Mesenchymal State Research Articles

MODL-40. CLONAL ARCHITECTURE, TRANSCRIPTIONAL CELLULAR STATES AND IN-VITRO DRUG RESPONSE IN GLIOBLASTOMA

Abstract Seventy-five multiregional tumors from 20 representative GBM patients (female/male ratio = 0.75, median age= 63 ys) were profiled using deep whole-exome sequencing with a nominal coverage of > 300x. The clonal complexity of each individual tumor was measured by calculating the molecular distance using the Euclidean distance of all possible paired regions based on somatic mutational profiles. Among them, fresh tumor slices of 4 patients were cultured in minimal essential media and treated with 4.2 Gy plus 200 µM temozolomide for 48 hours. Spatial Transcriptomics was performed on matched tumor slices with and without treatment. Molecular characterization of multiregional tumors revealed two patterns of evolutionary trajectory, linear and branched (linear: 8 cases, branched: 8 cases) among 16 patients. Tumors with branched evolution demonstrated considerable levels of molecular divergence, determined by high molecular distance and a low number of mutations that were shared among all regions. In-vitro drug response varied considerably between patients based on cell proliferation (minus 0.5 to 13.8 %), apoptotic rates (30.1 to 44.4 %), and cell viability (minus 7.8 to 34.1 %). Under treatment pressure an increase of mesenchymal cellular states with peculiar redistribution of transcriptional patterns and increased clonal complexity was observed. In sum, we report on an ongoing study in glioblastoma that seeks to link the clonal architecture with spatially-resolved transcriptional patterns and in-vitro drug response. Our study will provide new insights into the clinical relevance of the clonal composition of this deadly cancer.

TMIC-12. RAB27 REGULATION OF VASCULAR WALL INTEGRITY MEDIATES T-CELL INFILTERATION IN GLIOBLASTOMA

Abstract Endothelial secretome exerts a potent influence on the phenotype of cancer cells and immune microenvironment. Recently, we have reported that glioblastoma stem cells acquire a more aggressive mesenchymal state under influence of vascular endothelial cell derived extracellular vesicles (EVs). Since Rab27, GTPases involved in multiple aspects of EV (exosome) biogenesis, has been implicated in control of angiogenic pathways through regulation of VEGFR1 turnover and possibly other mechanisms, we interrogated the role of Rab27 in the function of normal and glioblastoma associated brain microvasculature. Using mice with disrupted Rab27a and Rab27b isoforms, we documented that these proteins control development and stability of the brain vasculature including pathological angiogenesis in glioblastoma. Thus, relative to wild-type C57bl/6 (WT) and double heterozygous Rab27a+/-;Rab27b+/- (dHET) mice, animals with Rab27a/b double knock-out (Rab27a/b-dKO) exhibit reduced microvascular density in brain cortices. When neoangiogensis was induced following orthotopic inoculation of mouse glioma cells (GL261), multiple morphological anomalies were observed in the tumour vasculature relative to tumours established in control mice. Moreover, vascular permeability of tumours in Rab27a/b-dKO mice markedly exceeded that of similar tumours in wild type or heterozygous controls resulting in augmented T-cell infiltration into the otherwise ‘cold’ brain tumor microenvironment. Our results suggest that key switches in the cellular secretome, such as Rab27a and Rab27b, may impact endothelial membrane dynamic and intercellular interactions capabilities, whereby they may play a critical role in ensuring the stability and sustained cellular barrier function of the vasculature during brain tumour progression. Therapeutic implications of these findings are being explored.

Abstract 13004: Blockade of Endothelial to Mesenchymal Transition (EndMT) by an AP-1 Inhibitor Attenuates Atherosclerosis in Diabetes

Background: Atherosclerotic cardiovascular disease is the leading cause of death in individuals with diabetes. Endothelial to mesenchymal transition (EndMT) is characterized by endothelial transformation to a mesenchymal like cellular state. EndMT-mediated endothelial cell dysfunction is known to be involved in cardiovascular complications of diabetes including atherosclerosis. Gene expression changes occur when endothelial cells transition to mesenchymal cells. These transcriptomic changes are regulated by multiple factors including transcription factors (TFs). Although, previous studies have implicated the role of EndMT in atherosclerosis, the role of AP-1 TF complex in EndMT in diabetes induced atherosclerosis is not known. Methods: Single cell transcriptomic (SCT) analysis of diabetic aorta in atheroprone Apoe -/- mice was performed using 10X Genomics platform. In vitro model mimicking diabetes-induced EndMT was established in human aortic endothelial cells (HAECs) using high glucose (25mM) and TNF- α ± a small molecular inhibitor of AP-1, T-5224. RNA and ChIP (chromatin Immunoprecipitation) sequencing was performed. Moreover, Apoe -/- mice made diabetic with streptozotocin, were treated with T-5224 (30 mg/kg daily for 5 weeks) in a 10-week intervention study. Results: SCT analysis of aorta identified increased expression of AP-1 members cFOS and Junb in diabetic endothelial cells in Apoe -/- mice. These genes were also upregulated in the HAECs-based in vitro EndMT model. Interestingly, AP-1 inhibition by T-5224 attenuated EndMT . RNA and ChIP sequencing identified several AP-1 target genes including Col1A2, Col4A1, TGFB, THBD, INSR, PIK3R3 (FDR <0.05, Log2 fold change (2x)) that were common to many enriched pathways including “leukocyte trans-endothelial migration” and “AGE-RAGE signaling pathways in diabetic complications” directly relevant to EndMT in diabetes. Importantly, T-5224 treatment blocked EndMT resulting in reduced atherosclerosis in diabetic Apoe -/- mice. Conclusion: This study identified the AP-1 inhibition with T-5224 as a novel treatment for the first time for the diabetes induced EndMT and atherosclerosis.

Diagnostically significant dysplastic feature identification as a tool for managing connective tissue risks in sports medicine

Aim: to identificate the diagnostically significant signs of connective tissue dysplasia, detection of which will allow adequate sports activity selection, injury prevention and effective rehabilitation.Materials and methods: clinical-somatometric examination of 117 persons aged 18 to 49 years (35.14 ± 5.63 years) with recurrent musculoskeletal injuries occurred during habitual physical training was carried out. Corresponding subgroups were formed by age and gender. Ranked by significance clinic-morphological dysplastic features, identified by T.I. Kadurina and L.N. Abbakumova (2008), were used to assess the mesenchymal tissue state. The control group, comparable to the main group by age and sex, was represented by 36 healthy young people.Results: persons with a predisposition to musculoskeletal injuries have a high dysplastic sign incidence. It was proved that clinically significant of them are moderate to high degree myopia, gothic palate, excessively soft auricles, “crunch” during movements in the temporomandibular joint, pathological spine kyphosis, joint hypermobility, chest deformities, O- and X-shaped legs and varicose lower limbs. Clinically significant markers of connective tissue dysplasia were found to be modified with age and to have characteristic gender features.Conclusions: the study results have particular importance for injury-free physical activity and medical-biological support for sports activities.

THU491 GRHL2 Overexpression In Breast Cancer Progression

Abstract Disclosure: C. Zheng: None. K.O. Allen: None. R. Reese: None. N.M. Solodin: None. J. Vera: None. S. Mcilwain: None. T. Liu: None. E.T. Alarid: None. Metastasis constitutes the permissive migration and growth of cancerous cells from the primary tumor to a distant location in the body. Epithelial to mesenchymal transition (EMT) is implicated in metastasis as it allows for cells to abandon an epithelial identity, adopt a mesenchymal phenotype, become migratory, and promote tumor progression. Grainyhead-like protein 2 (GRHL2) is a nuclear transcription factor responsible for driving epithelial cell fate. By regulating epithelial differentiation and integrity in estrogen receptor (ER) positive breast cancer, GRHL2 suppresses EMT, and as such, it was long thought to act primarily as a tumor suppressor. However, clinical evidence contradicts its tumor suppressive role in that patients expressing high levels of GRHL2 have a significantly lower probability of long-term recurrence free survival compared to low levels of GRHL2, and high levels of GRHL2 are also associated with high grade tumors. To better understand this discrepancy, a tetracycline (tet) inducible GRHL2 overexpression model in the ER-positive breast cancer MCF7 cell line was developed to assess cancer associated phenotypes and the transition between epithelial and mesenchymal states. Dose response and time course studies were performed to determine optimal GRHL2 induction in our tet-inducible model using immunofluorescent microscopy and Western blot analysis. Gene expression analyses was performed to assess representative genes associated with epithelial and mesenchymal states, and the intermediates between them known as hybrid or partial EMT. Interestingly, immunocytochemistry identified that the overexpression of GRHL2 induces the late EMT marker, vimentin, despite the inhibition of migration in the epithelial dominant MCF7 cell line. Together, these data indicates that GRHL2 overexpression leads to a complex phenotype that cannot be defined by a binary epithelial or mesenchymal state. Further analyses are aimed at understanding the pleiotropic roles GRHL2 plays in ER+ tumor progression. Presentation: Thursday, June 15, 2023

BULK RNA-SEQ DECONVOLUTION OF IMAGE-LOCALIZED HIGGRADE GLIOMA BIOPSIES REVEALS MEANINGFUL CELLULAR STATES

Abstract AIMS High-grade glioma continues to have dismal survival with current standard-of-care treatment, owing in part to its intra- and inter-patient heterogeneity. Typical diagnostic biopsies are taken from the dense tumor core to determine the presence of abnormal cells and the status of a few key genes (e.g. IDH1, MGMT). However, the tumor core is typically resected, leaving behind possibly genetically, transcriptomically and/or phenotypically distinct invasive margins that repopulate the disease. As these remaining populations are the ones ultimately being treated, it is important to know their compositional differences from the tumor core. We aim to identify the phenotypic niches defined by the relative composition of key cellular populations and understand their variation amongst patients. METHOD We have established an image-localized research biopsy study, that samples from both the invasive margin and tumor core. From this protocol, we currently have 202 samples from 58 patients with available bulk RNA-Seq, collected between Mayo Clinic and Barrow Neurological Institute. Using a single-cell reference dataset from our collaborators at Columbia University, we used CIBERSORTx, a deconvolution method, to predict relative abundances of 7 normal, 6 glioma, and 5 immune cell states for each sample. RESULTS We find that these cell state abundances connect to patient survival and show regional differences. For example, proneural glioma states were higher in invasive regions, whereas proliferative and mesenchymal states were higher in the tumor core. CONCLUSIONS Our analysis demonstrates a need to characterize the residual tissue following glioma resection to better understand the recurrent disease.

Enhancing Transcriptional Reprogramming of Mesenchymal Glioblastoma with Grainyhead-like 2 and HDAC Inhibitors Leads to Apoptosis and Cell-Cycle Dysregulation.

Glioblastoma (GBM) tumor cells exhibit mesenchymal properties which are thought to play significant roles in therapeutic resistance and tumor recurrence. An important question is whether impairment of the mesenchymal state of GBM can sensitize these tumors to therapeutic intervention. HDAC inhibitors (HDACi) are being tested in GBM for their ability promote mesenchymal-to-epithelial transcriptional (MET) reprogramming, and for their cancer-specific ability to dysregulate the cell cycle and induce apoptosis. We set out to enhance the transcriptional reprogramming and apoptotic effects of HDACi in GBM by introducing an epithelial transcription factor, Grainyhead-like 2 (GRHL2), to specifically counter the mesenchymal state. GRHL2 significantly enhanced HDACi-mediated MET reprogramming. Surprisingly, we found that inducing GRHL2 in glioma stem cells (GSCs) altered cell-cycle drivers and promoted aneuploidy. Mass spectrometry analysis of GRHL2 interacting proteins revealed association with several key mitotic factors, suggesting their exogenous expression disrupted the established mitotic program in GBM. Associated with this cell-cycle dysregulation, the combination of GRHL2 and HDACi induced elevated levels of apoptosis. The key implication of our study is that although genetic strategies to repress the mesenchymal properties of glioblastoma may be effective, biological interactions of epithelial factors in mesenchymal cancer cells may dysregulate normal homeostatic cellular mechanisms.

MicroRNA-200b-mediated reversion of a spectrum of epithelial-to-mesenchymal transition states in recessive dystrophic epidermolysis bullosa squamous cell carcinomas.

Cutaneous squamous cell carcinoma (SCC) is the leading cause of death in patients with recessive dystrophic epidermolysis bullosa (RDEB). However, the survival time from first diagnosis differs between patients; some tumours spread particularly fast, while others may remain localized for years. As treatment options are limited, there is an urgent need for further insights into the pathomechanisms of RDEB tumours, to foster therapy development and support clinical decision-making. To investigate differences in RDEB tumours of diverging aggressiveness at the molecular and phenotypic level, with a particular focus on epithelial-to-mesenchymal (EMT) transition states and thus microRNA-200b (miR-200b) as a regulator. Primary RDEB-SCC keratinocyte lines were characterized with respect to their EMT state. For this purpose, cell morphology was classified and the expression of EMT markers analysed using immunofluorescence, flow cytometry, semi-quantitative reverse transcriptase polymerase chain reaction and Western blotting. The motility of RDEB-SCC cells was determined and conditioned medium of RDEB-SCC cells was used to treat endothelial cells in an angiogenesis assay. In addition, we mined previously generated microRNA (miRNA) profiling data to identify a candidate with potential therapeutic relevance and performed transient miRNA transfection studies to investigate the candidate's ability to reverse EMT characteristics. We observed high variability in EMT state in the RDEB-SCC cell lines, which correlated with in situ analysis of two available patient biopsies and respective clinical disease course. Furthermore, we identified miR-200b-3p to be downregulated in RDEB-SCCs, and the extent of deregulation significantly correlated with the EMT features of the various tumour lines. miR-200b-3p was reintroduced into RDEB-SCC cell lines with pronounced EMT features, which resulted in a significant increase in epithelial characteristics, including cell morphology, EMT marker expression, migration and angiogenic potential. RDEB-SCCs exist in different EMT states and the level of miR-200b is indicative of how far an RDEB-SCC has gone down the EMT path. Moreover, the reintroduction of miR-200b significantly reduced mesenchymal features.

P02.24.B A DRUG DISCOVERY PLATFORM FOR COMBINATORIAL TARGETING OF CELL STATES AND ENTITIES

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited therapeutic options. Therapeutic resistance arises from the collaboration among heterogeneous and highly plastic cellular entities and states conserved across GBM patients and the tumor microenvironment.Tumor cells adapt in response to the current standard of care and infiltration of innate immune cells, recurrently acquiring a mesenchymal state that drives therapeutic resistance.Moreover, the blood-brain barrier (BBB) poses challenges to the effectiveness and bioavailability of approved therapeutics, as well as the discovery of new ones.Our lab has developed synthetic locus control regions (sLCRs) as transcriptional reporters for glioblastoma subtypes, enabling the study of cell identity and state transitions in vitro and in vivo. We have combined this tool with validated cellular models for glioblastoma mesenchymal transition and therapeutic resistance to establish an in vitro phenotypic screening platform. Currently, we are utilizing this platform to identify treatment combinations and dosing schemes that aim to overcome innate immunity-driven resistance and synergize with the standard of care. As a proof-of-concept for a new generation of brain tumor target discovery platforms, we will present the setup of our platform and preliminary results for a class of BBB-penetrant drugs.

RNA-Binding Proteins Regulate Post-Transcriptional Responses to TGF-β to Coordinate Function and Mesenchymal Activation of Murine Endothelial Cells.

Endothelial cells (ECs) are primed to respond to various signaling cues. For example, TGF (transforming growth factor)-β has major effects on EC function and phenotype by driving ECs towards a more mesenchymal state (ie, triggering endothelial to mesenchymal activation), a dynamic process associated with cardiovascular diseases. Although transcriptional regulation triggered by TGF-β in ECs is well characterized, post-transcriptional regulatory mechanisms induced by TGF-β remain largely unknown. Using RNA interactome capture, we identified global TGF-β driven changes in RNA-binding proteins in ECs. We investigated specific changes in the RNA-binding patterns of hnRNP H1 (heterogeneous nuclear ribonucleoprotein H1) and Csde1 (cold shock domain containing E1) using RNA immunoprecipitation and overlapped this with RNA-sequencing data after knockdown of either protein for functional insight. Using a modified proximity ligation assay, we visualized the specific interactions between hnRNP H1 and Csde1 and target RNAs in situ both in vitro and in mouse heart sections. Characterization of TGF-β-regulated RBPs (RNA-binding proteins) revealed hnRNP H1 and Csde1 as key regulators of the cellular response to TGF-β at the post-transcriptional level, with loss of either protein-promoting mesenchymal activation in ECs. We found that TGF-β drives an increase in binding of hnRNP H1 to its target RNAs, offsetting mesenchymal activation, but a decrease in Csde1 RNA-binding, facilitating this process. Both, hnRNP H1 and Csde1, dynamically bind and regulate specific subsets of mRNAs related to mesenchymal activation and endothelial function. Together, we show that RBPs play a key role in the endothelial response to TGF-β stimulation at the post-transcriptional level and that the RBPs hnRNP H1 and Csde1 serve to maintain EC function and counteract mesenchymal activation. We propose that TGF-β profoundly modifies RNA-protein interaction entailing feedback and feed-forward control at the post-transcriptional level, to fine-tune mesenchymal activation in ECs.

Preoperative Locoregional Therapy May Relate with Stemness and Distinct Transitions Between Epithelial and Mesenchymal States in Hepatocellular Carcinoma

Preoperative Locoregional Therapy May Relate with Stemness and Distinct Transitions Between Epithelial and Mesenchymal States in Hepatocellular Carcinoma

Transcriptomic Maps of Colorectal Liver Metastasis: Machine Learning of Gene Activation Patterns and Epigenetic Trajectories in Support of Precision Medicine.

The molecular mechanisms of the liver metastasis of colorectal cancer (CRLM) remain poorly understood. Here, we applied machine learning and bioinformatics trajectory inference to analyze a gene expression dataset of CRLM. We studied the co-regulation patterns at the gene level, the potential paths of tumor development, their functional context, and their prognostic relevance. Our analysis confirmed the subtyping of five liver metastasis subtypes (LMS). We provide gene-marker signatures for each LMS, and a comprehensive functional characterization that considers both the hallmarks of cancer and the tumor microenvironment. The ordering of CRLMs along a pseudotime-tree revealed a continuous shift in expression programs, suggesting a developmental relationship between the subtypes. Notably, trajectory inference and personalized analysis discovered a range of epigenetic states that shape and guide metastasis progression. By constructing prognostic maps that divided the expression landscape into regions associated with favorable and unfavorable prognoses, we derived a prognostic expression score. This was associated with critical processes such as epithelial-mesenchymal transition, treatment resistance, and immune evasion. These factors were associated with responses to neoadjuvant treatment and the formation of an immuno-suppressive, mesenchymal state. Our machine learning-based molecular profiling provides an in-depth characterization of CRLM heterogeneity with possible implications for treatment and personalized diagnostics.

ZHX2 deficiency enriches hybrid MET cells through regulating E-cadherin expression

Growing evidence indicates that the epithelial to mesenchymal (E/M) hybrid state plays a key role in tumorigenesis. Importantly, a hybrid mesenchymal to epithelial transition (MET) state in which individual cells express both epithelial and mesenchymal markers was recently identified in vivo, further strengthening the bonds between the hybrid EMT state and cancer progression. However, the role and the molecular mechanisms by which the hybrid MET state is maintained in triple-negative breast cancer cells (TNBC) remain elusive. Here, we find that loss of ZHX2 expression results in the hybrid MET phenotype in mesenchymal TNBC cells. Mechanistically, through directly binding to the CDH1 promoter, depletion of ZHX2 specifically reactivates expression of CDH1 encoding E-cadherin, an epithelial marker that is crucial for maintaining epithelial phenotype. Functionally, loss of ZHX2 expression enriches the hybrid MET cells and inhibits the migration and dissemination of TNBC cells or organoids, which could be reversed by restoration of E-cadherin. Moreover, depletion of ZHX2 suppresses lung metastasis in preclinical models of TNBC. In patients with TNBC, ZHX2 expression was amplified and negatively correlated with the expression of E-cadherin. These findings suggest that loss of ZHX2 promotes the hybrid MET state to impair TNBC progression.

Dual Role of CXCL8 in Maintaining the Mesenchymal State of Glioblastoma Stem Cells and M2-Like Tumor-Associated Macrophages.

The dynamic interplay between glioblastoma stem cells (GSC) and tumor-associated macrophages (TAM) sculpts the tumor immune microenvironment (TIME) and promotes malignant progression of glioblastoma (GBM). However, the mechanisms underlying this interaction are still incompletely understood. Here, we investigate the role of CXCL8 in the maintenance of the mesenchymal state of GSC populations and reprogramming the TIME to an immunosuppressive state. We performed an integrative multi-omics analyses of RNA sequencing, GBM mRNA expression datasets, immune signatures, and epigenetic profiling to define the specific genes expressed in the mesenchymal GSC subsets. We then used patient-derived GSCs and a xenograft murine model to investigate the mechanisms of tumor-intrinsic and extrinsic factor to maintain the mesenchymal state of GSCs and induce TAM polarization. We identified that CXCL8 was preferentially expressed and secreted by mesenchymal GSCs and activated PI3K/AKT and NF-κB signaling to maintain GSC proliferation, survival, and self-renewal through a cell-intrinsic mechanism. CXCL8 induced signaling through a CXCR2-JAK2/STAT3 axis in TAMs, which supported an M2-like TAM phenotype through a paracrine, cell-extrinsic pathway. Genetic- and small molecule-based inhibition of these dual complementary signaling cascades in GSCs and TAMs suppressed GBM tumor growth and prolonged survival of orthotopic xenograft-bearing mice. CXCL8 plays critical roles in maintaining the mesenchymal state of GSCs and M2-like TAM polarization in GBM, highlighting an interplay between cell-autonomous and cell-extrinsic mechanisms. Targeting CXCL8 and its downstream effectors may effectively improve GBM treatment.

Combinatorial targeting of a specific EMT/MET network by macroH2A variants safeguards mesenchymal identity.

Generation of induced pluripotent stem cells from specialized cell types provides an excellent model to study how cells maintain their stability, and how they can change identity, especially in the context of disease. Previous studies have shown that chromatin safeguards cell identity by acting as a barrier to reprogramming. We investigated mechanisms by which the histone macroH2A variants inhibit reprogramming and discovered that they work as gate keepers of the mesenchymal cell state by blocking epithelial transition, a step required for reprogramming of mouse fibroblasts. More specifically, we found that individual macroH2A variants regulate the expression of defined sets of genes, whose overall function is to stabilize the mesenchymal gene expression program, thus resisting reprogramming. We identified a novel gene network (MSCN, mesenchymal network) composed of 63 macroH2A-regulated genes related to extracellular matrix, cell membrane, signaling and the transcriptional regulators Id2 and Snai2, all of which function as guardians of the mesenchymal phenotype. ChIP-seq and KD experiments revealed a macroH2A variant-specific combinatorial targeting of the genes reconstructing the MSCN, thus generating robustness in gene expression programs to resist cellular reprogramming.

A Review of the Transition of Oral Keratinocytes from an Epithelial to a Mesenchymal State in Oral Submucous Fibrosis and the Potential Function of Apamin in Reversing This Transition

Epithelial–mesenchymal transition (EMT) causes polarized and cohesive epithelial cells to become motile and join the extracellular matrix (ECM). Embryonic development, wound healing, and tissue repair need it. Interestingly, the same mechanism may cause cancer, organ fibrosis, scarring, and organ failure. WNT, Notch, Hedgehog, and RTK signaling impact EMT. This process also includes nontranscriptional changes due to growth hormones, cytokines, hypoxia, and ECM interaction. The reason for the development of oral submucous fibrosis (OSF) is believed to be multifaceted; nevertheless, there is substantial evidence supporting the notion that it arises from dysregulation of collagen. EMT is a prominent phenomenon in the development of OSF, whereby myofibroblasts and keratinocytes are the cells most affected. The role of EMTs is crucial in both physiological and pathological processes. The significance of EMT involvement in the pathogenesis of OSF and the preceding inflammatory response suggests a promising avenue for further investigation. Transforming growth factor-1 (TGF-1) plays a crucial role in the EMT of oral keratinocytes that initiates the pathogenesis of OSF. The objective of this review is to analyze the events of EMT in OSF, along with the processes and molecular routes that regulate alterations in gene expression within the oral cavity. The potential for cancerous transformation is linked to OSF, prompting an examination of the involvement of apamin in the advancement of EMT triggered by TGF-1 in oral keratinocytes.

The discovery, function, and regulation of epithelial splicing regulatory proteins (ESRP) 1 and 2.

Alternative splicing is a broad and evolutionarily conserved mechanism to diversify gene expression and functionality. The process relies on RNA binding proteins (RBPs) to recognize and bind target sequences in pre-mRNAs, which allows for the inclusion or skipping of various alternative exons. One recently discovered family of RBPs is the epithelial splicing regulatory proteins (ESRP) 1 and 2. Here, we discuss the structure and physiological function of the ESRPs in a variety of contexts. We emphasize the current understanding of their splicing activities, using the classic example of fibroblast growth factor receptor 2 mutually exclusive splicing. We also describe the mechanistic roles of ESRPs in coordinating the splicing and functional output of key signaling pathways that support the maintenance of, or shift between, epithelial and mesenchymal cell states. In particular, we highlight their functions in the development of mammalian limbs, the inner ear, and craniofacial structure while discussing the genetic and biochemical evidence that showcases their conserved roles in tissue regeneration, disease, and cancer pathogenesis.

DIPG-08. THE LANDSCAPE OF TUMOR CELL STATES AND SPATIAL ORGANIZATION IN H3-K27M MUTANT DIFFUSE MIDLINE GLIOMA ACROSS AGE AND LOCATION

Abstract Histone 3 lysine27-to-methionine mutations (H3-K27M) frequently occur in childhood diffuse midline gliomas (DMGs) of the pons, thalamus, and spinal cord, presumed to be driven by the specific spatiotemporal context of these midline locations during postnatal development. While most common in the pons and at mid-childhood ages, the same oncohistone mutation is recurrently detected in adult DMGs and throughout different midline regions. The potential heterogeneity of tumors at different ages and in different anatomical locations of the midline are vastly understudied. Through dissecting the transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs - spanning the age range from 2-68 years and locations from spinal cord to thalamus - at single cell resolution, we delineate how age- and location-dependent contexts shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We identify that oligodendrocyte precursor (OPC)-like cells constitute the stem-like compartment in H3-K27M DMGs across all clinico-anatomical groups, however, depending on location, display varying levels of maturity resembling less differentiated pre-OPCs or more mature OPCs further differentiated along the oligodendroglial lineage. We further demonstrate increased mesenchymal cell states in adult tumors, which we link to age-related differences in glioma-associated immune cell compartments, in particular an increase of macrophages in adult compared to pediatric tumors. Furthermore, we resolve the spatial organization of H3-K27M DMG cell types and states in intact patient tissues, identifying a local niche of the oligodendroglial lineage. Our study provides a powerful resource for rational modeling and therapeutic frameworks taking into account determinants of age and location in this lethal glioma group.

Unpacking the Complexity of Epithelial Plasticity: From Master Regulator Transcription Factors to Non-Coding RNAs.

Cellular plasticity in cancer enables adaptation to selective pressures and stress imposed by the tumor microenvironment. This plasticity facilitates the remodeling of cancer cell phenotype and function (such as tumor stemness, metastasis, chemo/radio resistance), and the reprogramming of the surrounding tumor microenvironment to enable immune evasion. Epithelial plasticity is one form of cellular plasticity, which is intrinsically linked with epithelial-mesenchymal transition (EMT). Traditionally, EMT has been regarded as a binary state. Yet, increasing evidence suggests that EMT involves a spectrum of quasi-epithelial and quasi-mesenchymal phenotypes governed by complex interactions between cellular metabolism, transcriptome regulation, and epigenetic mechanisms. Herein, we review the complex cross-talk between the different layers of epithelial plasticity in cancer, encompassing the core layer of transcription factors, their interacting epigenetic modifiers and non-coding RNAs, and the manipulation of cancer immunogenicity in transitioning between epithelial and mesenchymal states. In examining these factors, we provide insights into promising therapeutic avenues and potential anti-cancer targets.

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential invivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells invivo.