Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis, and tumor suppression. Here, we ...report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL auto-regulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels.
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•PURPL is a p53-regulated lncRNA•p53 is upregulated upon loss of PURPL, inducing growth defects•PURPL associates with the p53 regulator MYBBP1A•PURPL suppresses p53 levels by inhibiting the p53-MYBBP1A interaction
For a cell to divide, the tumor suppressor protein p53 must be kept at low levels. Li et al. find that a long noncoding RNA PURPL allows cancer cells to divide by keeping p53 levels low. PURPL binds to the p53 regulator MYBBP1A to suppress p53 levels and facilitate cell proliferation.
Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to ...exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.
Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression ...of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.
Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS‐FLI1 is the primary oncogenic ...driver. The epigenome of EWS cells reflects EWS‐FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS‐FLI1 positively regulates the expression of proteins required for serine‐glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS‐FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell‐based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS‐FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one‐carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine‐glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS‐FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low‐ and high‐risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS‐FLI1 reprograms the metabolism of EWS cells and that serine‐glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type.
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in ...the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4
overexpression as murine myoblasts expressing FGFR4
undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4
were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4
tumor-derived cell and human RMS cell viability. FGFR4
-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC
values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
Given the paucity of druggable mutations in high-risk neuroblastoma (NB), we undertook chromatin-focused small interfering RNA and chemical screens to uncover epigenetic regulators critical for the ...differentiation block in high-risk NB. High-content Opera imaging identified 53 genes whose loss of expression led to a decrease in NB cell proliferation and 16 also induced differentiation. From these, the secondary chemical screen identified SETD8, the H4K20me1 methyltransferase, as a druggable NB target. Functional studies revealed that SETD8 ablation rescued the pro-apoptotic and cell-cycle arrest functions of p53 by decreasing p53K382me1, leading to activation of the p53 canonical pathway. In pre-clinical xenograft NB models, genetic or pharmacological (UNC0379) SETD8 inhibition conferred a significant survival advantage, providing evidence for SETD8 as a therapeutic target in NB.
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•We identified 53 epigenetic regulators that control NB growth and/or differentiation•SETD8 inhibition activates the p53 pathway by decreasing p53K382me1 levels•UNC0379 is a small-molecule inhibitor of SETD8 that activates the p53 pathway•SETD8 is validated as a druggable therapeutic target in NB
Veschi et al. perform both genetic and chemical screening to identify histone methyltransferase SETD8 as a potential target in neuroblastoma (NB). Chemical or genetic inhibition of SETD8 in NB leads to increased p53 activity and reduced tumor cell growth, resulting in prolonged survival in mouse models of NB.
Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van ...Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)—a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.
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•Wnt/PCP/VANGL2 are expressed in a majority of primary human rhabdomyosarcoma (RMS)•Vangl2 labels TPCs and expands their number in zebrafish embryonal RMS•VANGL2 knockdown inhibits growth and self-renewal of human RMS•RHOA functions downstream of VANGL2 to regulate growth and self-renewal
Hayes et al. find that Vangl2 specifically labels progenitors that sustain growth and self-renewal in both zebrafish and human rhabdomyosarcoma and is required for their maintenance. This work reveals direct regulation of stem cell programs and tumor growth by Vangl2/RhoA signaling, offering opportunities for direct assessment and therapeutic targeting.
The PAX3-FOXO1 fusion protein is the key oncogenic driver in fusion positive rhabdomyosarcoma (FP-RMS), an aggressive soft tissue malignancy with a particularly poor prognosis. Identifying key ...downstream targets of PAX3-FOXO1 will provide new therapeutic opportunities for treatment of FP-RMS. Herein, we demonstrate that Forkhead Box F1 (FOXF1) transcription factor is uniquely expressed in FP-RMS and is required for FP-RMS tumorigenesis. The PAX3-FOXO1 directly binds to FOXF1 enhancers and induces FOXF1 gene expression. CRISPR/Cas9 mediated inactivation of either FOXF1 coding sequence or FOXF1 enhancers suppresses FP-RMS tumorigenesis even in the presence of PAX3-FOXO1 oncogene. Knockdown or genetic knockout of FOXF1 induces myogenic differentiation in PAX3-FOXO1-positive FP-RMS. Over-expression of FOXF1 decreases myogenic differentiation in primary human myoblasts. In FP-RMS tumor cells, FOXF1 protein binds chromatin near enhancers associated with FP-RMS gene signature. FOXF1 cooperates with PAX3-FOXO1 and E-box transcription factors MYOD1 and MYOG to regulate FP-RMS-specific gene expression. Altogether, FOXF1 functions downstream of PAX3-FOXO1 to promote FP-RMS tumorigenesis.
Seven new arylpyrrole alkaloids (1–7), along with four known compounds, were isolated from an extract of a Dactylia sp. nov. marine sponge, and their structures were elucidated by interpretation of ...NMR and MS spectroscopic data. Denigrins D–G (1–4) have highly substituted pyrrole or pyrrolone rings in their core structures, while dactylpyrroles A–C (5–7) have tricyclic phenanthrene cores. Due to the proton-deficient nature of these scaffolds, key heteronuclear correlations from 1H–15N HMBC and LR-HSQMBC NMR experiments were used in the structure assignment of denigrin D (1). Dictyodendrin F (8), a previously described co-metabolite, inhibited transcription driven by the oncogenic PAX3-FOXO1 fusion gene with an IC50 value of 13 μM.
Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain ...unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene
. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity
and
. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of
. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.
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