Sustained low-dose treatment with the histone deacetylase inhibitor LBH589 induces terminal differentation of osteosarcoma cells

Histone deacetylase inhibitors (HDACi) were identified nearly four decades ago based on their ability to induce cellular differentiation. However, the clinical development of these compounds as cancer therapies has focused on their capacity to induce apoptosis in hematologic and lymphoid malignancies, often in combination with conventional cytotoxic agents. In many cases, HDACi doses necessary to induce these effects result in significant toxicity. Since osteosarcoma cells express markers of terminal osteoblast differentiation in response to DNA methyltransferase inhibitors, we reasoned that the epigenetic reprogramming capacity of HDACi might be exploited for therapeutic benefit. Here, we show that continuous exposure of osteosarcoma cells to low concentrations of HDACi LBH589 (Panobinostat) over a three-week period induces terminal osteoblast differentiation and irreversible senescence without inducing cell death. Remarkably, transcriptional profiling revealed that HDACi therapy initiated gene signatures characteristic of chondrocyte and adipocyte lineages in addition to marked upregulation of mature osteoblast markers. In a mouse xenograft model, continuous low dose treatment with LBH589 induced a sustained cytostatic response accompanied by induction of mature osteoblast gene expression. These data suggest that the remarkable capacity of osteosarcoma cells to differentiate in response to HDACi therapy could be exploited for therapeutic benefit without inducing systemic toxicity

UHRF1 is a mediator of KRAS driven oncogenesis in lung adenocarcinoma

Abstract KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention

Next-Generation Sequence Analysis of Cancer Xenograft Models

<div><p>Next-generation sequencing (NGS) studies in cancer are limited by the amount, quality and purity of tissue samples. In this situation, primary xenografts have proven useful preclinical models. However, the presence of mouse-derived stromal cells represents a technical challenge to their use in NGS studies. We examined this problem in an established primary xenograft model of small cell lung cancer (SCLC), a malignancy often diagnosed from small biopsy or needle aspirate samples. Using an <i>in silico</i> strategy that assign reads according to species-of-origin, we prospectively compared NGS data from primary xenograft models with matched cell lines and with published datasets. We show here that low-coverage whole-genome analysis demonstrated remarkable concordance between published genome data and internal controls, despite the presence of mouse genomic DNA. Exome capture sequencing revealed that this enrichment procedure was highly species-specific, with less than 4% of reads aligning to the mouse genome. Human-specific expression profiling with RNA-Seq replicated array-based gene expression experiments, whereas mouse-specific transcript profiles correlated with published datasets from human cancer stroma. We conclude that primary xenografts represent a useful platform for complex NGS analysis in cancer research for tumours with limited sample resources, or those with prominent stromal cell populations.</p></div

Genomic Characterisation of Small Cell Lung Cancer Patient-Derived Xenografts Generated from Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration Specimens

<div><p>Patient-derived xenograft (PDX) models generated from surgical specimens are gaining popularity as preclinical models of cancer. However, establishment of PDX lines from small cell lung cancer (SCLC) patients is difficult due to very limited amount of available biopsy material. We asked whether SCLC cells obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) could generate PDX lines that maintained the phenotypic and genetic characteristics of the primary tumor. Following successful EBUS-TBNA sampling for diagnostic purposes, we obtained an extra sample for cytologic analysis and implantation into the flanks of immunodeficient mice. Animals were monitored for engraftment for up to 6 months. Histopathologic and immunohistochemical analysis, and targeted next-generation re-sequencing, were then performed in both the primary sample and the derivative PDX line. A total of 12 patients were enrolled in the study. EBUS-TBNA aspirates yielded large numbers of viable tumor cells sufficient to inject between 18,750 and 1,487,000 cells per flank, and to yield microgram quantities of high-quality DNA. Of these, samples from 10 patients generated xenografts (engraftment rate 83%) with a mean latency of 104 days (range 63–188). All but one maintained a typical SCLC phenotype that closely matched the original sample. Identical mutations that are characteristic of SCLC were identified in both the primary sample and xenograft line. EBUS-TBNA has the potential to be a powerful tool in the development of new targeting strategies for SCLC patients by providing large numbers of viable tumor cells suitable for both xenografting and complex genomic analysis.</p></div

Summary of the results produced by the proposed strategy to isolate and identify species-specific NGS reads in human xenografts.

<p>For each read category, the proportion (%) of the total number of reads is specified.</p

Copy number variations, inter and intra-chromosomal rearrangements and B allele frequencies of NCI-H209 cell line and a xenograft tumour derived from it.

<p>(A) Circos plot representing copy number variations, inter and intra-chromosomal rearrangements of NCI-H209 cell line and a xenograft tumour derived from it. Copy number variations (red, gain; green, loss) were calculated based on coverage using the correspondent peripheral blood as control. Inter and intra-chromosomal rearrangements are represented in blue (inter-chromosomal) and dark blue (intra-chromosomal). (B, C) Detailed profile of copy number variations and B-allele frequencies of chromosome 1 from the analysed cell line and xenograft. As described above, the correspondent peripheral blood was used as control for both type of analysis. Copy number profiles are shown in red (gain), green (loss) and grey (no change). LOH are shown light blue.</p

Overview of the steps followed to identify and isolate common and species-specific sequence reads, including gene identification and pathway analysis.

<p>The software components utilized in each step are also specified. Solid lines represent the principal analytical path followed and dashed lines represent auxiliary steps.</p

Comprehensive correlation analysis between the RNA-Seq and Affymetrix expression array platforms.

<p>(<b>A</b>) Comparison of gene expression detected by RNA-Seq and Affymetrix expression array platforms for identical SCLC samples (mean, n = 3, P&lt;0.01). (<b>B</b>) Comparison of the gene expression between SCLC primary tumours <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074432#pone.0074432-Peifer1" target="_blank">[34]</a> (Y axis, mean, n = 15) and primary xenografts (X axis, mean, n = 3) (P&lt;0.01). (<b>C</b>) Comparison of gene expression detected by Affymetrix array of micro-dissected human cancer stroma <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074432#pone.0074432-Casey1" target="_blank">[19]</a> (Y axis, mean, n = 28) and mouse-specific RNA-Seq expression data in the SCLC xenograft models (X axis, mean, n = 3) (P&lt;0.01).</p

Features of specimen LX104 and its derivative xenograft.

<p><b>A.</b> Diff-Quick stained cytology smear of diagnostic EBUS-TBNA sample. Scale bar = 15 µm. <b>B.</b> Diff-Quick stained cytology smear of experimental EBUS-TBNA sample. Scale bar = 15 µm. <b>C.</b> Haematoxylin and eosin stained section of the diagnostic cell block. Scale bar = 30 µm. <b>D.</b> Diagnostic cell block stained for CD56. Scale bar = 30 µm. <b>E.</b> Haematoxylin and eosin stained section of the derivative xenograft. Scale bar = 300 µm. <b>F.</b> Haematoxylin and eosin stained section of the derivative xenograft. Scale bar = 30 µm. <b>G.</b> Section of the derivative xenograft stained for CD56. Scale bar = 30 µm. <b>H.</b> Section of the derivative xenograft stained for TTF1. Scale bar = 30 µm.</p

Immunohistochemical analysis of EBUS-TBNA diagnostic and research specimens and derivative xenografts.

<p>Scale bar = 30 µm. Blank squares indicate that the sample was not available.</p