Enhancer Landscape Research Articles

Enhancer Profiling Reveals a Protective Role of RXRα Against Calcium Oxalate-Induced Crystal Deposition and Kidney Injury.

During the formation of kidney stones, the interaction between crystals and tubular epithelial cells (TECs) leads to tubular injury and dysfunction, which in turn promote stone formation. However, the molecular mechanisms underlying these changes in TECs remain elusive. Drug screening revealed that JQ1 inhibited the adhesion of calcium oxalate (CaOx) crystals to TECs. Its therapeutic effect is further confirmed in a glyoxylic acid-induced CaOx crystal deposition mouse model. Utilizing epigenomic and transcriptomic profiling, dynamic enhancer landscape and gene expression program associated with nephrolithiasis are charted. Bioinformatic analysis pinpointing the RXRα as a central transcription factor (TF) modulating enhancer activity. Importantly, the animal studies revealed that RXRα deletion promoted the CaOx crystal deposition, while its activation by Bexarotene (Bex), an FDA-approved drug, mitigated this progression. Mechanistically, under normal circumstances, RXRα inhibited nephrolithiasis-promoting genes by recruiting the HDAC3/SMART complex to repress enhancer activity. Yet, with the progression of CaOx crystal deposition, RXRα expression decreased, leading to enhancer activation and subsequent upregulation of nephrolithiasis-promoting genes. In summary, the work illustrates an epigenetic mechanism underlying TECs fate transition during CaOx crystal deposition and highlights the therapeutic potential of JQ1 and Bex in managing kidney stone diseases.

Mutation of SMARCA4 Induces Cancer Cell-Intrinsic Defects in the Enhancer Landscape and Resistance to Immunotherapy.

Cancer genomic studies have identified frequent alterations in genes encoding components of the SWI/SNF chromatin remodeling complex, including SMARCA4 and ARID1A. Importantly, clinical reports indicate that SMARCA4-mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring SMARCA4 deficiency. Specifically, models with SMARCA4 loss showed decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells and CD4+ T cells into the tumor microenvironment. SMARCA4 loss in tumor cells led to profound downregulation of STING1, IL1B, and other components of the innate immune system, as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. The deregulation of gene expression was caused by cancer cell-intrinsic reprogramming of the enhancer landscape with marked loss of chromatin accessibility at enhancers of genes involved in innate immune response, such as STING1, IL1B, type I interferon, and inflammatory cytokines. Interestingly, the transcription factor NF-κB binding motif was enriched in enhancers that lose accessibility upon SMARCA4 deficiency. Furthermore, SMARCA4 and NF-κB co-occupied the same genomic loci on enhancers associated with STING1 and IFNB1, indicating a functional interplay between SMARCA4 and NF-κB. Taken together, these findings provide the mechanistic basis for the poor response of SMARCA4-mutant tumors to immunotherapy and establish a functional link between SMARCA4 and NF-κB in innate immune and inflammatory gene expression regulation.

Abstract A020: Treatment of neuroblastoma with retinoic acid plus a KAT6A/B inhibitor induces sustained growth arrest and increases GD2 expression as a target for immunotherapy

Abstract Neuroblastoma is a malignancy of the peripheral sympathetic nervous system, accounting for 15% of childhood cancer deaths. Despite multi-modal treatment with systemic chemotherapy, surgery, radiation, two cycles of high-dose chemotherapy requiring stem cell rescue, and immunotherapy with anti-GD2 antibody treatment combined with retinoic acid, overall survival for patients is only about 50% and treatment -associated toxicity is immense. Thus, less toxic and more effective treatment options are desperately needed. Retinoic acid is clinically used to induce growth arrest of neuroblastoma cells and epigenetic rewiring of the enhancer landscape, leading to suppression of oncogenic MYCN expression. However, the epigenetic and growth suppressing effects of retinoic acid are completely reversible, leading to tumor re-growth. Here, we sought to identify epigenetic modifiers that enhance the antiproliferative effects of retinoids in neuroblastoma. In a drug screen with 452 compounds targeting epigenetic modifiers, we identified PF-9363, an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, as synergistically enhancing neuroblastoma growth arrest in combination with retinoic acid, with a BLISS synergy score of 17 when both compounds are administered at 1µM each. Importantly, the combination treatment with retinoic acid plus PF-9363 resulted in durable growth arrest of neuroblastoma, which persisted beyond retinoid withdrawal in in vitro models, in contrast to single retinoid treatment. Durable growth arrest was due to sustained downregulation of MYCN and the essential transcription factors PHOX2B and GATA3, which was caused by gene expression silencing through deposition of H3K27me3 across their regulatory elements. In xenograft models of neuroblastoma in NSG mice, the KAT6A/B inhibitor PF-9363 alone demonstrated significant anti-tumor activity when given at 5 mg/kg daily by oral gavage and tumor volumes were significantly different compared to mice treated with vehicle starting at day 4 (p<0.01) through the end of treatment on day 28 (p<0.01). Moreover, the combination treatment with retinoic acid and PF-9363 also induced significant growth suppression starting from day 4 onward, compared to the vehicle control (p<0.01), and exhibited the most sustained growth suppressing effect among all treatment conditions even after retinoic acid was withdrawn on day 14, which was significantly different from retinoid treatment alone (p<0.05), where drug withdrawal led to rapid tumor proliferation. Furthermore, expression of GD2, a cell surface target for antibody therapy, was induced on GD2low neuroblastoma through the treatment with retinoic acid plus PF-9363 after 14 days of treatment determined by flow cytometry in vitro and immunofluorescence in vivo, rendering neuroblastoma cells more susceptible for anti-GD2 immunotherapy. Our studies nominate KAT6A/B inhibition as a novel approach to enhance the effectiveness of differentiation and immunotherapies in neuroblastoma and may be relevant for other tumors of neuro-ectodermal origin. Citation Format: Mark Zimmerman, Adam Durbin, Brian Abraham, Thomas Look, Francesca Alvarez-Calderon, Ulrike Gerdemann, Nina Weichert-Leahey. Treatment of neuroblastoma with retinoic acid plus a KAT6A/B inhibitor induces sustained growth arrest and increases GD2 expression as a target for immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A020.

Palmitic acid alters enhancers/super-enhancers near inflammatory and efferocytosis-associated genes in human monocytes.

Free fatty acids like palmitic acid (PA) are elevated in obesity and diabetes and dysregulate monocyte and macrophage functions, contributing to enhanced inflammation in these cardiometabolic diseases. Epigenetic mechanisms regulating enhancer functions play key roles in inflammatory gene expression, but their role in PA-induced monocyte/macrophage dysfunction is unknown. We found that PA treatment altered the epigenetic landscape of enhancers and super-enhancers (SEs) in human monocytes. Integration with RNA-seq data revealed that PA-induced enhancers/SEs correlated with PA-increased expression of inflammatory and immune response genes, while PA-inhibited enhancers correlated with downregulation of phagocytosis and efferocytosis genes. These genes were similarly regulated in macrophages from mouse models of diabetes and accelerated atherosclerosis, human atherosclerosis, and by infectious agents. PA-regulated enhancers/SEs harbored SNPs associated with diabetes, obesity, and body mass index indicating disease relevance. We verified increased chromatin interactions between PA-regulated enhancers/SEs and inflammatory gene promoters, and reduced interactions at efferocytosis genes. PA-induced gene expression was reduced by inhibitors of BRD4, and NF-κB. PA treatment inhibited phagocytosis and efferocytosis in human macrophages. Together, our findings demonstrate that PA-induced enhancer dynamics at key monocyte/macrophage enhancers/SEs regulate inflammatory and immune genes and responses. Targeting these PA-regulated epigenetic changes could provide novel therapeutic opportunities for cardiometabolic disorders.

Microglial plasticity governed by state-specific enhancer landscapes.

Single-cell transcriptomic studies have identified distinct microglial subpopulations with shared and divergent gene signatures across development, aging and disease. Whether these microglial subsets represent ontogenically separate lineages of cells, or they are manifestations of plastic changes of microglial states downstream of some converging signals is unknown. Furthermore, despite the well-established role of enhancer landscapes underlying the identity of microglia, to what extent histone modifications and DNA methylation regulate microglial state switches at enhancers have not been defined. Here, using genetic fate mapping, we demonstrate the common embryonic origin of proliferative-region-associated microglia (PAM) enriched in developing white matter, and track their dynamic transitions into disease-associated microglia (DAM) and white matter-associated microglia (WAM) states in disease and aging contexts, respectively. This study links spatiotemporally discrete microglial states through their transcriptomic and epigenomic plasticity, while revealing state-specific histone modification profiles that govern state switches in health and disease.

Motif distribution and DNA methylation underlie distinct Cdx2 binding during development and homeostasis

Transcription factors guide tissue development by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of transcription factors. However, how transcription factors navigate chromatin features to selectively bind a small subset of all the possible genomic target loci remains poorly understood. Here we show that Cdx2—a lineage defining transcription factor that binds distinct targets in developing versus adult intestinal epithelial cells—has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic Cdx2 targets and methylated state of the CpG during development enables selective Cdx2 binding and activation of developmental enhancers and genes. In adult cells, demethylation at these enhancers prevents ectopic Cdx2 binding, instead directing Cdx2 to its canonical motif without a CpG. This shift in Cdx2 binding facilitates Ctcf and Hnf4 recruitment, establishing super-enhancers during development and homeostatic enhancers in adult cells, respectively. Induced DNA methylation in adult mouse epithelium or cultured cells recruits Cdx2 to developmental targets, promoting corecruitment of partner transcription factors. Thus, Cdx2’s differential CpG motif preferences enable it to navigate distinct DNA methylation profiles, activating genes specific to appropriate developmental stages.

DOP134 Uncovering genetic archetypes to explain patient heterogeneity in IBD

Abstract Background Genome-wide association studies (GWAS) have identified 300 loci linked to inflammatory bowel disease (IBD) risk, underscoring the genetic complexity of the disease. But these findings do not explain the significant clinical heterogeneity observed across IBD patients. Without clear biomarkers to define subtypes, the clinical impact of these genetic variants remains limited. We hypothesized that distinct genetic archetypes underlie the variability in symptoms observed among patients. We developed a battery of polygenic risk scores to capture these genetic profiles and explore their potential for more precise patient stratification. Methods We applied a three-step strategy to develop partitioned polygenic risk scores (PRS) to characterize distinct genetic profiles. First, we used a phenome-wide association approach to classify genetic risk variants into non-overlapping clusters based on their association with traits related with IBD. Second, we used functional genomics datasets to identify partitioned PRSs based on similarity in gene enhancer landscapes, gene expression changes across IBD-relevant tissues, co-expression networks in patients, and publicly available gene ontologies and biological pathways. Finally, we used a genetic algorithm to integrate the abovementioned molecularly-based PRSs and assign patients into independent clusters. This algorithm utilized artificial intelligence principles based on genetic programming, focusing on optimizing the discovery of groups of patients with most genetic differences among them. Results Our analyses identified distinct genetic archetypes in IBD patients, revealing clusters associated with specific biological pathways. The phenome-wide approach classified genome-wide significant variants into seven non-overlapping clusters, some linked to inflammatory (e.g. multiple sclerosis) and behavioral traits (e.g. sedentary behaviour) that are relevant to the comorbidity patterns observed in each patient. Through functionally-informed partitioning of genetic risk, we proposed 18,470 novel pathway-specific risk scores adapted for IBD. Notably, 25% of them significantly differentiated patients from controls. Finally, the genetic algorithm integrating these pathway-specific PRSs uncovered patient clusters with unique genetic profiles, highlighting the diverse biological underpinnings of IBD heterogeneity. Conclusion By combining GWAS results, functional genomics, and AI algorithms, we uncovered distinct genetic archetypes in IBD patients that reflect diverse biological pathways, including inflammatory and behavioral traits. This strategy improves our understanding of the genetic underpinnings of IBD heterogeneity and may serve to lay the groundwork for more precise patient stratification.

MICAL2 Promotes Pancreatic Cancer Growth and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers; thus, identifying more effective therapies is a major unmet need. In this study, we characterized the super enhancer (SE) landscape of human PDAC to identify drivers of the disease that might be targetable. This analysis revealed MICAL2 as a super enhancer-associated gene in human PDAC, which encodes the flavin monooxygenase MICAL2 that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin-related transcription factors (MRTF-A and MRTF-B). MICAL2 was overexpressed in PDAC, and high MICAL2 expression correlated with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, MICAL2 promoted both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 also inhibited macropinocytosis. MICAL2, MRTF-A, and MRTF-B influenced PDAC cell proliferation and migration and promoted cell cycle progression in vitro. Importantly, MICAL2 supported in vivo tumor growth and metastasis. Interestingly, MRTF-B, but not MRTF-A, phenocopied MICAL2-driven phenotypes in vivo. This study highlights the multiple ways in which MICAL2 impacts PDAC biology and provides a foundation for future investigations into the potential of targeting MICAL2 for therapeutic intervention.

High-resolution dissection of human cell type-specific enhancers in cis and trans activities.

High-resolution dissection of human cell type-specific enhancers in cis and trans activities.

Epigenomics and single cell CRISPR screening to investigate the risk‐modifying role of microglia in Alzheimer’s disease and multiple sclerosis

AbstractBackgroundAlzheimer’s disease (AD) and other neurodegenerative diseases (NDs) cause substantial health‐related and economic burdens, but progress towards preventative or ameliorative treatments has been limited. Genome‐wide association studies have identified hundreds of risk loci containing single nucleotide polymorphisms (SNPs) that alter risk for these diseases, but >90% of these SNPs are in noncoding regions, which are cell type‐specific and difficult to study. To address this gap, we have characterized the epigenomes of iPSC‐derived neuronal and glial cells and performed CRISPRi single cell screening to dissect the molecular and cellular mechanisms underlying 10 ND risk loci.MethodWe generated neural progenitors, excitatory neurons, astrocytes and microglia from male and female iPSCs and performed RNA‐seq, H3K27ac ChIP‐seq, and Promoter Capture Hi‐C. We compared these data to those from primary uncultured (ex vivo) human neurons, astrocytes and microglia to compare their enhancer landscapes and ND risk SNP enrichment patterns. To perform CRISPRi single cell screening we integrated a dCas9‐KRAB transgene into an iPSC safe harbor locus and developed a novel lentiviral method that allows for efficient and well‐tolerated delivery of sgRNAs to iPSC‐derived microglia‐like cells (iMGLs).ResultiMGLs displayed the strongest correlation of their enhancer landscapes with those of their ex vivo counterparts, as well as highly consistent enrichment of AD and multiple sclerosis (MS) risk SNPs in microglia‐specific enhancers. Enhancer/promoter interactions in iMGLs also overlapped significantly with those from ex vivo microglia. As these results suggest that iMGLs are a suitable model for studying ND risk loci, we performed a CRISPRi single cell screen to identify the target genes and pathways affected at 10 AD and MS risk loci.ConclusionWhile the enhancer landscapes of iPSC‐derived neuronal and glial cells vary in similarity to their ex vivo counterparts, they display similar ND risk SNP enrichments in cell type‐specific enhancers, suggesting that disease risk SNP mechanisms are largely recapitulated in iPSC‐derived cells. The iMGL epigenome is notably similar to ex vivo microglia, and CRISPRi screening of AD and MS risk loci in these cells will advance our understanding of these diseases and nominate potential therapeutic targets.

Leveraging dynamic enhancer landscapes to decode microglia phenotypes in Alzheimer’s disease

AbstractBackgroundMicroglia are the major innate immune cells of the brain and play diverse roles in brain development and homeostasis. In the context of Alzheimer’s disease, microglia acquire new phenotypes that can exert protective or pathogenic roles. Single cell and single nuclei RNA sequencing experiments have defined molecular signatures of different disease‐associated microglia states associated with protective or pathogenic functions, but the mechanisms driving these transitions are not known. As a general approach to this question, we are performing a quantitative analysis of the epigenetic landscapes of mouse and human microglia subsets in models of amyloid pathology.MethodIn collaboration with the Butovsky and Henna laboratories, we defined amyloid responsive enhancers in aged APP/PS1 mice using ATAC seq. In collaboration with the Blurton‐Jones laboratory, we used ATAC‐seq to perform quantitative analysis of enhancers in homeostatic, MHCII‐high and CD9‐high human microglia engrafted into humanized 5XFAD mice. We integrated these findings with patterns of transcription factor expression and changes in gene expression.ResultThe responses of both mouse and human microglia to amyloid pathology in vivo was associated with significant increases in ATAC‐seq signal at more than 1400 genomic locations. A large fraction of these locations correspond to putative enhancers. More than ten distinct transcription factor motifs were identified in both mouse and human amyloid responsive ATAC‐seq peaks. There was a near complete overlap of these motifs in both mouse and human microglia, which were associated with a similar set of expressed transcription factors. A subset of these factors was evaluated for their potential roles in regulating amyloid responsive genes using human iPSC derived microglia and mouse macrophage systems. Although the transcription factor motifs identified in amyloid responsive ATAC‐seq peaks were nearly identical between mouse and human microglia, there was substantial divergence in the downstream genes they regulated.ConclusionThese studies indicate that the response to amyloid involves many transcription factors, nominates specific factors for further study, and reinforces the value of engrafting human microglia into disease models as a means of investigating the impact of human‐specific non coding regulatory elements.

Mediator Subunit Med4 Enforces Metastatic Dormancy in Breast Cancer.

Long term survival of breast cancer patients is limited due to recurrence from metastatic dormant cancer cells. However, the mechanisms by which these dormant breast cancer cells survive and awaken remain poorly understood. Our unbiased genome-scale genetic screen in mice identified Med4 as a novel cancer-cell intrinsic gatekeeper in metastatic reactivation. MED4 haploinsufficiency is prevalent in metastatic breast cancer patients and correlates with poorer prognosis. Syngeneic xenograft models revealed that Med4 enforces breast cancer dormancy. Contrary to the canonical function of the Mediator complex in activating gene expression, Med4 maintains 3D chromatin compaction and enhancer landscape, by preventing enhancer priming or activation through the suppression of H3K4me1 deposition. Med4 haploinsufficiency disrupts enhancer poise and reprograms the enhancer dynamics to facilitate extracellular matrix (ECM) gene expression and integrin-mediated mechano-transduction, driving metastatic growth. Our findings establish Med4 as a key regulator of cellular dormancy and a potential biomarker for high-risk metastatic relapse.

Genetic and Epigenetic Dysregulation of Transcriptional Enhancers in Cancer

Enhancers are noncoding DNA sequences responsible for orchestrating gene expression programs by interacting with transcription factors and chromatin regulators within complex genome structures. However, their fundamental functions can be disrupted by genetic and epigenetic alterations, leading to aberrant enhancer activation or rewiring that contributes to oncogenesis. Analyzing dysregulated enhancer landscapes reveals new subtype-defining genomic features, such as enhancer hijacking, and identifies disease-relevant transcriptional regulators as potential targets for developing enhancer-targeting therapeutic strategies. Here, we delve into evolving concepts and technologies for studying enhancers; discuss how genetic, epigenetic, and topological alterations disrupt enhancer regulation to promote oncogenic gene expression; and underscore the importance of understanding the regulatory principles governing enhancer function to guide therapeutic strategies. Furthermore, we highlight key challenges and emerging opportunities in targeting enhancers and their regulators to improve cancer classification, prognosis, and treatment.

Pharmacologic Blockade of a Pioneer Transcription Factor.

Cancers frequently co-opt lineage-specific transcription factors (TF) utilized in normal development to sustain proliferation. However, the effects of these TFs on tumor development depend considerably on where in the genome they bind. A new article by Taylor and colleagues expands on previously developed diamidine compounds that obstruct the DNA binding sites of the pioneer TF PU.1 (SPI1) in acute myeloid leukemia. Immobilization and sequencing of genomic DNA targeted by these compounds revealed that these inhibitors alter the genomic binding patterns of PU.1. The authors report that their strategy constrains the genomic binding preferences of PU.1, leading to redistribution of PU.1 to promoters and other gene-proximal regions with elevated guanine/cytosine content. In this study, we discuss recent developments for targeting PU.1 in hematologic malignancies. We also explore the shared functional roles of PU.1 and SWI/SNF ATP-dependent chromatin remodeling complexes, which not only work together to sustain the enhancer landscape needed for tumor cell proliferation but also play key roles in nontumor settings.

Enhancer landscape of lung neuroendocrine tumors reveals regulatory and developmental signatures with potential theranostic implications

Well-differentiated low-grade lung neuroendocrine tumors (lung carcinoids or LNETs) are histopathologically classified as typical and atypical LNETs, but each subtype is still heterogeneous at both the molecular level and its clinical manifestation. Here, we report genome-wide profiles of primary LNETs' cis-regulatory elements by H3K27ac ChIP-seq with matching RNA-seq profiles. Analysis of these regulatory landscapes revealed three regulatory subtypes, independent of the typical/atypical classification. We identified unique differentiation signals that delineate each subtype. The "proneuronal" subtype emerges under the influence of ASCL1, SOX4, and TCF4 transcription factors, embodying a pronounced proneuronal signature. The "luminal-like" subtype is characterized by gain of acetylation at markers of luminal cells and GATA2 activation and loss of LRP5 and OTP. The "HNF+" subtype is characterized by a robust enhancer landscape driven by HNF1A, HNF4A, and FOXA3, with notable acetylation and expression of FGF signaling genes, especially FGFR3 and FGFR4, pivotal components of the FGF pathway. Our findings not only deepen the understanding of LNETs' regulatory and developmental diversity but also spotlight the HNF+ subtype's reliance on FGFR signaling. We demonstrate that targeting this pathway with FGF inhibitors curtails tumor growth both in vitro and in xenograft models, unveiling a potential vulnerability and paving the way for targeted therapies. Overall, our work provides an important resource for studying LNETs to reveal regulatory networks, differentiation signals, and therapeutically relevant dependencies.

Massively parallel reporter assays identify enhancer elements in oesophageal Adenocarcinoma.

Cancer is a disease underpinned by aberrant gene expression. Enhancers are regulatory elements that play a major role in transcriptional control and changes in active enhancer function are likely critical in the pathogenesis of oesophageal adenocarcinoma (OAC). Here, we utilise STARR-seq to profile the genome-wide enhancer landscape in OAC and identify hundreds of high-confidence enhancer elements. These regions are enriched in enhancer-associated chromatin marks, are actively transcribed and exhibit high levels of associated gene activity in OAC cells. These characteristics are maintained in human patient samples, demonstrating their disease relevance. This relevance is further underlined by their responsiveness to oncogenic ERBB2 inhibition and increased activity compared to the pre-cancerous Barrett's state. Mechanistically, these enhancers are linked to the core OAC transcriptional network and in particular KLF5 binding is associated with high level activity, providing further support for a role of this transcription factor in defining the OAC transcriptome. Our results therefore uncover a set of enhancer elements with physiological significance, that widen our understanding of the molecular alterations in OAC and point to mechanisms through which response to targeted therapy may occur.

Enhancer reprogramming underlies therapeutic utility of a SMARCA2 degrader in SMARCA4 mutant cancer.

Enhancer reprogramming underlies therapeutic utility of a SMARCA2 degrader in SMARCA4 mutant cancer.

MICAL2 Is a Super Enhancer Associated Gene that Promotes Pancreatic Cancer Growth and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.

The primitive endoderm supports lineage plasticity to enable regulative development

The primitive endoderm supports lineage plasticity to enable regulative development

SMARCA4 mutation induces tumor cell-intrinsic defects in enhancer landscape and resistance to immunotherapy.

Cancer genomic studies have identified frequent alterations in components of the SWI/SNF (SWItch/Sucrose Non- Fermenting) chromatin remodeling complex including SMARCA4 and ARID1A . Importantly, clinical reports indicate that SMARCA4 -mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. However, the mechanistic basis of immunotherapy resistance is unknown. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring SMARCA4 deficiency. Specifically, we show that SMARCA4 loss caused decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells (DCs) and CD4+ T cells into the tumor microenvironment (TME). Mechanistically, we show that SMARCA4 loss in tumor cells led to profound downregulation of STING, IL1β and other components of the innate immune system as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. We establish that this deregulation of gene expression is caused by cancer cell-intrinsic reprogramming of the enhancer landscape with marked loss of chromatin accessibility at enhancers of genes involved in innate immune response such as STING, IL1β, type I IFN and inflammatory cytokines. Interestingly, we observed that transcription factor NF-κB binding motif was highly enriched in enhancers that lose accessibility upon SMARCA4 deficiency. Finally, we confirmed that SMARCA4 and NF-κB co-occupy the same genomic loci on enhancers associated with STING and IL1β, indicating a functional interplay between SMARCA4 and NF-κB. Taken together, our findings provide the mechanistic basis for the poor response of SMARCA4 -mutant tumors to anti-PD1 immunotherapy and establish a functional link between SMARCA4 and NF-κB on innate immune and inflammatory gene expression regulation.