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ZFTA (C11orf95)—a gene of unknown function—partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation—including ZFTA zinc fingers and partner gene transactivation domains—thereby unmasking vulnerabilities for therapeutic targeting.Significance:Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting.This article is highlighted in the In This Issue feature, p. 2113

ZFTA-RELAFUS1 domain interactomes

ZFTAFUS-sig

Background Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. Methods Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y–box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. Results Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. Conclusion These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. Key Points 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma. 2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors., info:eu-repo/semantics/published

WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care (SOC) surgical resection, radiotherapy, and chemotherapy. Unfortunately, while curative, this treatment is associated with major, long-term, debilitating motor, developmental, and neuroendocrine side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. Similarities have been demonstrated between cancer cell lysosomes and those of patients with Niemann-Pick, a lysosomal storage disease characterised by lysosomal fragility and sphingomyelin accumulation. A class of drugs known as Functional Inhibitors of Acid Sphingomyelinase (FIASMAs), increase lysosomal sphingomyelin and destabilise the cancer cell’s more fragile lysosomal membrane which leads to the induction of cell-death pathways via lysosomal membrane permeabilisation. Loratadine, an antihistamine with high FIASMA activity, consistently induced lysosomal membrane permeabilisation, leading to increased cell-death, in our panel of mouse and human WNT-medulloblastoma lines. Loratadine exhibited no detrimental effect on normal mouse embryonic stem cells from the lower rhombic lip – the putative cell of origin in WNT-medulloblastoma. Luciferase-expressing mouse WNT-medulloblastoma cells were orthotopically implanted into CD1-nude mice and monitored for tumour development via bioluminescent imaging. Upon tumour engraftment, mice were subjected to reduced SOC (radiotherapy and adjuvant vincristine) plus a clinically relevant dose of loratadine. Response and survival were compared to mice treated with full SOC (radiotherapy, vincristine, cisplatin, and etoposide). Mice treated with 2mg/kg/day of loratadine following reduced SOC demonstrated increased survival when compared to those treated with full SOC (p=0.02) along with a significant reduction in weight loss during treatment (p=

ZFTA (C11orf95)—a gene of unknown function—partners with a variety of transcriptional coactivators in translocations that drive supratentorial ependymoma, a frequently lethal brain tumor. Understanding the function of ZFTA is key to developing therapies that inhibit these fusion proteins. Here, using a combination of transcriptomics, chromatin immunoprecipitation sequencing, and proteomics, we interrogated a series of deletion-mutant genes to identify a tripartite transformation mechanism of ZFTA-containing fusions, including: spontaneous nuclear translocation, extensive chromatin binding, and SWI/SNF, SAGA, and NuA4/Tip60 HAT chromatin modifier complex recruitment. Thereby, ZFTA tethers fusion proteins across the genome, modifying chromatin to an active state and enabling its partner transcriptional coactivators to promote promiscuous expression of a transforming transcriptome. Using mouse models, we validate further those elements of ZFTA-fusion proteins that are critical for transformation—including ZFTA zinc fingers and partner gene transactivation domains—thereby unmasking vulnerabilities for therapeutic targeting. Significance: Ependymomas are hard-to-treat brain tumors driven by translocations between ZFTA and a variety of transcriptional coactivators. We dissect the transforming mechanism of these fusion proteins and identify protein domains indispensable for tumorigenesis, thereby providing insights into the molecular basis of ependymoma tumorigenesis and vulnerabilities for therapeutic targeting. This article is highlighted in the In This Issue feature, p. 2113

YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C′-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1–TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma—the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes.

Photosensitizers (PS) are used in photodynamic therapy to treat several cancers. The efficacy of photodynamic therapy (PDT) could be further improved by overcoming aggregation-dependent quenching of PS and by improving the biodistribution of the PS. In this work we attempted to overcome these issues by conjugating a PS with a lipid molecule and tested the liposomes prepared with this PS conjugated lipid for PDT. A novel lipid–porphyrin conjugate (1 : 1) was synthesized by attaching a PS, 5-(4-methoxycarbonylphenyl)-10,15,20-triphenyl-21H,23H-porphine, to the head group of a glutamide lipid. Two liposomal preparations, with egg phosphatidylcholine as the bulk lipid, were prepared viz. liposomes with PS conjugated lipid (LPSL) and PS entrapped in liposomes (PSL). At equimolar concentrations of the PS, both liposomal preparations were found to generate comparable amounts of reactive oxygen species as free PS upon light exposure. Electron micrographs and dynamic light scattering measurements indicated uniform and circular liposomes of 150 nm in size and near neutral zeta potential. Uptake of these liposomes by the human ovarian carcinoma cell line, SK-OV-3, was shown by FACS and confocal microscopy. Upon light exposure, the LPSL, i.e., with the conjugate lipid, have shown a substantial decrease (>4 times) in the PS requirement compared to PSL or free PS in its ability to cause light mediated cell death of SK-OV-3 cells. The light mediate cell death by LPSL was shown to be not dependent on the bulk properties of the lipid. Our data suggest a potential benefit of conjugating PS with a lipid in improving the efficiency of PDT.

This article is derived from historical research applied to the environmental problem of dryland salinity. The research evaluates the validity of the dominant model used to explain dryland salinity...