An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Cancer-Specific Retargeting of BAF Complexes by a Prion-like Domain.

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs.

Epigenome editing of microsatellite repeats defines tumor-specific enhancer functions and dependencies.

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies.

IMPs: an RNA-binding protein family that provides a link between stem cell maintenance in normal development and cancer.

IMPs, also known as insulin-like growth factor 2 (IGF2) messenger RNA (mRNA)-binding proteins (IGF2BPs), are highly conserved oncofetal RNA-binding proteins (RBPs) that regulate RNA processing at several levels, including localization, translation, and stability. Three mammalian IMP paralogs (IMP1-3) have been identified that are expressed in most organs during embryogenesis, where they are believed to play an important role in cell migration, metabolism, and stem cell renewal. Whereas some IMP2 expression is retained in several adult mouse organs, IMP1 and IMP3 are either absent or expressed at very low levels in most tissues after birth. However, all three paralogs can be re-expressed upon malignant transformation and are found in a broad range of cancer types where their expression often correlates with poor prognosis. IMPs appear to resume their physiological functions in malignant cells, which not only contribute to tumor progression but participate in the establishment and maintenance of tumor cell hierarchies. This review summarizes our current understanding of the functions of IMPs during normal development and focuses on a series of recent observations that have provided new insight into how their physiological functions enable IMPs to play a potentially key role in cancer stem cell maintenance and tumor growth.

Mesenchymal stromal cells in cancer: a review of their immunomodulatory functions and dual effects on tumor progression.

Mesenchymal stem or stromal cells (MSCs) are pluripotent cells implicated in a broad range of physiological events, including organogenesis and maintenance of tissue homeostasis as well as tissue regeneration and repair. Because their current definition is somewhat loose - based primarily on their ability to differentiate into a variety of mesenchymal tissues, adhere to plastic, and express, or lack, a handful of cell surface markers - MSCs likely encompass several subpopulations, which may have diverse properties. Their diversity may explain, at least in part, the pleiotropic functions that they display in different physiological and pathological settings. In the context of tissue injury, MSCs can respectively promote and attenuate inflammation during the early and late phases of tissue repair. They may thereby act as sensors of the inflammatory response and secrete mediators that boost or temper the response as required by the stage of the reparatory and regenerative process. MSCs are also implicated in regulating tumor development, in which they are increasingly recognized to play a complex role. Thus, MSCs can both promote and constrain tumor progression by directly affecting tumor cells via secreted mediators and cell-cell interactions and by modulating the innate and adaptive immune response. This review summarizes our current understanding of MSC involvement in tumor development and highlights the mechanistic underpinnings of their implication in tumor growth and progression. © 2020 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells.

Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.

Systematic identification of genomic markers of drug sensitivity in cancer cells.

Clinical responses to anticancer therapies are often restricted to a subset of patients. In some cases, mutated cancer genes are potent biomarkers for responses to targeted agents. Here, to uncover new biomarkers of sensitivity and resistance to cancer therapeutics, we screened a panel of several hundred cancer cell lines--which represent much of the tissue-type and genetic diversity of human cancers--with 130 drugs under clinical and preclinical investigation. In aggregate, we found that mutated cancer genes were associated with cellular response to most currently available cancer drugs. Classic oncogene addiction paradigms were modified by additional tissue-specific or expression biomarkers, and some frequently mutated genes were associated with sensitivity to a broad range of therapeutic agents. Unexpected relationships were revealed, including the marked sensitivity of Ewing's sarcoma cells harbouring the EWS (also known as EWSR1)-FLI1 gene translocation to poly(ADP-ribose) polymerase (PARP) inhibitors. By linking drug activity to the functional complexity of cancer genomes, systematic pharmacogenomic profiling in cancer cell lines provides a powerful biomarker discovery platform to guide rational cancer therapeutic strategies.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells.

Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.

Recruitment of Matrix Metalloproteinase-9 (MMP-9) to the Fibroblast Cell Surface by Lysyl Hydroxylase 3 (LH3) Triggers Transforming Growth Factor-ß (TGF-ß) Activation and Fibroblast Differentiation.

Solid tumor growth triggers a wound healing response. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts (also referred to as cancer-associated fibroblasts) primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among proteases implicated in stroma remodeling, matrix metalloproteinases (MMPs), including MMP-9, play a prominent role. Recent evidence indicates that MMP-9 recruitment to the tumor cell surface enhances tumor growth and invasion. In the present work, we addressed the potential relevance of MMP-9 recruitment to and activity at the surface of fibroblasts. We show that recruitment of MMP-9 to the fibroblast cell surface occurs through its fibronectin-like (FN) domain and that the molecule responsible for the recruitment is lysyl hydroxylase 3 (LH3). Functional assays suggest that both pro- and active MMP-9 trigger α-smooth muscle actin expression in cultured fibroblasts, reflecting myofibroblast differentiation, possibly as a result of TGF-ß activation. Moreover, the recombinant FN domain inhibited both MMP-9-induced TGF-ß activation and α-smooth muscle actin expression by displacing MMP-9 from the fibroblast cell surface. Together our results uncover LH3 as a new docking receptor of MMP-9 on the fibroblast cell surface and demonstrate that the MMP-9 FN domain is essential for the interaction. They also show that the recombinant FN domain inhibits MMP-9-induced TGF-ß activation and fibroblast differentiation, providing a potentially attractive therapeutic reagent toward attenuating tumor progression where MMP-9 activity is strongly implicated.

CIC-DUX4 Chromatin Profiling Reveals New Epigenetic Dependencies and Actionable Therapeutic Targets in CIC-Rearranged Sarcomas.

CIC-DUX4-rearranged sarcoma (CDS) is a rare and aggressive soft tissue tumor that occurs most frequently in young adults. The key oncogenic driver of this disease is the expression of the CIC-DUX4 fusion protein as a result of chromosomal rearrangements. CIC-DUX4 displays chromatin binding properties, and is therefore believed to function as an aberrant transcription factor. However, the chromatin remodeling events induced by CIC-DUX4 are not well understood, limiting our ability to identify new mechanism-based therapeutic strategies for these patients. Here, we generated a genome-wide profile of CIC-DUX4 DNA occupancy and associated chromatin states in human CDS cell models and primary tumors. Combining chromatin profiling, proximity ligation assays, as well as genetic and pharmacological perturbations, we show that CIC-DUX4 operates as a potent transcriptional activator at its binding sites. This property is in contrast with the repressive function of the wild-type CIC protein, and is mainly mediated through the direct interaction of CIC-DUX4 with the acetyltransferase p300. In keeping with this, we show p300 to be essential for CDS tumor cell proliferation; additionally, we find its pharmacological inhibition to significantly impact tumor growth in vitro and in vivo. Taken together, our study elucidates the mechanisms underpinning CIC-DUX4-mediated transcriptional regulation.

Securin and separase modulate membrane traffic by affecting endosomal acidification.

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.

Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity.

Cell fate transitions observed in embryonic development involve changes in three-dimensional genomic organization that provide proper lineage specification. Whether similar events occur within tumor cells and contribute to cancer evolution remains largely unexplored. We modeled this process in the pediatric cancer Ewing sarcoma and investigated high-resolution looping and large-scale nuclear conformation changes associated with the oncogenic fusion protein EWS-FLI1. We show that chromatin interactions in tumor cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Conversely, EWS-FLI1 depletion led to the disassembly of these looping networks and a widespread nuclear reorganization through the establishment of new looping patterns and large-scale compartment configuration matching those observed in mesenchymal stem cells, a candidate Ewing sarcoma progenitor. Our data demonstrate that major architectural features of nuclear organization in cancer cells can depend on single oncogenes and are readily reversed to reestablish latent differentiation programs.

Necrotic debris and STING exert therapeutically relevant effects on tumor cholesterol homeostasis.

Malignant tumors commonly display necrosis, which invariably triggers an inflammatory response that supports tumor growth. However, the effect on tumor cells of necrotic debris, or damage-associated molecular patterns (DAMPs) released by dying cells is unknown. Here, we addressed the effect of DAMPs on primary Ewing sarcoma (EwS) cells and cell lines grown in 3D (spheroids) and 2D culture. We show that DAMPs promote the growth of EwS spheroids but not 2D cultures and that the underlying mechanism implicates an increase in cholesterol load in spheroids. In contrast, stimulation of the nucleic acid sensor signaling platform STING by its ligand cyclic GMP-AMP decreases the tumor cell cholesterol load and reduces their tumor initiating ability. Overexpression of STING or stimulation with cyclic GMP-AMP opposes the growth stimulatory effect of DAMPs and synergizes with the cholesterol synthesis inhibitor simvastatin to inhibit tumor growth. Our observations show that modulation of cholesterol homeostasis is a major effect of necrotic cell debris and STING and suggest that combining STING agonists with statins may help control tumor growth.

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia.

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD+) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Transportin regulates nuclear import of CD44.

CD44 is a facultative cell surface proteoglycan that serves as the principal cell surface receptor for hyaluronan (HA). Studies have shown that in addition to participating in numerous signaling pathways, CD44 becomes internalized upon engagement by ligand and that a portion of its intracellular domain can translocate to the nucleus where it is believed to play a functional role in cell proliferation and survival. However, the mechanisms whereby fragments of CD44 enter the nucleus have not been elucidated. Here we show that CD44 interacts with two import receptors of the importin ß superfamily, importin ß itself and transportin. Inhibition of importin ß-dependent transport failed to block CD44 accumulation in the nucleus. By contrast, inhibition of the transportin-dependent pathway abrogated CD44 import. Mutagenesis of the intracellular domain of CD44 revealed that the 20 membrane-proximal residues contain sequences required for transportin-mediated nuclear transport. Our observations provide evidence that CD44 interacts with importin family members and identify the transportin-dependent pathway as the mechanism whereby full-length CD44 enters the nucleus.

A live single-cell reporter assay links intratumor heterogeneity to metastatic proclivity in Ewing sarcoma.

Targeting of the most aggressive tumor cell subpopulations is key for effective management of most solid malignancies. However, the metastable nature of tumor heterogeneity, which allows cells to transition between strong and weak tumorigenic phenotypes, and the lack of reliable markers of tumor-promoting properties hamper identification of the most relevant cells. To overcome these obstacles, we designed a functional microRNA (miR)-based live-cell reporter assay to identify highly tumorigenic cells in xenotransplants of primary Ewing sarcoma (EwS) 3D cultures. Leveraging the inverse relationship between cell pluripotency and miR-145 expression, we successfully separated highly tumorigenic, metastasis-prone (miR-145low) cells from poorly tumorigenic, nonmetastatic (miR-145high) counterparts. Gene expression and functional studies of the two cell populations identified the EPHB2 receptor as a prognostic biomarker in patients with EwS and a major promoter of metastasis. Our study provides a simple and powerful means to identify and isolate tumor cells that display aggressive behavior.

The chromatin landscape of primary synovial sarcoma organoids is linked to specific epigenetic mechanisms and dependencies.

Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent.