PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis. Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation

Biosemiotic Entropy of the Genome: Mutations and Epigenetic Imbalances Resulting in Cancer

Biosemiotic entropy involves the deterioration of biological sign systems. The genome is a coded sign system that is connected to phenotypic outputs through the interpretive functions of the tRNA/ribosome machinery. This symbolic sign system (semiosis) at the core of all biology has been termed “biosemiosis”. Layers of biosemiosis and cellular information management are analogous in varying degrees to the semiotics of computer programming, spoken, and written human languages. Biosemiotic entropy — an error or deviation from a healthy state — results from errors in copying functional information (mutations) and errors in the appropriate context or quantity of gene expression (epigenetic imbalance). The concept of biosemiotic entropy is a deeply imbedded assumption in the study of cancer biology. Cells have a homeostatic, preprogrammed, ideal or healthy state that is rooted in genomics, strictly orchestrated by epigenetic regulation, and maintained by DNA repair mechanisms. Cancer is an eminent illustration of biosemiotic entropy, in which the corrosion of genetic information via substitutions, deletions, insertions, fusions, and aberrant regulation results in malignant phenotypes. However, little attention has been given to explicitly outlining the paradigm of biosemiotic entropy in the context of cancer. Herein we distill semiotic theory (from the familiar and well understood spheres of human language and computer code) to draw analogies useful for understanding the operation of biological semiosis at the genetic level. We propose that the myriad checkpoints, error correcting mechanisms, and immunities are all systems whose primary role is to defend against the constant pressure of biosemiotic entropy, which malignancy must shut down in order to achieve advanced stages. In lieu of the narrower tumor suppressor/oncogene model, characterization of oncogenesis into the biosemiotic framework of sign, index, or object entropy may allow for more effective explanatory hypotheses for cancer diagnosis, with consequence in improving profiling and bettering therapeutic outcomes

NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy

NuRD subunit CHD4 regulates super-enhancer accessibility in rhabdomyosarcoma and represents a general tumor dependency

The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy.ISSN:2050-084

BET Bromodomain Degradation Disrupts Function but Not 3D Formation of RNA Pol2 Clusters

Fusion-positive rhabdomyosarcoma (FP-RMS) is driven by a translocation that creates the chimeric transcription factor PAX3-FOXO1 (P3F), which assembles de novo super enhancers to drive high levels of transcription of other core regulatory transcription factors (CRTFs). P3F recruits co-regulatory factors to super enhancers such as BRD4, which recognizes acetylated lysines via BET bromodomains. In this study, we demonstrate that inhibition or degradation of BRD4 leads to global decreases in transcription, and selective downregulation of CRTFs. We also show that the BRD4 degrader ARV-771 halts transcription while preserving RNA Polymerase II (Pol2) loops between super enhancers and their target genes, and causes the removal of Pol2 only past the transcriptional end site of CRTF genes, suggesting a novel effect of BRD4 on Pol2 looping. We finally test the most potent molecule, inhibitor BMS-986158, in an orthotopic PDX mouse model of FP-RMS with additional high-risk mutations, and find that it is well tolerated in vivo and leads to an average decrease in tumor size. This effort represents a partnership with an FP-RMS patient and family advocates to make preclinical data rapidly accessible to the family, and to generate data to inform future patients who develop this disease

Histone Deacetylase Inhibitors Equipped with Estrogen Receptor Modulation Activity

We describe a set of novel histone deacetylase inhibitors (HDACi) equipped with either an antagonist or an agonist of the estrogen receptor (ER) to confer selective activity against breast cancers. These bifunctional compounds potently inhibit HDAC at nanomolar concentrations and either agonize or antagonize ERα and ERβ. The ER antagonist activities of tamoxifen–HDACi conjugates (Tam-HDACi) are nearly identical to those of tamoxifen. Conversely, ethynyl-estradiol–HDACi conjugates (EED-HDACi) have attenuated ER agonist activities relative to the parent ethynyl-estradiol. In silico docking analysis provides structural basis for the trends of ER agonism/antagonism and ER subtype selectivity. Excitingly, lead Tam-HDACi conjugates show anticancer activity that is selectively more potent against MCF-7 (ERα positive breast cancer) compared to MDA-MB-231 (triple negative breast cancer), DU145 (prostate cancer), or Vero (noncancerous cell line). This dual-targeting approach illustrates the utility of designing small molecules with an emphasis on cell-type selectivity, not merely improved potency, working toward a higher therapeutic index at the earliest stages of drug development

Antiandrogen Gold Nanoparticles Dual-Target and Overcome Treatment Resistance in Hormone-Insensitive Prostate Cancer Cells

Prostate cancer is the most commonly diagnosed cancer among men in the developed countries. One in six males in the U.S. and one in nine males in the U.K. will develop the disease at some point during their lifetime. Despite advances in prostate cancer screening, more than a quarter of a million men die from the disease every year due primarily to treatment-resistance and metastasis. Colloidal nanotechnologies can provide tremendous enhancements to existing targeting/treatment strategies for prostate cancer to which malignant cells are less sensitive. Here, we show that antiandrogen gold nanoparticlesmultivalent analogues of antiandrogens currently used in clinical therapy for prostate cancerselectively engage two distinct receptors, androgen receptor (AR), a target for the treatment of prostate cancer, as well as a novel G-protein coupled receptor, GPRC6A, that is also upregulated in prostate cancer. These nanoparticles selectively accumulated in hormone-insensitive and chemotherapy-resistant prostate cancer cells, bound androgen receptor with multivalent affinity, and exhibited greatly enhanced drug potency versus monovalent antiandrogens currently in clinical use. Further, antiandrogen gold nanoparticles selectively stimulated GPRC6A with multivalent affinity, demonstrating that the delivery of nanoscale antiandrogens can also be facilitated by the transmembrane receptor in order to realize increasingly selective, increasingly potent therapy for treatment-resistant prostate cancers