Protein destabilization underlies pathogenic missense mutations in ARID1B.

ARID1B is a SWI/SNF subunit frequently mutated in human Coffin-Siris syndrome (CSS) and it is necessary for proliferation of ARID1A mutant cancers. While most CSS ARID1B aberrations introduce frameshifts or stop codons, the functional consequence of missense mutations found in ARID1B is unclear. We here perform saturated mutagenesis screens on ARID1B and demonstrate that protein destabilization is the main mechanism associated with pathogenic missense mutations in patients with Coffin-Siris Syndrome

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

AbstractMYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in &gt; 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.</jats:p

Fibroblast growth factor receptors as novel therapeutic targets in malignant rhabdoid tumors

Malignant rhabdoid tumors (MRTs) are highly aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. In this study, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumor samples and that re-expression of SNF5 in MRT cell lines causes a striking repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblast. In vivo, treatment with NVP-BGJ398, a novel selective, pan-specific FGFR inhibitor, blocks progression of a MRT allograft derived from a SNF5-deficient mouse model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs

A novel potent oral series of VEGFR2 inhibitors abrogate tumor growth by inhibiting angiogenesis

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays the compounds exhibit single digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the vasculature as demonstrated by ELISA for TIE-2 in lysates, or by immunohistochemical analysis. This novel series of compounds shows potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role

FGFR1 and FGFR2 expression in human primary MRTs.

<p>Scatter plot showing expression of <i>FGFR1</i> (left panel) and <i>FGFR2</i> (right panel) versus <i>SNF5</i> levels among human primary sarcoma samples. MRTs are indicated in red. Dashed line indicates threshold of 10^th percentile of highest <i>FGFR1</i> or <i>FGFR2</i> expression in sample set.</p

Re-expression of SNF5 in MRT lines abrogates FGFR expression.

<p>(A) Immunoblot analysis (left panel) of FGFR2 protein expression in G401 cells five days post retroviral transduction of SNF5. β-Tubulin expression was used to monitor equal loading. <i>FGFR2</i> mRNA levels (right panel) were determined by qRT-PCR. Expression is shown as relative levels to control infected cells. Data are given as average with SEM (n = 3). <i>FGFR2</i> mRNA expression values were normalized to <i>GAPDH</i> mRNA copies. Data were compared by unpaired Student’s t test; *p<0.05. (B) Analysis of FGFR1 protein and mRNA expression in G402 cells upon re-expression of SNF5 as described in (A).</p

SNF5 is recruited to the FGFR2 promoter in BJ cells.

<p>(A) Schematic overview of the human FGFR2 promoter. Amplicons of primer pairs used for ChIP are shown as red squares and location is indicated relative to the transcriptional start site (TSS, +1). Exons are shown as blue boxes. (B) FGFR2 promoter occupancy by SNF5 in BJ cells. Fold enrichment from chromatin immunoprecititaions (ChIP) with a SNF5-specific antibody compared to an IgG control was analyzed by qPCR using the primer pairs indicated in (A). (C) Fold enrichment of the negative control locus IGX1A and the promoter region of the known SNF5 target gene CDKN1A. Fold enrichment is given as average with SEM (n≥3). Data were compared by unpaired Student’s t test with respect to the fold enrichment of the IGX1A locus; *p<0.05.</p

A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role

SNF5 loss of function induces FGFR2 expression in human fibroblasts.

<p>(A) Effect of siRNA-mediated knockdown of SNF5 on FGFR2 expression in BJ cells. <i>SNF5</i> and <i>FGFR2</i> expression levels were analyzed by qRT-PCR at 72 h post siRNA transfection. Expression is shown as relative levels to cells transfected with non-targeting control siRNA and is given as average with SEM (n≥3). <i>E</i>xpression values were normalized to <i>GAPDH</i> mRNA copies. (B) Immunoblot analysis of FGFR2 expression upon knockdown of SNF5 in BJ cells as described in (A). β-Tubulin expression was used to monitor equal loading. (C) Effect of siRNA-mediated knockdown of BRG1 on FGFR2 expression in BJ cells as described in (A).</p

FGFR expression levels of the MRT cell lines A204, G401 and G402.

<p>(A) Scatter plot showing expression and copy number levels for <i>FGFR1</i> (left panel) and <i>FGFR2</i> (right panel) within the CCLE. MRT lines A204, G401 and G402 are indicated in red. (B) Quantitative RT-PCR (qRT-PCR) analysis of <i>FGFR1</i> and <i>FGFR2</i> mRNA expression in MRT cell lines and soft tissue cancer lines SKLMS1 and SKUT1. Expression values are given as average with standard errors of the mean (SEM) (n≥3) with respect to <i>GAPDH</i> mRNA levels (arbitrarily set as 100). (C) qRT-PCR and immunoblot analysis of SNF5-deficiency in MRT lines. SKLMS1 and SKUT1 cells were used as positive controls for SNF5 expression. <i>SNF5</i> mRNA expression is given as average with SEM (n≥3) with respect to <i>GAPDH</i> mRNA levels (arbitrarily set as 100). β-Tubulin expression was used to monitor equal loading.</p