmed a hyper‐acetylated state of AD iNs. This affects several splicing factors, triggering an isoform switch to PKM that ultimately leads to a loss of neuronal fate and maturity. Inhibition of the resulting citrate shunt to acetylCoA modulates the isoform switch in favor of PKM1, and re‐instates a mature and more resilient neuronal state.ConclusionWe conclude that age‐equivalent adult‐like iNs from patients are a useful model to study age‐dependent pathological signatures of AD. This iN model identifies AD‐related neuronal changes as part of an active cellular program that impairs neuronal cell identity and neuronal resilience by utilizing cancer‐related signaling cascades. We further reason that the AD‐specific metabolic switch and fate loss in neurons regulates, and is reinforced by, alternative splicing of PKM.

Abstract B048: Multiplex analysis of pancreatic, oesophageal and rectal adenocarcinoma: A cross-cancer approach on the impact of neoadjuvant therapy on the tumour microenvironment

Abstract Introduction: The prognosis of gastrointestinal malignancies has improved significantly with the introduction of neoadjuvant therapy (neoTx). For rectal (RCa) and esophageal adenocarcinoma (OCa), neoTx has become the standard of care, resulting in tumor-downsizing and significantly prolonged patient survival. However, the effects of neoTx on the topographical interactions between different populations of tumor-infiltrating immune cells, the degree of intratumoral immune infiltration, the distance to tumor cells, as well as their spatial interactions with other important features of the tumor microenvironment (TME), are still unknown. In this cross-cancer histopathological profiling of the microenvironment of rectal and esophageal tumors, we aim to investigate the local effects of cytotoxic agents and determine whether these correspond to the pathological changes observed in pancreatic cancer (PCa). Methods In this study, we employed multiplex immunohistochemistry (mIHC) based on tyramide signal amplification (TSA) using OPAL® dyes and a customized 6-plex approach to characterize tumor-associated stromal features (CD11c, CD34, NCAM, PGP9.5, aSMA, PanCK) as well as tumor-infiltrating lymphoid (CD3, CD4, CD8, CD20, FOXP3, PanCK) and myeloid cell subsets (CD11b, CD33, CD68, CD208, HLA-DR, PanCK). FFPE samples from 60 neoadjuvantly treated RCa and 40 OCa patients and a matched cohort of primary resected patients were included in the analysis. Results In PCa, neoTx alters the TME by depleting pro-tumorigenic immune cells, such as myeloid-derived suppressor cells (MDSC) and regulatory T cells, and suppressing tumor-associated stromal activation, neural invasion, and microangiogenesis. In our current study, we generated three standardized 6-plex panels for comprehensive histopathologic analysis of the immune architecture and associated stromal features of neoadjuvantly treated RCa and OCa patients compared with primary resected patients. Lymphoid subpopulations included Th-cells, cytotoxic T-cells, regulatory T-cells, and B-cells, while MDSC, dendritic cells, M1- and M2-macrophages were analyzed in the myeloid panel within the same FFPE slide. Conclusion: Our findings highlight new differential cell identities in neoTx-treated PCa patients and confirm their suitability for optimizing future clinical immunotherapy trials. Our current approach validates multiplex-IHC approaches for histological profiling of FFPE tumor samples in RCa and OCa and demonstrates the importance of a deep topographic characterization to understand the TME composition of gastrointestinal malignancies after neoTx. Citation Format: Leonard Richter. Multiplex analysis of pancreatic, oesophageal and rectal adenocarcinoma: A cross-cancer approach on the impact of neoadjuvant therapy on the tumour microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B048.

LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial ‘LIM code’ to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea . We found that lim homeobox ( lhx ) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and - lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.1 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding ( lhx6/8 ), axial patterning ( islet1 , lhx1/5-1, lmx1a/b-3 ), head/body proportions ( islet2 ) and stem cell proliferation ( lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3 ). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

Super-enhancers include classical enhancers and facilitators to fully activate gene expression

Super-enhancers are compound regulatory elements that control expression of key cell identity genes. They recruit high levels of tissue-specific transcription factors and co-activators such as the Mediator complex and contact target gene promoters with high frequency. Most super-enhancers contain multiple constituent regulatory elements, but it is unclear whether these elements have distinct roles in activating target gene expression. Here, by rebuilding the endogenous multipartite α-globin super-enhancer, we show that it contains bioinformatically equivalent but functionally distinct element types: classical enhancers and facilitator elements. Facilitators have no intrinsic enhancer activity, yet in their absence, classical enhancers are unable to fully upregulate their target genes. Without facilitators, classical enhancers exhibit reduced Mediator recruitment, enhancer RNA transcription, and enhancer-promoter interactions. Facilitators are interchangeable but display functional hierarchy based on their position within a multipartite enhancer. Facilitators thus play an important role in potentiating the activity of classical enhancers and ensuring robust activation of target genes.

Aging in the dermis: Fibroblast senescence and its significance.

Skin aging is characterized by changes in its structural, cellular, and molecular components in both the epidermis and dermis. Dermal aging is distinguished by reduced dermal thickness, increased wrinkles, and a sagging appearance. Due to intrinsic or extrinsic factors, accumulation of excessive reactive oxygen species (ROS) triggers a series of aging events, including imbalanced extracellular matrix (ECM) homeostasis, accumulation of senescent fibroblasts, loss of cell identity, and chronic inflammation mediated by senescence-associated secretory phenotype (SASP). These events are regulated by signaling pathways, such as nuclear factor erythroid 2-related factor 2 (Nrf2), mechanistic target of rapamycin (mTOR), transforming growth factor beta (TGF-β), and insulin-like growth factor 1 (IGF-1). Senescent fibroblasts can induce and accelerate age-related dysfunction of other skin cells and may even cause systemic inflammation. In this review, we summarize the role of dermal fibroblasts in cutaneous aging and inflammation. Moreover, the underlying mechanisms by which dermal fibroblasts influence cutaneous aging and inflammation are also discussed.

Abstract A152: The oncogene MYC as a driver of circadian clock disruption and dedifferentiation in the lung: implications in early lung carcinogenesis

Abstract Circadian rhythms (24-hour cycles) are disrupted in many human cancers, supporting cancer growth. Non-small cell lung cancers (NSCLCs) exhibit a disrupted molecular circadian clock, and associated alterations in circadian gene expression indicate a poor prognosis. However, it remains unclear how clock disruption occurs in NSCLCs and what NSCLCs gain from this disturbance. The oncogene MYC has been functionally implicated in circadian disruption in a variety of cancer cell lines. In NSCLCs, the MYC or one of its related family members is found mutated or amplified in more than 50% of the cases and its expression correlates with worse survival. BMAL1 is a non-redundant component of the circadian clock and is essential for the overall functionality of this machinery and the circadian regulation of gene expression. Using publicly available data we observed an inverse correlation between the expression of MYC and BMAL1 in LUAD. However, it is unknown if MYC drives clock disruption in the lung and whether clock disruption is functionally required for MYC oncogenic effects. Using an organoid model generated from isolated alveolar type II cells (ATII), the cell of origin lung adenocarcinoma (LUAD), the most prevalent type of NSCLC, we showed that MYC suppresses the expression of BMAL1. In parallel, MYC decreased the expression of specific ATII cell-lineage markers, suggesting the activation of a dedifferentiation program. Using an in vivo mouse model of NSCLC with MYC-inducible expression in ATII cells, we further demonstrated that MYC overexpression for 3 days induces an overall suppression of molecular clock genes, including BMAL1. We also observed that MYC increased the expression of proliferative markers such as Ki67 and promoted the loss of cell-lineage markers in ATII cells, which are early events in lung carcinogenesis. However, it is unclear if these effects are applicable to human LUAD. In human LUAD, we found that molecular subtypes that lack epithelial identity markers and are correlated with worse survival, show lower BMAL1 expression. These data corroborate with a potential implication of the molecular clock on the control of differentiation programs and tumor progression. Circadian clock pharmacological restoration in vivo, using the RORα agonist SR1078, attenuated the expression of the proliferation marker Ki67 induced by MYC in the lung, suggesting that clock disruption potentially mediates MYC oncogenic effects to some extent. Additionally, we performed CUT&TAG in a mouse lung epithelial cell line (MLE12) and identified that BMAL1 binds to the promoter of ATII identity genes (Sftpb, Abca3) and cell cycle genes (Cdk4), suggesting that the molecular clock potentially regulates lung epithelial cell identity and proliferation. Overall, our data identify MYC as a driver of circadian clock disruption in lung alveolar cells which is a potential mechanism implicated in MYC-driven proliferation and dedifferentiation in early-lung cancer. Circadian clock restoration might be a valuable tool to target MYC-amplified tumors. Citation Format: Juliana Cazarin de Menezes, Jamison Burchett, Siti Noor Ain Binti Ahmad Shahidan, Rachel E DeRollo, Brian Altman. The oncogene MYC as a driver of circadian clock disruption and dedifferentiation in the lung: implications in early lung carcinogenesis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A152.

Partial cellular reprogramming: A deep dive into an emerging rejuvenation technology.

Aging and age-associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging-related benefits of partial cellular reprogramming while examining limitations and future directions for the field.

Abstract A056: Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer

Abstract The Androgen Receptor (AR) cistrome is critical in the development of prostate cell identity while its misregulation promotes prostate cancer development. The pioneer transcription factor Forkhead box A1 (FOXA1) has been shown to be essential for the recruitment of AR to androgen response elements (AREs) allowing for reprogramming of the AR cistrome resulting in prostate cell transformation. FHD-286 is a BRM/BRG1 dual ATPase inhibitor currently in clinical trials for AML. Here, we show that treatment of prostate cancer cell lines with FHD-286 results in ablation of FOXA1 mediated AREs at tumor-associated AR binding sites (T-ARBS). Dual ATPase treatment subsequently reduces the expression level of various oncogenic AR target genes, leading to a decrease in tumor cell viability. Both patient derived organoid and In vivo studies provide further validation by showing a decrease in tumor growth. Strikingly, inhibition of BAF complex activity bypasses AR resistance mechanisms commonly seen after castration and Enzalutamide treatment as cell lines containing AR-V7 splice variants and neuroendocrine organoids display sensitivity. Taken together, our data illustrates a novel mechanism of treating AR mediated prostate cancer though the inhibition tumor associated AREs by treatment with FHD-286. Citation Format: Gabriel J Sandoval, Richard C Centore, Sal Topal, Dave Lahr, GiNell Elliott, Heena Gandevia, Ammar Adam, Jessica Piel, Holly M Nguyen, Eva Corey, Yu Chen, Steven Bellon, Murphy Hentemann. Treatment with dual BRG1/BRM (SMARCA4/2) inhibitor FHD-286 ablates tumor-associated androgen response elements (AREs) in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A056.