Transcriptional Dysregulation Research Articles

Cockayne Syndrome Patient iPSC-Derived Brain Organoids and Neurospheres Show Early Transcriptional Dysregulation of Biological Processes Associated with Brain Development and Metabolism.

Cockayne syndrome (CS) is a rare hereditary autosomal recessive disorder primarily caused by mutations in Cockayne syndrome protein A (CSA) or B (CSB). While many of the functions of CSB have been at least partially elucidated, little is known about the actual developmental dysregulation in this devasting disorder. Of particular interest is the regulation of cerebral development as the most debilitating symptoms are of neurological nature. We generated neurospheres and cerebral organoids utilizing Cockayne syndrome B protein (CSB)-deficient induced pluripotent stem cells derived from two patients with distinct severity levels of CS and healthy controls. The transcriptome of both developmental timepoints was explored using RNA-Seq and bioinformatic analysis to identify dysregulated biological processes common to both patients with CS in comparison to the control. CSB-deficient neurospheres displayed upregulation of the VEGFA-VEGFR2 signalling pathway, vesicle-mediated transport and head development. CSB-deficient cerebral organoids exhibited downregulation of brain development, neuron projection development and synaptic signalling. We further identified the upregulation of steroid biosynthesis as common to both timepoints, in particular the upregulation of the cholesterol biosynthesis branch. Our results provide insights into the neurodevelopmental dysregulation in patients with CS and strengthen the theory that CS is not only a neurodegenerative but also a neurodevelopmental disorder.

Paternal aging impacts expression and epigenetic markers as early as the first embryonic tissue lineage differentiation

BackgroundAdvanced paternal age (APA) is associated with adverse outcomes to offspring health, including increased risk for neurodevelopmental disorders. The aim of this study was to investigate the methylome and transcriptome of the first two early embryonic tissue lineages, the inner cell mass (ICM) and the trophectoderm (TE), from human blastocysts in association with paternal age and disease risk. High quality human blastocysts were donated with patient consent from donor oocyte IVF cycles from either APA (≥ 50 years) or young fathers. Blastocysts were mechanically separated into ICM and TE lineage samples for both methylome and transcriptome analyses.ResultsSignificant differential methylation and transcription was observed concurrently in ICM and TE lineages of APA-derived blastocysts compared to those from young fathers. The methylome revealed significant enrichment for neuronal signaling pathways, as well as an association with neurodevelopmental disorders and imprinted genes, largely overlapping within both the ICM and TE lineages. Significant enrichment of neurodevelopmental signaling pathways was also observed for differentially expressed genes, but only in the ICM. In stark contrast, no significant signaling pathways or gene ontology terms were identified in the trophectoderm. Despite normal semen parameters in aged fathers, these significant molecular alterations can adversely contribute to downstream impacts on offspring health, in particular neurodevelopmental disorders like autism spectrum disorder and schizophrenia.ConclusionsAn increased risk for neurodevelopmental disorders is well described in children conceived by aged fathers. Using blastocysts derived from donor oocyte IVF cycles to strategically control for maternal age, our data reveals evidence of methylation dysregulation in both tissue lineages, as well as transcription dysregulation in neurodevelopmental signaling pathways associated with APA fathers. This data also reveals that embryos derived from APA fathers do not appear to be compromised for initial implantation potential with no significant pathway signaling disruption in trophectoderm transcription. Collectively, our work provides insights into the complex molecular mechanisms that occur upon paternal aging during the first lineage differentiation in the preimplantation embryo. Early expression and epigenetic markers of APA-derived preimplantation embryos highlight the susceptibility of the future fetus to adverse health outcomes.

Abstract 940: Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background Liquid biopsies based on cell-free DNA (cfDNA) analysis provide non-invasive clinical diagnostic insights. Recently, cfDNA fragmentomics has emerged as a tool for inferring epigenomic and transcriptional information from tumor-derived cfDNA. We conducted a comprehensive profiling of cfDNA fragmentomics, whole transcriptome sequencing (WTS), and genome-wide DNA methylation on two metastatic castration-resistant prostate cancer (mCRPC) samples to systematically investigate molecular aberrations in mCRPC. Methods Two mCRPC patient plasma samples were extensively profiled using high-depth 150x whole-genome sequencing (WGS), 30x whole-genome methylation, and cfRNA WTS. CfDNA fragmentomics analysis was performed to assess androgen receptor (AR) transcriptional activity. We calculated fragment coverage around AR binding sites (ARBS) and quantified ARBS nucleosome profiling abnormality scores (ARBS scores) by comparing centric fragment coverage with normal plasma background. Additionally, we calculated fragment size entropy around AR promoter and enhancer regions to reflect AR gene expression activity. Fragmentomics-inferred gene activity was validated through promoter-targeted panels and WTS profiling of matched mCRPC samples. Results Both mCRPC plasma samples exhibited significant ARBS nucleosome depletion. Sample A showed a higher ARBS score compared to Sample B. Interestingly, AR enhancer activity inferred from high-depth WGS and targeted panels was high in both samples, while AR promoter activity was high only in Sample A. Genome-wide DNA methylation revealed hypo-methylation in AR enhancer and ARBS regions in both samples, with Sample A showing more pronounced hypo-methylation. Copy number variation analysis indicated AR amplification in Sample A and RB1 loss in Sample B. Collectively, these findings suggest that Sample A may represent androgen receptor-dependent prostate cancer (ARPC), while Sample B may have developed resistance to androgen therapy, possibly indicating neuroendocrine prostate cancer (NEPC). Conclusions This study, to our knowledge, is the first to combine high-depth WGS, promoter-targeted panels, WTS, and genome-wide DNA methylation to systematically study epigenomic and transcriptional dysregulation in mCRPC. The integration of mutation, copy number variation, fragmentomics, and DNA methylation profiling holds potential clinical utility for inferring prostate cancer subtypes, facilitating patient stratification, and guiding treatment selection. Citation Format: Chao Dai, Lisha Zhu, Yong Huang, Giancarlo Bonora, Kemin Zhou, Shidong Jia, Pan Du. Integrated analysis of cfDNA fragmentomics, DNA methylation and cfRNA transcription in metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 940.

Abstract 3018: Transcriptional dysregulation in germline TP53-driven breast cancers

Abstract Background: Pathogenic germline variants (PGVs) in TP53 is associated with multicancer risk syndrome known as Li-Fraumeni Syndrome (LFS) and females have 80-90% risk of breast cancer at very early age compared to sporadic breast cancers. The mechanism of p53 tumor suppression relies on its function as a transcriptional activator; however, there is little data on the specific transcriptional defects in human breast tumors driven by germline mutant p53. Methods: To investigate the specific transcriptional defects due to TP53 PGVs in human breast, we performed bulk RNA-seq on invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS) and surrounding and contralateral normal breast tissue in LFSBC compared to non-LFS sporadic BC. We also compared our results to breast tumors from The Cancer Genome Atlas (TCGA) with and without acquired TP53 mutations. Results: Our RNA-seq analysis revealed significant changes in the expression of key pathways in LFSBC compared to non-LFS breast cancer. Notably, cancer hallmark pathways such as apoptosis and reactive oxygen species were downregulated, while androgen response and cholesterol homeostasis pathways were upregulated in LFSBC compared to non-LFSBC. Expression of downstream targets of p53 such as BAX, tumor suppressors such as CDKN1A (p21) and BTG2, were significantly down in LFSBC suggesting impaired transcriptional activity of mutated TP53 in LFS patients. We also observed downregulation of cell cycle regulators in LFS breast tumors compared to their surrounding and contralateral tissues. Although, there were no significant transcriptional differences as the tumor progressed from LFS DCIS to IDC, indicating that the damage caused by the mutated p53 had already occurred at the early stage. Additionally, we also investigated the immune cell composition in LFS-BC and non-LFSBC. LFSBC exhibited a higher relative proportion of CD4+ T cells, CD20+ B cells, NK cells, macrophages and monocytes compared to all sporadic non-LFSBC subtypes (TCGA) and age matched non-LFSBC. This providing novel insights into the immune landscape associated with LFS-driven breast cancer. Conclusion: Our findings suggest that breast tissues with an inherited TP53 mutation undergo a defective transcriptional state, shifting away from apoptosis, and experiencing dysregulation of cell cycle at an early stage as DCIS, potentially contributing to the early onset of LFS breast cancer. The preliminary analysis of immune repertoire in LFSBC opens avenues for deeper investigations into spatial transcriptomic profiles of germline TP53-mutated breast tumors also the immune cells. Overall, this study provides valuable insights into the TP53 mutation driven breast cancers in humans. Citation Format: Nabamita Boruah, Renyta Moses, Ryan Hausler, Heena Desai, Anh Le, Gregory Kelly, Ashvathi Raghavakaimal, Mohana Narasimhamurthy, Anupma Nayak, Arnold J. Levine, Kara N. Maxwell. Transcriptional dysregulation in germline TP53-driven breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3018.

Abstract 141: A human induced pluripotent stem cell model of KMT2A rearranged leukemia

Abstract Acute lymphoblastic leukemia in infants (iALL) is a high-risk subtype of childhood leukemia with poor survival outcomes despite intensive therapies. Rearrangement of KMT2A (KMT2A-r) occurs in 70% of cases and is associated with refractoriness to therapy, early relapse, and rapid leukemia progression. KMT2A-r generates a driver fusion oncogene, most commonly KMT2A::AFF1 in iALL, which leads to epigenetic dysregulation of target gene transcription. iALL with KMT2A::AFF1 is an unusual cancer in that other somatic mutations are uncommon, there are no known genetic risk factors, and phenotypic switch from lymphoid to myeloid, mixed, or undifferentiated phenotype is common during treatment and particularly resistant to therapies. The cell of origin is thought to be a very early hematopoietic precursor, with transcriptomic studies of iALL blasts showing similarities to hematopoietic stem and progenitor cells (HSPCs), multipotent progenitors and early lymphoid progenitors (ELPs). Unfortunately, research into this rare but devastating disease has been hindered by a lack of appropriate, representative models. Interestingly, KMT2A-r originates in HSPCs in utero and the age of onset of iALL is a strong prognostic factor, with age younger than six months associated with the highest risk of relapse. However, little is known regarding how KMT2A-r subverts early hematopoiesis or drives the severe disease phenotype. In an effort to understand the role of the developmental state of the cell of origin in iALL, we have created a highly controlled induced pluripotent stem (iPS) cell model system of KMT2A::AFF1 leukemia. Specifically, we engineered human iPS cell lines with doxycycline regulatable expression of KMT2A::AFF1 fusion and we are employing CRISPR gene editing technology to introduce clinically identified variants of interest. Utilizing directed differentiation, we have produced functional HSPCs from iPS cells. Notably, this model recapitulates hematopoietic ontogeny, with the ability to control iALL induction at specific developmental stages. Following doxycycline treatment, we demonstrate that the cells express KMT2A::AFF1 fusion transcripts. We are utilizing single cell genomics to investigate transcriptomic changes during hematopoietic differentiation of our KMT2A::AFF1 iPS cells. We will compare these results to published data sets of single cell RNA sequencing data from healthy fetal HSPCs and to our existing iALL and pediatric KMT2A-r ALL single cell sequencing data sets. Our iPS cell based iALL model system provides the opportunity to investigate a range of critical and outstanding questions of iALL disease initiation, progression, and treatment. Citation Format: Erin Guest, Meagan Vacek, Jacqelyn Nemechek, Irina Pushel, Bradley T. Thornton, Molly Leyda, Midhat S. Farooqi, John M. Perry, Jay L. Vivian. A human induced pluripotent stem cell model of KMT2A rearranged leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 141.

Abstract 5631: YAP1 is a critical factor for FUS::DDIT3/BAF/c-Jun complex assembly in myxoid liposarcoma

Abstract Introduction: FUS::DDIT3 is the dominant fusion protein in myxoid liposarcoma (MLS), resulting from a disease-defining chromosomal translocation. Acting as an aberrant transcription factor, FUS::DDIT3 disturbs gene regulatory networks interfering with adipogenic differentiation, while driving oncogenic gene expression and proliferation. Functionally, FUS::DDIT3 has been shown to cooperate with the transcriptional coregulator YAP1 and to interact with the BAF chromatin remodeling complex. However, mechanistic details on FUS::DDIT3’s function in transcriptional dysregulation leading to proliferative effects and the required interactome are incompletely understood. This study was performed to investigate transcriptional and epigenetic players recruited by FUS::DDIT3 to drive MLS proliferation and tumorigenesis. Experimental Procedures: To study protein-protein interactions between FUS::DDIT3 and its interacting partners, co-immunoprecipitation experiments of nuclear extracts and proximity ligation assays were performed using human MLS cell lines. To further investigate dependencies of nuclear complex assembly, combined RNA interference (RNAi) experiments were conducted. MLS cell viability and proliferation were assessed using MTT assays and xCELLigence real-time analysis, respectively. RT-qPCRs were performed to analyze the expression of FUS::DDIT3 target genes. The expression of FUS::DDIT3’s interacting proteins was evaluated by immunohistochemistry in primary human MLS specimens. Results: Co-immunoprecipitation and proximity ligation assays demonstrated nuclear interactions between FUS::DDIT3, YAP1, and the canonical BAF (cBAF) complex in MLS cell lines. In addition, a novel interaction of FUS::DDIT3 with the AP-1 transcription factor c-Jun was identified. Knockdown of the cBAF-specific subunits ARID1A and ARID1B resulted in reduced MLS cell viability and proliferation. Similarly, RNAi-mediated silencing of c-Jun impaired MLS cell viability and led to decreased expression of FUS::DDIT3 target genes. Strikingly, RNAi-mediated silencing of YAP1 resulted in decreased interaction of FUS::DDIT3 with cBAF components and c-Jun in MLS cells. Conclusions: Our results identify YAP1 as a key factor mediating the nuclear interaction of FUS::DDIT3 with the cBAF complex and the AP-1 transcription factor c-Jun, both shown to be essential for MLS cell proliferation. Citation Format: Ruth Berthold, Ilka Isfort, Lorena Heinst, Eva Wardelmann, Pierre Åman, Claudia Scholl, Marcel Trautmann, Stefan Fröhling, Wolfgang Hartmann. YAP1 is a critical factor for FUS::DDIT3/BAF/c-Jun complex assembly in myxoid liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5631.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI >= |.20|, p < 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p < 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Abstract 5491: Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level

Abstract Immune exclusion and evasion are central barriers to the success of immunotherapies and cell therapies in solid tumors. Here we applied single cell spatial and perturbational transcriptomics alongside clinical,histological, and genomic profiling to elucidate immune exclusion and evasion in high-grade serous tubo-ovarian cancer (HGSC). Using high-plex spatial transcriptomics we profiled more than 1.3 million cells from 95 tumors and 60 patients, revealing generalizable principles in HGSC tumor tissue organization.Our data demonstrates that effector T cells resist stroma-mediated trapping and sequestration. However, upon infiltration into the tumor, T cells, as well as Natural Killer (NK) cells, preferentially co-localize only with a subset of malignant cells that manifest a distinct transcriptional cell state. The latter consists of dozens of co-regulated genes and is repressed under various copy number alterations. Performing CRISPR Perturb-seq screens in ovarian cancer cells, we identified functionally diverse genetic perturbations - including knockout of the insulin sensing repressor PTPN1 and the epigenetic regulator ACTR8 - that de-repress the proposed immunogenic malignant cell state identified in patients and indeed sensitize ovarian cancer cells to T cell and NK cell cytotoxicity. Taken together, our study uncovered a profound connection between somatic genetic aberrations, malignant cell transcriptional dysregulation, and immune evasion at the cellular and tissue level, allowing us to identify targets that reprogram malignant cell states as an avenue to unleash anti-tumor immune responses. Citation Format: Karmen Aguirre, Christine Yiwen-Yeh, Olivia Laveroni. Mapping ovarian cancer spatial organization uncovers immune evasion drivers at the genetic, cellular, and tissue level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5491.

Abstract 963: Comprehensive epigenomic profiling from plasma to inform therapy selection: A proof-of-concept study in cancer

Abstract BACKGROUND: Analysis of genomic alterations in circulating tumor DNA is gaining traction in clinical oncology and can provide insights into tumor biology without the need for invasive tissue sampling. In addition to genetic alterations, epigenomic reprogramming leads to transcriptional dysregulation and drives cancer phenotypes including molecular subtypes, histologic subtypes, and mechanisms resistance. As current liquid biopsy assays fail to effectively capture transcriptional biology, new approaches are needed to examine the transcriptional status of therapeutic gene targets and pathways including those relevant to rapidly emerging classes such as antibody-drug conjugates (ADC) and radio-conjugates. METHODS: We developed a novel multimodal assay that reveals genome-wide transcriptional activity of promoters, enhancers from chromatin and surveys the DNA methylome using 1ml of patient plasma. We applied this assay to patient plasma samples from across 15 tumor types generating 7000 genome-wide transcriptional profiles. RESULTS: We confirmed a significant correlation between epigenomic signals of transcriptional activation and gene expression from RNA-seq (Promoter vs RNA-seq: ⍴ =0.95, p<0.05; DNA methylation vs RNA-seq: ⍴=-0.16, p<0.05). We identified transcriptional activation in cancer patient plasma of well-established cancer-associated biomarkers (e.g. MET, DLL3, PSMA) in a tumor-specific manner. Epigenomic profiling simultaneously inferred the expression of multiple pertinent ADC targets currently under investigation (MET, HER2, HER3, TROP2, B7H4 in breast cancer), and SLFN11, a known biomarker for sensitivity to these agents. Unlike targeted panels, the platform assesses transcriptional regulation genome-wide, enabling the assessment of transcriptional activity of select genes as well as related pathways. For example, a multimodal classifier robustly predicted HER2 status across metastatic breast cancer plasma samples based on features both within the HER2 amplicon as well as HER2-distal genes that are activated in HER2+ cancers (AUC = 0.93, 0.84-1.0 95% CI). The multimodal classifier out-performed individual analyte-based classifiers (AUCPromoter+Enhancer = 0.85, 0.72-0.98 95% CI; AUCDNA-methylation = 0.69, 0.48-0.90 95% CI). Epigenomic signatures were also used to delineate multiple mechanisms of resistance to androgen receptor signaling inhibition in prostate cancer, including reactivation of the androgen receptor, glucocorticoid signaling bypass, and histological transformation to a neuroendocrine subtype. CONCLUSION: This comprehensive epigenomic liquid biopsy assay provides a platform for simultaneous genome-wide transcriptional assessment of disease and therapy relevant genes and pathways, representing a paradigm shift in our ability to longitudinally monitor cancer in individual patients. Citation Format: Travis Clark, Ji-Heui Seo, Anthony D'Ippolito, Aparna Gorthi, Jonathan Beagan, Jamey Guess, Amy Donahue, Mandy Greene, Alyssa Lau, Fernando Ramirez, Kristian Cibulskis, Hyun-Hwan Jeong, Matthew Davidsohn, Gitanjali Lakshminarayanam, Michael Coyne, Baovy Tran, Tyrone Tamakloe, Hathairat Sawaengsri, Marc Eid, Karl Semaan, Brad Fortunato, Corrie A. Painter, Juliann Chmielecki, Toni K. Choueiri, J Carl Barrett, Geoff Otto, Matthew L. Freedman, Matthew L. Eaton, Sylvan C. Baca. Comprehensive epigenomic profiling from plasma to inform therapy selection: A proof-of-concept study in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 963.

Abstract 1686: Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis

Abstract The transcription process is controlled by regulatory elements that are often located in non-coding regions. Among regulatory elements, activities of enhancers that are bound by cell-type specific transcription factors (TFs) are tightly linked to cell identity. Enhancers loop to interact with the promoters of target genes at a distance to drive different cell fates. Evidently, transcriptional dysregulation linked to enhancers that are differentially activated in cancer is an underlying factor in tumorigenesis. To identify enhancers linked to prostate tumorigenesis, we analyzed hundreds of in-house and public H3K27ac ChIP-seq data generated from normal prostate and prostate cancer cell lines and tissues. We identified thousands of prostate cancer-specific enhancers (PSEs) that were activated exclusively in cancer conditions. To further identify the target genes of PSEs, we performed 3C-derived assays, such as Hi-C and Micro-C, and mapped genome-wide interactions. However, they were limited by the resolution to detect enhancer-promoter interactions. The recent development of Region Capture Micro-C (RCMC), a hybridization-based capture Micro-C method, allowed us to deeply map the highly nested interactions between regulatory elements. By utilizing RCMC, we generated high-resolution, focal contact maps that revealed comprehensive enhancer-promoter interactions near PSEs in prostate cancer cells. Interestingly, we found that PSEs loop to other PSEs clustered in the proximal genomic region, in addition to promoters of significantly upregulated genes in cancer compared to normal cells. We termed these regions as multi-connected "3D enhancer-promoter hubs”. To mechanistically dissect these hubs, we deleted 5+ PSEs one by one using the CRISPR/Cas9 system and performed multi-omics analyses, including RNA-seq and ChIP-seq. We found that single deletions of a subset of PSEs led to the downregulation of multiple genes looped to the deleted PSE as well as to other PSEs. Moreover, we found that deletion reduced the activity of other connected PSEs. When we further evaluated the chromatin contacts of regulatory elements in the PSE-deleted cells, we observed that many chromatin contacts remained unaffected. Finally, we identified TFs that bind to PSEs, and we are currently in the process of investigating enhancer-promoter hub connectivity by performing knockdown experiments on these TFs. Furthermore, we are using molecular assays to identify PSEs that could exert a phenotypic shift in cancer cell identity to drive prostate tumorigenesis. Overall, our study not only provides mechanistic insights into the spatial regulation of 3D enhancer-promoter hubs but also identifies new non-coding drivers in prostate tumorigenesis. Citation Format: Huan Cao, Zexun Wu, Leonardo Gonzalez-Smith, Seolyn Yang, Suhn K. Rhie. Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1686.

Abstract 4400: Transcriptional dysregulation of chromosomes localized into the micronucleus

Abstract Chromosomal Instability (CIN) is a characteristic of the most aggressive and drug-resistant cancers. Chromosomally unstable cells form micronuclei (MN), a structure with an unstable nuclear membrane that spontaneously surrounds missegregating chromosomes upon mitotic exit. Several studies have demonstrated that CIN drives drug resistance and metastasis through the karyotypic diversity caused by copy number alterations. Beyond facilitating karyotypic diversity, CIN has recently been demonstrated to cause durable epigenetic alterations including reduction of the activating histone H3K27ac mark in chromosomes localized to micronuclei. This reduction in histone H3K27 acetylation persists after the relocalization of the missegregated chromosome to the primary nucleus (PN). Given the loss of transcription-associated epigenetic modifications, we hypothesized that MN would exhibit transcriptional impairment. Indeed, by using nascent RNA assays and immunofluorescence, we found that chromosomes localized into the MN are less transcriptionally active and exhibit lower levels of active RNA polymerase II (RNAP2). Transcriptional inhibitors targeting RNAP2 largely abrogate MN transcription. Conversely, class-specific histone deacetylase inhibitors (HDACi) cause increased hyperacetylation of the MN relative to the PN, which persists beyond MN reincorporation into the PN. We also hypothesized that transcription in MN depends on the manner of MN biogenesis. Preliminary data shows that MN formed from breakage-fusion-bridge (BFB) events may cause less transcriptionally active MN than ones generated from treatment with the MPS1 inhibitor, reversine. To better understand the mechanisms that regulate transcription in the MN, our ongoing work is focused on an unbiased investigation of the micronuclear transcriptome. Citation Format: Danguole Norkunaite, Duaa Al-Rawi, Samuel Bakhoum. Transcriptional dysregulation of chromosomes localized into the micronucleus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4400.

Activation of Bivalent Gene POU4F1 Promotes and Maintains Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is the most aggressive molecular subtype of breast cancer with worse prognosis and fewer treatment options. The underlying mechanisms upon BLBC transcriptional dysregulation and its upstream transcription factors (TFs) remain unclear. Here, among the hyperactive candidate TFs of BLBC identified by bioinformatic analysis, POU4F1 is uniquely upregulated in BLBC and is associated with poor prognosis. POU4F1 is necessary for the tumor growth and malignant phenotypes of BLBC through regulating G1/S transition by direct binding at the promoter of CDK2 and CCND1. More importantly, POU4F1 maintains BLBC identity by repressing ERα expression through CDK2-mediated EZH2 phosphorylation and subsequent H3K27me3 modification in ESR1 promoter. Knocking out POU4F1 in BLBC cells reactivates functional ERα expression, rendering BLBC sensitive to tamoxifen treatment. In-depth epigenetic analysis reveals that the subtype-specific re-configuration and activation of the bivalent chromatin in the POU4F1 promoter contributes to its unique expression in BLBC, which is maintained by DNA demethylase TET1. Together, these results reveal a subtype-specific epigenetically activated TF with critical role in promoting and maintaining BLBC, suggesting that POU4F1 is a potential therapeutic target for BLBC.

Transcription factor EHF interacting with coactivator AJUBA aggravates malignancy and acts as a therapeutic target for gastroesophageal adenocarcinoma

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy.

Predicting mechanism of immune response in microsatellite instability colorectal cancer

Colorectal cancer (CRC), also known as colon cancer, is the third most common cancer and the fourth most cause of cancer-related death in the world. CRC can be classified into two major subtypes, including microsatellite instability (MSI) and microsatellite stability (MSS), which showed different characteristics in immunotherapy. Low sensitivity of diagnostic biomarkers and metastasis are still the principal cause of mortality, especially in MSI. Here, applying computational programs, we identified recurring expression programs based on single cell RNA sequencing (scRNA-Seq) data of CRC cell lines. Notably, three MSI specific recurring modules were identified by non-negative matrix factorization (NMF). High NMF score genes enriched in the function of metabolism and inflammatory response. Focusing on top specific active transcription factor (TF), RUNX3 (Runt-related transcription factor 3), our results suggest that T cell infiltration was increased in RUNX3 high MSI CRC samples. Unbiased Gene Set Enrichment Analysis (GSEA) showed that RUNX3 was strongly associated with immune and metastasis related functions, such as Interferon Gamma (IFN-γ) and EPITHELIAL MESENCHYMAL TRANSITION (EMT). In addition, RUNX3 shows specific highly activated at epigenetic level in MSI compared with other gastrointestinal carcinomas. Positive correlation between RUNX3 and most immune checkpoints further confirmed RUNX3 might have crucial roles in MSI cancer progression and immunotherapy. Taken together, these results indicate significant tumor heterogeneity of two CRC subtypes at single-cell level and epigenetic modification level. These results also linked transcriptional dysregulation with immune infiltration at single-cell level in MSI, which may advance the application of scRNA-Seq technology in immunotherapy and contribute to developing novel biomarkers of this malignancy.

DNA Hypomethylation–Mediated Transcription Dysregulation Participates in Pathogenesis of Polycystic Ovary Syndrome

Polycystic ovary syndrome is a highly heterogenous and genetically complex endocrine disorder. Although the etiology remains mostly elusive, growing evidences suggested abnormal changes of DNA methylation correlate well with systemic and tissue-specific dysfunctions in polycystic ovary syndrome. Dehydroepiandrosterone-induced polycystic ovary syndrome-like mouse model was generated, which has similar metabolic and reproductive phenotype as human polycystic ovary syndrome patients, and used to experimentally validate potential role of aberrant DNA methylation in polycystic ovary syndrome in this study. Integrated DNA methylation and transcriptome analysis revealed potential role of genomic DNA hypomethylation in transcription regulation of polycystic ovary syndrome and identified several key candidate genes, including BMP4, Adcy7, Tnfaip3, and Fas, which were regulated by aberrant DNA hypomethylation. Moreover, intraperitoneal injection of S-adenosylmethionine increased overall DNA methylation level of polycystic ovary syndrome-like mice and restored expression of the candidate genes to similar levels as the control, alleviating reproductive and metabolic abnormalities in polycystic ovary syndrome-like mice. These findings provided direct evidence showing the importance of normal DNA methylation in epigenetic regulation of polycystic ovary syndrome and potential targets for diagnosis and treatment of the disease.

Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Environmentally relevant concentrations of 2,3,7,8-TCDD induced inhibition of multicellular alternative splicing and transcriptional dysregulation

Alternative splicing (AS), found in approximately 95 % of human genes, significantly amplifies protein diversity and is implicated in disease pathogenesis when dysregulated. However, the precise involvement of AS in the toxic mechanisms induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains incompletely elucidated. This study conducted a thorough global AS analysis in six human cell lines following TCDD exposure. Our findings revealed that environmentally relevant concentration (0.1 nM) of TCDD significantly suppressed AS events in all cell types, notably inhibiting diverse splicing events and reducing transcript diversity, potentially attributed to modifications in the splicing patterns of the inhibitory factor family, particularly hnRNP. And we identified 151 genes with substantial AS alterations shared among these cell types, particularly enriched in immune and metabolic pathways. Moreover, TCDD induced cell-specific changes in splicing patterns and transcript levels, with increased sensitivity notably in THP-1 monocyte, potentially linked to aberrant expression of pivotal genes within the spliceosome pathway (DDX5, EFTUD2, PUF60, RBM25, SRSF1, and CRNKL1). This study extends our understanding of disrupted alternative splicing and its relation to the multisystem toxicity of TCDD. It sheds light on how environmental toxins affect post-transcriptional regulatory processes, offering a fresh perspective for toxicology and disease etiology investigations.

CDCA7-associated global aberrant DNA hypomethylation translates to localized, tissue-specific transcriptional responses.

Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.

Impact of heterozygous ALK1 mutations on the transcriptomic response to BMP9 and BMP10 in endothelial cells from hereditary hemorrhagic telangiectasia and pulmonary arterial hypertension donors

Heterozygous activin receptor-like kinase 1 (ALK1) mutations are associated with two vascular diseases: hereditary hemorrhagic telangiectasia (HHT) and more rarely pulmonary arterial hypertension (PAH). Here, we aimed to understand the impact of ALK1 mutations on BMP9 and BMP10 transcriptomic responses in endothelial cells. Endothelial colony-forming cells (ECFCs) and microvascular endothelial cells (HMVECs) carrying loss of function ALK1 mutations were isolated from newborn HHT and adult PAH donors, respectively. RNA-sequencing was performed on each type of cells compared to controls following an 18 h stimulation with BMP9 or BMP10. In control ECFCs, BMP9 and BMP10 stimulations induced similar transcriptomic responses with around 800 differentially expressed genes (DEGs). ALK1-mutated ECFCs unexpectedly revealed highly similar transcriptomic profiles to controls, both at the baseline and upon stimulation, and normal activation of Smad1/5 that could not be explained by a compensation in cell-surface ALK1 level. Conversely, PAH HMVECs revealed strong transcriptional dysregulations compared to controls with > 1200 DEGs at the baseline. Consequently, because our study involved two variables, ALK1 genotype and BMP stimulation, we performed two-factor differential expression analysis and identified 44 BMP9-dysregulated genes in mutated HMVECs, but none in ECFCs. Yet, the impaired regulation of at least one hit, namely lunatic fringe (LFNG), was validated by RT-qPCR in three different ALK1-mutated endothelial models. In conclusion, ALK1 heterozygosity only modified the BMP9/BMP10 regulation of few genes, including LFNG involved in NOTCH signaling. Future studies will uncover whether dysregulations in such hits are enough to promote HHT/PAH pathogenesis, making them potential therapeutic targets, or if second hits are necessary.

Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.