USP1 Is Required for Replication Fork Protection in BRCA1-Deficient Tumors.

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.

Allosteric Activation of Ubiquitin-Specific Proteases by ß-Propeller Proteins UAF1 and WDR20.

Ubiquitin-specific proteases (USPs) constitute the largest family of deubiquitinating enzymes, whose catalytic competency is often modulated by their binding partners through unknown mechanisms. Here we report on a series of crystallographic and biochemical analyses of an evolutionarily conserved deubiquitinase, USP12, which is activated by two ß-propeller proteins, UAF1 and WDR20. Our structures reveal that UAF1 and WDR20 interact with USP12 at two distinct sites far from its catalytic center. Without increasing the substrate affinity of USP12, the two ß-propeller proteins potentiate the enzyme through different allosteric mechanisms. UAF1 docks at the distal end of the USP12 Fingers domain and induces a cascade of structural changes that reach a critical ubiquitin-contacting loop adjacent to the catalytic cleft. By contrast, WDR20 anchors at the base of this loop and remotely modulates the catalytic center of the enzyme. Our results provide a mechanistic example for allosteric activation of USPs by their regulatory partners.

Interplay between the DNA Damage Response and Immunotherapy Response in Cancer.

Genome instability and immune evasion are both defining hallmarks of cancer. Tumorigenesis is frequently initiated when there is DNA damage to a proto-oncogene or tumor suppressor gene and DNA repair mechanisms are lost or insufficient to correct the damage; immune evasion then prevents the host immune system from recognizing these transformed cells. Therapies targeting genomic instability and immune evasion have been effectively used to treat cancer. Genotoxic therapies such as chemoradiation have been employed in cancer treatments for several decades, while immunotherapy is a relatively new class of cancer therapy that has led to disease regression even in patients with advanced cancer. Several recent studies have shown synergy between both classes of therapy targeting these two defining hallmarks of cancer, and different mechanisms are proposed to be involved. Here, we review the different classes of DNA damage, their links to cancer, and their contribution to immunotherapy responses, as well as the different models that are currently being used to study tumor-immune interactions.

Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases.

Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD) and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF). The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO), small interfering RNA (siRNA), and microRNA (miRNA) are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir) has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

Establishment and characterization of a patient-derived solitary fibrous tumor/hemangiopericytoma cell line model.

Solitary fibrous tumor/Hemangiopericytoma (SFT/HPC) is a rare subtype of soft tissue sarcoma harboring NAB2-STAT6 gene fusions. Mechanistic studies and therapeutic development on SFT/HPC are impeded by scarcity and lack of system models. In this study, we established and characterized a novel SFT/HPC patient-derived cell line (PDC), SFT-S1, and screened for potential drug candidates that could be repurposed for the treatment of SFT/HPC. Immunohistochemistry profiles of the PDC was consistent with the patient's tumor sample (CD99+/CD34+/desmin-). RNA sequencing, followed by Sanger sequencing confirmed the pathognomonic NAB2exon3-STAT6exon18 fusion in both the PDC and the original tumor. Transcriptomic data showed strong enrichment for oncogenic pathways (epithelial-mesenchymal transition, FGF, EGR1 and TGFß signaling pathways) in the tumor. Whole genome sequencing identified potentially pathogenic somatic variants such as MAGEA10 and ABCA2. Among a panel of 14 targeted agents screened, dasatinib was identified to be the most potent small molecule inhibitor against the PDC (IC50, 473 nM), followed by osimertinib (IC50, 730 nM) and sunitinib (IC50, 1765 nM). Methylation profiling of the tumor suggests that this specific variant of SFT/HPC could lead to genome-wide hypomethylation. In conclusion, we established a novel PDC model of SFT/HPC with comprehensive characterization of its genomic, epigenomic and transcriptomic landscape, which can facilitate future preclinical studies of SFT/HPC, such as in vitro drug screening and in vivo drug testing.

Therapeutic and immunomodulatory potential of pazopanib in malignant phyllodes tumor.

Malignant phyllodes tumors (PT) are rare aggressive fibroepithelial neoplasms with high metastatic potential and lack effective therapy. We established a patient-derived xenograft (PDX) and cell line model (designated MPT-S1) of malignant PT which demonstrated clinical response to pazopanib. Whole exome sequencing identified somatic mutations in TP53, RB1, MED12, and KMT2D. Immunohistochemistry and genomic profiles of the tumor, PDX and cell line were concordant. In keeping with clinical observation, pazopanib reduced cell viability in a dose-dependent manner and evoked apoptosis, and led to significant abrogation of in vivo tumor growth. Whole transcriptomic analysis revealed that pazopanib decreased expression of genes involved in oncogenic and apoptosis signaling. We also observed decreased expression of ENPP1, with known roles in cancer invasion and metastasis, as well as STING pathway upregulation. Accordingly, pazopanib induced micronuclei formation, and evoked phospho-TBK1 and PD-L1 expression. In an additional cohort of malignant PT (n = 14), six (42.9%) showed comparable or higher levels of ENPP1 relative to MPT-S1, highlighting its potential role as a therapeutic target. In conclusion, we established MPT-S1, a new PDX and cell line model, and provided evidence for the clinical efficacy of pazopanib in malignant PT.

Disruption of the monocarboxylate transporter-4-basigin interaction inhibits the hypoxic response, proliferation, and tumor progression.

We have previously shown that glioblastoma stem cells (GSCs) are enriched in the hypoxic tumor microenvironment, and that monocarboxylate transporter-4 (MCT4) is critical for mediating GSC signaling in hypoxia. Basigin is involved in many physiological functions during early stages of development and in cancer and is required for functional plasma membrane expression of MCT4. We sought to determine if disruption of the MCT-Basigin interaction may be achieved with a small molecule. Using a cell-based drug-screening assay, we identified Acriflavine (ACF), a small molecule that inhibits the binding between Basigin and MCT4. Surface plasmon resonance and cellular thermal-shift-assays confirmed ACF binding to basigin in vitro and in live glioblastoma cells, respectively. ACF significantly inhibited growth and self-renewal potential of several glioblastoma neurosphere lines in vitro, and this activity was further augmented by hypoxia. Finally, treatment of mice bearing GSC-derived xenografts resulted in significant inhibition of tumor progression in early and late-stage disease. ACF treatment inhibited intratumoral expression of VEGF and tumor vascularization. Our work serves as a proof-of-concept as it shows, for the first time, that disruption of MCT binding to their chaperon, Basigin, may be an effective approach to target GSC and to inhibit angiogenesis and tumor progression.

Arsenic trioxide inhibits Hedgehog, Notch and stem cell properties in glioblastoma neurospheres.

BACKGROUND: Notch and Hedgehog signaling have been implicated in the pathogenesis and stem-like characteristics of glioblastomas, and inhibitors of the pathways have been suggested as new therapies for these aggressive tumors. It has also been reported that targeting both pathways simultaneously can be advantageous in treating glioblastoma neurospheres, but this is difficult to achieve in vivo using multiple agents. Since arsenic trioxide has been shown to inhibit both Notch and Hedgehog in some solid tumors, we examined its effects on these pathways and on stem cell phenotype in glioblastoma. RESULTS: We found that arsenic trioxide suppresses proliferation and promotes apoptosis in three stem-like glioblastoma neurospheres lines, while inhibiting Notch and Hedgehog target genes. Importantly, arsenic trioxide markedly reduced clonogenic capacity of the tumor neurospheres, and the stem-like CD133-positive fraction was also diminished along with expression of the stem cell markers SOX2 and CD133. CONCLUSIONS: Our results suggest that arsenic trioxide may be effective in targeting stem-like glioblastoma cells in patients by inhibiting Notch and Hedgehog activity.

Benzylidene-indolinones are effective as multi-targeted kinase inhibitor therapeutics against hepatocellular carcinoma.

Effective pharmacological intervention of advanced hepatocellular carcinoma (HCC) is currently lacking. Despite the use of tyrosine kinase inhibitors (TKIs) for the targeted therapy of several malignancies, no agent has been developed to specifically interfere with the oncogenic tyrosine kinase signaling aberrations found in HCC. Therefore, we adopted an orthogonal biological phenotypic screening approach to uncover candidate compounds: based on a potent cytotoxicity toward HCC-derived cell lines, and minimal toxicity toward normal liver cells. Given the success of indolinone as a chemical scaffold in deriving potent multi-kinase inhibitors (e.g. sunitinib), we screened a group of newly synthesized benzylidene-indolinones. Among the candidates, E/Z 6-Chloro-3-(3-trifluoromethyl-benzyliden)-1,3-dihydroindol-2-one (compound 47) exhibited potent anti-proliferative, anti-migratory, pro-apoptotic properties and good safety profile as compared to known multi-targeted tyrosine kinase inhibitors sunitinib and sorafenib. Additionally, an accompanying suppression of alpha-fetoprotein (AFP) transcription, an HCC tumor marker, implies a favorable selectivity and efficacy on HCC. The in vivo efficacy was demonstrated in an HCC xenograft where 47 was administered once weekly (60 mg/kg) and suppressed tumor burden to the same extent as sorafenib (30 mg/kg daily). A receptor tyrosine kinase (RTK) array study revealed promising inhibition of multiple tyrosine kinases such as IGF-1R, Tyro3 and EphA2 phosphorylation. Gene silencing of these targets ameliorated the cytotoxic potential of 47 on the HuH7 cell line, thereby implicating their contribution to the tumorigenicity of HCC. Hence, 47 exhibits potent anti-cancer effects on HCC cell lines, and is a suitable lead for developing multi-targeted kinase inhibitors of relevance to HCC.

Hypoxia promotes uveal melanoma invasion through enhanced Notch and MAPK activation.

The transcriptional response promoted by hypoxia-inducible factors has been associated with metastatic spread of uveal melanoma. We found expression of hypoxia-inducible factor 1α (HIF-1α) protein in well-vascularized tumor regions as well as in four cell lines grown in normoxia, thus this pathway may be important even in well-oxygenated uveal melanoma cells. HIF-1α protein accumulation in normoxia was inhibited by rapamycin. As expected, hypoxia (1% pO2) further induced HIF-1α protein levels along with its target genes VEGF and LOX. Growth in hypoxia significantly increased cellular invasion of all 5 uveal melanoma lines tested, as did the introduction of an oxygen-insensitive HIF-1α mutant into Mel285 cells with low HIF-1α baseline levels. In contrast, HIF-1α knockdown using shRNA significantly decreased growth in hypoxia, and reduced by more than 50% tumor invasion in four lines with high HIF-1α baseline levels. Pharmacologic blockade of HIF-1α protein expression using digoxin dramatically suppressed cellular invasion both in normoxia and in hypoxia. We found that Notch pathway components, including Jag1-2 ligands, Hes1-Hey1 targets and the intracellular domain of Notch1, were increased in hypoxia, as well as the phosphorylation levels of Erk1-2 and Akt. Pharmacologic and genetic inhibition of Notch largely blocked the hypoxic induction of invasion as did the pharmacologic suppression of Erk1-2 activity. In addition, the increase in Erk1-2 and Akt phosphorylation by hypoxia was partially reduced by inhibiting Notch signaling. Our findings support the functional importance of HIF-1α signaling in promoting the invasive capacity of uveal melanoma cells in both hypoxia and normoxia, and suggest that pharmacologically targeting HIF-1α pathway directly or through blockade of Notch or Erk1-2 pathways can slow tumor spread.

Pathological and molecular advances in pediatric low-grade astrocytoma.

Pediatric low-grade astrocytomas are the most common brain tumors in children. They can have similar microscopic and clinical features, making accurate diagnosis difficult. For patients whose tumors are in locations that do not permit full resection, or those with an intrinsically aggressive biology, more effective therapies are required. Until recently, little was known about the molecular changes that drive the initiation and growth of pilocytic and other low-grade astrocytomas beyond the association of a minority of cases, primarily in the optic nerve, with neurofibromatosis type 1. Over the past several years, a wide range of studies have implicated the BRAF oncogene and other members of this signaling cascade in the pathobiology of pediatric low-grade astrocytoma. In this review, we attempt to summarize this rapidly developing field and discuss the potential for translating our growing molecular knowledge into improved diagnostic and prognostic biomarkers and new targeted therapies.

BRAF activation induces transformation and then senescence in human neural stem cells: a pilocytic astrocytoma model.

PURPOSE: BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. EXPERIMENTAL DESIGN: The constitutively active BRAF(V600E) allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16(INK4a) levels in pilocytic astrocytoma. RESULTS: BRAF(V600E) expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAF(V600E)-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic ß-galactosidase, PAI-1, and p16(INK4a) whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic ß-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16(INK4a) immunoreactivity in the majority of cases, but patients with tumors negative for p16(INK4a) had significantly shorter overall survival. CONCLUSIONS: BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16(INK4a) expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

Fibroblast growth factor receptor 4 regulates proliferation, anti-apoptosis and alpha-fetoprotein secretion during hepatocellular carcinoma progression and represents a potential target for therapeutic intervention.

BACKGROUND/AIMS: FGFR4, a member of the fibroblast growth factor receptor family, has been recently associated with progression of melanoma, breast and head and neck carcinoma. Given its uniquely high expression in the liver, we investigated its contributory role to hepatocellular carcinoma (HCC). METHODS: We performed a comprehensive sequencing of full-length FGFR4 transcript in 57 tumor/normal HCC tissue pairs, and quantified their mRNA expressions. Notable mutations and expression patterns were correlated with patient data. Clinically significant trends were examined in in vitro models. RESULTS: We found eight genetic alterations including two highly frequent polymorphisms (V10I and G338R). Secretion of alpha-fetoprotein (AFP), a HCC biomarker, was increased among patients bearing homozygous Arg388 alleles. One-third of these patients exhibited increased FGFR4 mRNA expression in the matched tumor/normal tissue. Subsequent in vitro perturbation of FGFR4 signaling through both FGF19-stimulation and FGFR4 silencing confirmed a mechanistic link between FGFR4 activities and tumor aggressiveness. More importantly, inhibition of FGFR activity with PD173074 exquisitely blocked HuH7 (high FGFR4 expression) proliferation as compared to control cell lines. CONCLUSIONS: FGFR4 contributes significantly to HCC progression by modulating AFP secretion, proliferation and anti-apoptosis. Its frequent overexpression in patients renders its inhibition a novel and much needed pharmacological approach against HCC.