High-grade serous ovarian carcinoma organoids as models of chromosomal instability.

High-grade serous ovarian carcinoma (HGSOC) is the most genomically complex cancer, characterized by ubiquitous TP53 mutation, profound chromosomal instability, and heterogeneity. The mutational processes driving chromosomal instability in HGSOC can be distinguished by specific copy number signatures. To develop clinically relevant models of these mutational processes we derived 15 continuous HGSOC patient-derived organoids (PDOs) and characterized them using bulk transcriptomic, bulk genomic, single-cell genomic, and drug sensitivity assays. We show that HGSOC PDOs comprise communities of different clonal populations and represent models of different causes of chromosomal instability including homologous recombination deficiency, chromothripsis, tandem-duplicator phenotype, and whole genome duplication. We also show that these PDOs can be used as exploratory tools to study transcriptional effects of copy number alterations as well as compound-sensitivity tests. In summary, HGSOC PDO cultures provide validated genomic models for studies of specific mutational processes and precision therapeutics.

Clonal somatic copy number altered driver events inform drug sensitivity in high-grade serous ovarian cancer.

Chromosomal instability is a major challenge to patient stratification and targeted drug development for high-grade serous ovarian carcinoma (HGSOC). Here we show that somatic copy number alterations (SCNAs) in frequently amplified HGSOC cancer genes significantly correlate with gene expression and methylation status. We identify five prevalent clonal driver SCNAs (chromosomal amplifications encompassing MYC, PIK3CA, CCNE1, KRAS and TERT) from multi-regional HGSOC data and reason that their strong selection should prioritise them as key biomarkers for targeted therapies. We use primary HGSOC spheroid models to test interactions between in vitro targeted therapy and SCNAs. MYC chromosomal copy number is associated with in-vitro and clinical response to paclitaxel and in-vitro response to mTORC1/2 inhibition. Activation of the mTOR survival pathway in the context of MYC-amplified HGSOC is statistically associated with increased prevalence of SCNAs in genes from the PI3K pathway. Co-occurrence of amplifications in MYC and genes from the PI3K pathway is independently observed in squamous lung cancer and triple negative breast cancer. In this work, we show that identifying co-occurrence of clonal driver SCNA genes could be used to tailor therapeutics for precision medicine.

FOXM1 binds directly to non-consensus sequences in the human genome.

BACKGROUND: The Forkhead (FKH) transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in most types of cancer. FOXM1, similar to other FKH factors, binds to a canonical FKH motif in vitro. However, genome-wide mapping studies in different cell lines have shown a lack of enrichment of the FKH motif, suggesting an alternative mode of chromatin recruitment. We have investigated the role of direct versus indirect DNA binding in FOXM1 recruitment by performing ChIP-seq with wild-type and DNA binding deficient FOXM1. RESULTS: An in vitro fluorescence polarization assay identified point mutations in the DNA binding domain of FOXM1 that inhibit binding to a FKH consensus sequence. Cell lines expressing either wild-type or DNA binding deficient GFP-tagged FOXM1 were used for genome-wide mapping studies comparing the distribution of the DNA binding deficient protein to the wild-type. This shows that interaction of the FOXM1 DNA binding domain with target DNA is essential for recruitment. Moreover, analysis of the protein interactome of wild-type versus DNA binding deficient FOXM1 shows that the reduced recruitment is not due to inhibition of protein-protein interactions. CONCLUSIONS: A functional DNA binding domain is essential for FOXM1 chromatin recruitment. Even in FOXM1 mutants with almost complete loss of binding, the protein-protein interactions and pattern of phosphorylation are largely unaffected. These results strongly support a model whereby FOXM1 is specifically recruited to chromatin through co-factor interactions by binding directly to non-canonical DNA sequences.

Suppression of the FOXM1 transcriptional programme via novel small molecule inhibition.

The transcription factor FOXM1 binds to sequence-specific motifs on DNA (C/TAAACA) through its DNA-binding domain (DBD) and activates proliferation- and differentiation-associated genes. Aberrant overexpression of FOXM1 is a key feature in oncogenesis and progression of many human cancers. Here--from a high-throughput screen applied to a library of 54,211 small molecules--we identify novel small molecule inhibitors of FOXM1 that block DNA binding. One of the identified compounds, FDI-6 (NCGC00099374), is characterized in depth and is shown to bind directly to FOXM1 protein, to displace FOXM1 from genomic targets in MCF-7 breast cancer cells, and induce concomitant transcriptional downregulation. Global transcript profiling of MCF-7 cells by RNA-seq shows that FDI-6 specifically downregulates FOXM1-activated genes with FOXM1 occupancy confirmed by ChIP-PCR. This small molecule-mediated effect is selective for FOXM1-controlled genes with no effect on genes regulated by homologous forkhead family factors.

IL-2 therapy promotes suppressive ICOS+ Treg expansion in melanoma patients.

High-dose (HD) IL-2 therapy in patients with cancer increases the general population of Tregs, which are positive for CD4, CD25, and the Treg-specific marker Foxp3. It is unknown whether specific subsets of Tregs are activated and expanded during HD IL-2 therapy or whether activation of any particular Treg subset correlates with clinical outcome. Here, we evaluated Treg population subsets that were induced in patients with melanoma following HD IL-2 therapy. We identified a Treg population that was positive for CD4, CD25, Foxp3, and the inducible T cell costimulator (ICOS). This Treg population increased more than any other lymphocyte subset during HD IL-2 therapy and had an activated Treg phenotype, as indicated by high levels of CD39, CD73, and TGF-ß. ICOS(+) Tregs were the most proliferative lymphocyte population in the blood after IL-2 therapy. Patients with melanoma with enhanced expansion of ICOS(+) Tregs in blood following the first cycle of HD IL-2 therapy had worse clinical outcomes than patients with fewer ICOS(+) Tregs. However, there was no difference in total Treg expansion between HD IL-2 responders and nonresponders. These data suggest that increased expansion of the ICOS(+) Treg population following the first cycle of HD IL-2 therapy may be predictive of clinical outcome.

Clinical responses to vemurafenib in patients with metastatic papillary thyroid cancer harboring BRAF(V600E) mutation.

BACKGROUND: Clinical benefit from cytotoxic chemotherapy for metastatic papillary thyroid carcinoma (PTC) is disappointing, and effective therapeutic approaches for these patients are urgently needed. Because kinase-activating mutations in the BRAF proto-oncogene commonly occur in advanced PTC, and inhibition of BRAF(V600E) has shown promising clinical activity in melanoma, BRAF inhibitor therapy may be an effective strategy to treat metastatic PTC. METHODS: The dose escalation portion of a first-in-human, phase I study of vemurafenib, a selective RAF inhibitor, included three patients with metastatic PTC harboring the BRAF(V600E) mutation. Vemurafenib was initially dosed at 240-360 mg twice a day, later escalated to 720 mg twice a day. Response evaluation was performed every 8 weeks per Response Evaluation Criteria in Solid Tumors (RECIST). RESULTS: Among the three patients, one had a confirmed partial response with reduction of pulmonary target lesions by 31%, and the duration of response was 7.6 months before the disease progressed in the lungs and the bones. The time to progression was 11.7 months. The other two patients had stable disease, and the time to progression was 13.2 and 11.4 months, respectively. CONCLUSIONS: Vemurafenib appears to have a promising clinical activity in patients with metastatic PTC, and our data suggest that the BRAF(V600E) mutant kinase is a relevant target for therapy in this patient population. Further investigation of inhibitors of mutated BRAF kinase in patients with PTC in a phase II study is warranted.

Long-term stabilization of leptomeningeal disease with whole-brain radiation therapy in a patient with metastatic melanoma treated with vemurafenib: a case report.

We present a patient with metastatic BRAF-mutated melanoma who achieved long-term stabilization of leptomeningeal disease (LMD) with sequential whole-brain radiation therapy and vemurafenib. A 53-year-old woman with melanoma that harbored the BRAF V600E mutation and had that metastasized to multiple lymph nodes, lungs, breast, and subcutaneous tissue had developed symptomatic LMD 16 months after starting vemurafenib treatment despite achieving a substantial response at the existing metastatic sites. Vemurafenib was discontinued for 7 days, she received whole-brain radiation therapy (30 Gy in 10 fractions), and 7 days after completing the radiation therapy, she resumed vemurafenib therapy. The neurologic symptoms improved significantly, and a cerebrospinal fluid examination revealed disappearance of melanoma cells. She remained alive with radiologically stable LMD for at least 18 months after the whole-brain radiation therapy.

Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells.

BACKGROUND: The forkhead transcription factor FOXM1 is a key regulator of the cell cycle. It is frequently over-expressed in cancer and is emerging as an important therapeutic target. In breast cancer FOXM1 expression is linked with estrogen receptor (ERα) activity and resistance to endocrine therapies, with high levels correlated with poor prognosis. However, the precise role of FOXM1 in ER positive breast cancer is not yet fully understood. RESULTS: The study utilizes chromatin immunoprecipitation followed by high-throughput sequencing to map FOXM1 binding in both ERα-positive and -negative breast cancer cell lines. The comparison between binding site distributions in the two cell lines uncovered a previously undescribed relationship between binding of FOXM1 and ERα. Further molecular analyses demonstrated that these two factors can bind simultaneously at genomic sites and furthermore that FOXM1 regulates the transcriptional activity of ERα via interaction with the coactivator CARM1. Inhibition of FOXM1 activity using the natural product thiostrepton revealed down-regulation of a set of FOXM1-regulated genes that are correlated with patient outcome in clinical breast cancer samples. CONCLUSIONS: These findings reveal a novel role for FOXM1 in ERα transcriptional activity in breast cancer and uncover a FOXM1-regulated gene signature associated with ER-positive breast cancer patient prognosis.

Pyridostatin analogues promote telomere dysfunction and long-term growth inhibition in human cancer cells.

The synthesis, biophysical and biological evaluation of a series of G-quadruplex interacting small molecules based on a N,N'-bis(quinolinyl)pyridine-2,6-dicarboxamide scaffold is described. The synthetic analogues were evaluated for their ability to stabilize telomeric G-quadruplex DNA, some of which showed very high stabilization potential associated with high selectivity over double-stranded DNA. The compounds exhibited growth arrest of cancer cells with detectable selectivity over normal cells. Long-time growth arrest was accompanied by senescence, where telomeric dysfunction is a predominant mechanism together with the accumulation of restricted DNA damage sites in the genome. Our data emphasize the potential of a senescence-mediated anticancer therapy through the use of G-quadruplex targeting small molecules based on the molecular framework of pyridostatin.

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets.

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.

The transcription factor FOXM1 is a cellular target of the natural product thiostrepton.

Transcription factors are proteins that bind specifically to defined DNA sequences to promote gene expression. Targeting transcription factors with small molecules to modulate the expression of certain genes has been notoriously difficult to achieve. The natural product thiostrepton is known to reduce the transcriptional activity of FOXM1, a transcription factor involved in tumorigenesis and cancer progression. Herein we demonstrate that thiostrepton interacts directly with FOXM1 protein in the human breast cancer cells MCF-7. Biophysical analyses of the thiostrepton-FOXM1 interaction provide additional insights on the molecular mode of action of thiostrepton. In cellular experiments, we show that thiostrepton can inhibit the binding of FOXM1 to genomic target sites. These findings illustrate the potential druggability of transcription factors and provide a molecular basis for targeting the FOXM1 family with small molecules.

G-quadruplex-binding benzo[a]phenoxazines down-regulate c-KIT expression in human gastric carcinoma cells.

There is considerable interest in the structure and function of G-quadruplex nucleic acid secondary structures, their cellular functions, and their potential as therapeutic targets. G-Quadruplex sequence motifs are prevalent in gene promoter regions and it has been hypothesized that G-quadruplex structure formation is associated with the transcriptional status of the downstream gene. Using a functional cell-based assay, we have identified two novel G-quadruplex ligands that reduce the transcription of a luciferase reporter driven from the G-quadruplex-containing c-KIT promoter. We have further shown that endogenous c-KIT expression in a human gastric carcinoma cell line is also reduced on treatment with these molecules. Biophysical analysis using surface plasmon resonance has shown that these molecules preferentially bind with high affinity to one of the two G-quadruplex sequences in the c-KIT promoter over double-stranded DNA. This work highlights the utility of cell-based reporter assays to identify new G-quadruplex binding molecules that modulate transcription and identifies benzo[a]phenoxazine derivatives as potential antitumor agents.

MMP1 bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms.

BACKGROUND: Identifying the functional importance of the millions of single nucleotide polymorphisms (SNPs) in the human genome is a difficult challenge. Therefore, a reverse strategy, which identifies functionally important SNPs by virtue of the bimodal abundance across the human population of the SNP-related mRNAs will be useful. Those mRNA transcripts that are expressed at two distinct abundances in proportion to SNP allele frequency may warrant further study. Matrix metalloproteinase 1 (MMP1) is important in both normal development and in numerous pathologies. Although much research has been conducted to investigate the expression of MMP1 in many different cell types and conditions, the regulation of its expression is still not fully understood. RESULTS: In this study, we used a novel but straightforward method based on agglomerative hierarchical clustering to identify bimodally expressed transcripts in human umbilical vein endothelial cell (HUVEC) microarray data from 15 individuals. We found that MMP1 mRNA abundance was bimodally distributed in un-treated HUVECs and showed a bimodal response to inflammatory mediator treatment. RT-PCR and MMP1 activity assays confirmed the bimodal regulation and DNA sequencing of 69 individuals identified an MMP1 gene promoter polymorphism that segregated precisely with the MMP1 bimodal expression. Chromatin immunoprecipitation (ChIP) experiments indicated that the transcription factors (TFs) ETS1, ETS2 and GATA3, bind to the MMP1 promoter in the region of this polymorphism and may contribute to the bimodal expression. CONCLUSIONS: We describe a simple method to identify putative bimodally expressed RNAs from transcriptome data that is effective yet easy for non-statisticians to understand and use. This method identified bimodal endothelial cell expression of MMP1, which appears to be biologically significant with implications for inflammatory disease. (271 Words).

Soluble FLT1 sensitizes endothelial cells to inflammatory cytokines by antagonizing VEGF receptor-mediated signalling.

AIMS: Pre-eclampsia affects 5-7% of pregnancies, and is a major cause of maternal and foetal death. Elevated serum levels of placentally derived splice variants of the vascular endothelial growth factor (VEGF) receptor, soluble fms-like tyrosine kinase-1 (sFLT1), are strongly implicated in the pathogenesis but, as yet, no underlying mechanism has been described. An excessive inflammatory-like response is thought to contribute to the maternal endothelial cell dysfunction that characterizes pre-eclampsia. We hypothesized that sFLT1 antagonizes autocrine VEGF-A signalling, rendering endothelial cells more sensitive to pro-inflammatory factors also released by the placenta. We tested this by manipulating VEGF receptor signalling and treating endothelial cells with low doses of tumour necrosis factor-α (TNF-α). METHODS AND RESULTS: Application of recombinant sFLT1 alone did not activate human umbilical vein endothelial cells (HUVECs). However, antagonizing the autocrine actions of endothelial VEGF-A and/or placenta growth factor (PlGF) by pre-incubation with recombinant sFLT1, anti-FLT1, anti-VEGF receptor 2 (KDR), anti-VEGF-A, VEGF receptor tyrosine kinase inhibitor SU5614, or knocking-down FLT1 or KDR transcripts rendered cells more sensitive to low doses of TNF-α. Each treatment increased activation, as measured by increases in endothelial intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), endothelin 1 (ET-1), von Willebrand factor (vWF), and leucocyte adhesion, and led to reduction in AKT Ser47³ and endothelial nitric oxide synthase (eNOS) Ser¹¹77 phosphorylation. CONCLUSIONS: Our data describe a mechanism by which sFLT1 sensitizes endothelial cells to pro-inflammatory factors, providing an explanation for how placental stress may precipitate the pre-eclamptic syndrome.

Targeting the c-Kit Promoter G-quadruplexes with 6-Substituted Indenoisoquinolines.

Herein, we demonstrate the design, synthesis, biophysical properties, and preliminary biological evaluation of 6-substituted indenoisoquinolines as a new class of G-quadruplex stabilizing small molecule ligands. We have synthesized 6-substituted indenoisoquinolines 1a-e in two steps from commercially available starting materials with excellent yields. The G-quadruplex stabilization potential of indenoisoquinolines 1a-e was evaluated by fluorescence resonance energy transfer-melting analysis, which showed that indenoisoquinolines show a high level of stabilization of various G-quadruplex DNA structures. Indenoisoquinolines demonstrated potent inhibition of cell growth in the GIST882 patient-derived gastrointestinal stromal tumor cell line, accompanied by inhibition of both c-Kit transcription and KIT oncoprotein levels.

Analysis of PPARalpha-dependent and PPARalpha-independent transcript regulation following fenofibrate treatment of human endothelial cells.

Fenofibrate is a synthetic ligand for the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha and has been widely used in the treatment of metabolic disorders, especially hyperlipemia, due to its lipid-lowering effect. The molecular mechanism of lipid-lowering is relatively well defined: an activated PPARalpha forms a PPAR-RXR heterodimer and this regulates the transcription of genes involved in energy metabolism by binding to PPAR response elements in their promoter regions, so-called "trans-activation". In addition, fenofibrate also has anti-inflammatory and anti-athrogenic effects in vascular endothelial and smooth muscle cells. We have limited information about the anti-inflammatory mechanism of fenofibrate; however, "trans-repression" which suppresses production of inflammatory cytokines and adhesion molecules probably contributes to this mechanism. Furthermore, there are reports that fenofibrate affects endothelial cells in a PPARalpha-independent manner. In order to identify PPARalpha-dependently and PPARalpha-independently regulated transcripts, we generated microarray data from human endothelial cells treated with fenofibrate, and with and without siRNA-mediated knock-down of PPARalpha. We also constructed dynamic Bayesian transcriptome networks to reveal PPARalpha-dependent and -independent pathways. Our transcriptome network analysis identified growth differentiation factor 15 (GDF15) as a hub gene having PPARalpha-independently regulated transcripts as its direct downstream children. This result suggests that GDF15 may be PPARalpha-independent master-regulator of fenofibrate action in human endothelial cells.

Unraveling dynamic activities of autocrine pathways that control drug-response transcriptome networks.

Some drugs affect secretion of secreted proteins (e.g. cytokines) released from target cells, but it remains unclear whether these proteins act in an autocrine manner and directly effect the cells on which the drugs act. In this study, we propose a computational method for testing a biological hypothesis: there exist autocrine signaling pathways that are dynamically regulated by drug response transcriptome networks and control them simultaneously. If such pathways are identified, they could be useful for revealing drug mode-of-action and identifying novel drug targets. By the node-set separation method proposed, dynamic structural changes can be embedded in transcriptome networks that enable us to find master-regulator genes or critical paths at each observed time. We then combine the protein-protein interaction network with the estimated dynamic transcriptome network to discover drug-affected autocrine pathways if they exist. The statistical significance (p-values) of the pathways are evaluated by the meta-analysis technique. The dynamics of the interactions between the transcriptome networks and the signaling pathways will be shown in this framework. We illustrate our strategy by an application using anti-hyperlipidemia drug, Fenofibrate. From over one million protein-protein interaction pathways, we extracted significant 23 autocrine-like pathways with the Bonferroni correction, including VEGF-NRP1-GIPC1-PRKCA-PPARalpha, that is one of the most significant ones and contains PPARalpha, a target of Fenofibrate.

Dermatofibrosarcoma protuberans and giant cell fibroblastoma exhibit CD99 positivity.

According to most authors, dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) represent the adult and juvenile forms, respectively, of the same disease entity, as evidenced by similar morphology, an identical chromosomal translocation, and CD34 positivity. It has been shown that DFSP and nuchal-type fibroma (NTF) (which is also CD34-positive) are related lesions, and that there might possibly be a continuum between the two. In addition, NTF exhibits CD99 positivity. It was therefore, hypothesized that both DFSP and GCF would show similar immunopositivity for CD99. Archives of pathology at several institutions were searched for DFSP and GCF tissue blocks. A total of 29 DFSP and 5 GCF were analyzed by immunohistochemistry for expression of CD99. Twenty-three of 29 DFSP (79%) and 2 of 5 GCP (40%) expressed CD99. Comparison of CD99 and CD34 showed that the non-tumoral periphery of DFSP was less probable to be CD99 positive, but this finding was not statistically significant.

Computational strategy for discovering druggable gene networks from genome-wide RNA expression profiles.

We propose a computational strategy for discovering gene networks affected by a chemical compound. Two kinds of DNA microarray data are assumed to be used: One dataset is short time-course data that measure responses of genes following an experimental treatment. The other dataset is obtained by several hundred single gene knock-downs. These two datasets provide three kinds of information; (i) A gene network is estimated from time-course data by the dynamic Bayesian network model, (ii) Relationships between the knocked-down genes and their regulatees are estimated directly from knock-down microarrays and (iii) A gene network can be estimated by gene knock-down data alone using the Bayesian network model. We propose a method that combines these three kinds of information to provide an accurate gene network that most strongly relates to the mode-of-action of the chemical compound in cells. This information plays an essential role in pharmacogenomics. We illustrate this method with an actual example where human endothelial cell gene networks were generated from a novel time course of gene expression following treatment with the drug fenofibrate, and from 270 novel gene knock-downs. Finally, we succeeded in inferring the gene network related to PPAR-alpha, which is a known target of fenofibrate.