Martijn Terpstra

Long noncoding RNAs as a novel component of the Myc transcriptional network

Myc is a well‐known transcription factor with important roles in cell cycle, apoptosis, and cellular transformation. Long noncoding RNAs (lncRNAs) have recently emerged as an important class of regulatory RNAs. Here, we show that lncRNAs are a main component of the Myc‐regulated transcriptional program using the P493‐6 tetracycline‐repressible myc model. We demonstrate that both Myc‐induced mRNAs and lncRNAs are significantly enriched for Myc binding sites. In contrast to Myc‐repressed mRNAs, Myc‐repressed lncRNAs are significantly enriched for Myc binding sites. Subcellular localization analysis revealed that compared to mRNAs, lncRNAs more often have a specific subcellular localization with a markedly higher percentage of nuclear enrichment within the Myc‐repressed lncRNA set. Parallel analysis of differentially expressed lncRNAs and mRNAs identified 105 juxtaposed lncRNA‐mRNA pairs, indicative for regulation in cis. To support the potential relevance of the Myc‐regulated lncRNAs in cellular transformation, we analyzed their expression in primary Myc‐high and Myc‐low B‐cell lymphomas. In total, 54% of the lncRNAs differentially expressed between the lymphoma subsets were identified as Myc‐regulated in P493‐6 cells. This study is the first to show that lncRNAs are an important factor within the Myc‐regulated transcriptional program and indicates a marked difference between Myc‐repressed lncRNAs and mRNAs.—Winkle, M., van den Berg, A., Tayari, M., Sietzema, J., Terpstra, M., Kortman, G., de Jong, D., Visser, L., Diepstra, A., Kok, K., Kluiver, J. Long noncoding RNAs as a novel component of the Myc transcriptional network. FASEB J. 29, 2338‐2346 (2015). www.fasebj.org

Prolonged re-expression of the hypermethylated gene EPB41L3 using artificial transcription factors and epigenetic drugs

Epigenetic silencing of tumor suppressor genes (TSGs) is considered a significant event in the progression of cancer. For example, EPB41L3, a potential biomarker in cervical cancer, is often silenced by cancer-specific promoter methylation. Artificial transcription factors (ATFs) are unique tools to re-express such silenced TSGs to functional levels; however, the induced effects are considered transient. Here, we aimed to improve the efficiency and sustainability of gene re-expression using engineered zinc fingers fused to VP64 (ZF-ATFs) or DNA methylation modifiers (ZF-Tet2 or ZF-TDG) and/or by co-treatment with epigenetic drugs [5-aza-2′-deoxycytidine or Trichostatin A (TSA)]. The EPB41L3-ZF effectively bound its methylated endogenous locus, as also confirmed by ChIP-seq. ZF-ATFs reactivated the epigenetically silenced target gene EPB41L3 (∼10-fold) in breast, ovarian, and cervical cancer cell lines. Prolonged high levels of EPB41L3 (∼150-fold) induction could be achieved by short-term co-treatment with epigenetic drugs. Interestingly, for otherwise ineffective ZF-Tet2 or ZF-TDG treatments, TSA facilitated re-expression of EPB41L3 up to twofold. ATF-mediated re-expression demonstrated a tumor suppressive role for EPB41L3 in cervical cancer cell lines. In conclusion, epigenetic reprogramming provides a novel way to improve sustainability of re-expression of epigenetically silenced promoters.

Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell–Specific Long Noncoding RNAs

Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker.

Re-expression of selected epigenetically silenced candidate tumor suppressor genes in cervical cancer by TET2-directed demethylation

DNA hypermethylation is extensively explored as therapeutic target for gene expression modulation in cancer. Here, we re-activated hypermethylated candidate tumor suppressor genes (TSGs) (C13ORF18, CCNA1, TFPI2, and Maspin) by TET2-induced demethylation in cervical cancer cell lines. To redirect TET2 to hypermethylated TSGs, we engineered zinc finger proteins (ZFPs), which were first fused to the transcriptional activator VP64 to validate effective gene re-expression and confirm TSG function. ChIP-Seq not only revealed enriched binding of ZFPs to their intended sequence, but also considerable off-target binding, especially at promoter regions. Nevertheless, results obtained by targeted re-expression using ZFP-VP64 constructs were in line with cDNA overexpression; both revealed strong growth inhibition for C13ORF18 and TFPI2, but not for CCNA1 and Maspin. To explore effectivity of locus-targeted demethylation, ZFP-TET2 fusions were constructed which efficiently demethylated genes with subsequent gene re-activation. Moreover, targeting TET2 to TFPI2 and C13ORF18, but not CCNA1, significantly decreased cell growth, viability, and colony formation in cervical cancer cells compared to a catalytically inactive mutant of TET2. These data underline that effective re-activation of hypermethylated genes can be achieved through targeted DNA demethylation by TET2, which can assist in realizing sustained re-expression of genes of interest.

Genomic Aberrations in Crizotinib Resistant Lung Adenocarcinoma Samples Identified by Transcriptome Sequencing

ALK-break positive non-small cell lung cancer (NSCLC) patients initially respond to crizotinib, but resistance occurs inevitably. In this study we aimed to identify fusion genes in crizotinib resistant tumor samples. Re-biopsies of three patients were subjected to paired-end RNA sequencing to identify fusion genes using deFuse and EricScript. The IGV browser was used to determine presence of known resistance-associated mutations. Sanger sequencing was used to validate fusion genes and digital droplet PCR to validate mutations. ALK fusion genes were detected in all three patients with EML4 being the fusion partner. One patient had no additional fusion genes. Another patient had one additional fusion gene, but without a predicted open reading frame (ORF). The third patient had three additional fusion genes, of which two were derived from the same chromosomal region as the EML4-ALK. A predicted ORF was identified only in the CLIP4-VSNL1 fusion product. The fusion genes validated in the post-treatment sample were also present in the biopsy before crizotinib. ALK mutations (p.C1156Y and p.G1269A) detected in the re-biopsies of two patients, were not detected in pre-treatment biopsies. In conclusion, fusion genes identified in our study are unlikely to be involved in crizotinib resistance based on presence in pre-treatment biopsies. The detection of ALK mutations in post-treatment tumor samples of two patients underlines their role in crizotinib resistance.

Mutation patterns in small cell and non-small cell lung cancer patients suggest a different level of heterogeneity between primary and metastatic tumors.

Several studies have shown heterogeneity in lung cancer, with parallel existence of multiple subclones characterized by their own specific mutational landscape. The extent to which minor clones become dominant in distinct metastasis is not clear. The aim of our study was to gain insight in the evolution pattern of lung cancer by investigating genomic heterogeneity between primary tumor and its distant metastases. Whole exome sequencing (WES) was performed on 24 tumor and five normal samples of two small cell lung carcinoma (SCLC) and three non-SCLC (NSCLC) patients. Validation of somatic variants in these 24 and screening of 33 additional samples was done by single primer enrichment technology. For each of the three NSCLC patients, about half of the mutations were shared between all tumor samples, whereas for SCLC patients, this percentage was around 95. Independent validation of the non-ubiquitous mutations confirmed the WES data for the vast majority of the variants. Phylogenetic trees indicated more distance between the tumor samples of the NSCLC patients as compared to the SCLC patients. Analysis of 30 independent DNA samples of 16 biopsies used for WES revealed a low degree of intra-tumor heterogeneity of the selected sets of mutations. In the primary tumors of all five patients, variable percentages (19-67%) of the seemingly metastases-specific mutations were present albeit at low read frequencies. Patients with advanced NSCLC have a high percentage of non-ubiquitous mutations indicative of branched evolution. In contrast, the low degree of heterogeneity in SCLC suggests a parallel and linear model of evolution.

Involvement of MicroRNAs in the Aging-Related Decline of CD28 Expression by Human T Cells

Loss of CD28 is a characteristic feature of T cell aging, but the underlying mechanisms of this loss are elusive. As differential expression of microRNAs (miRNAs) has been described between CD28+ and CD28− T cells, we hypothesized that altered miRNA expression contributes to the age-associated downregulation of CD28. To avoid the confounding effects of age-associated changes in the proportions of T cells at various differentiation stages in vivo, an experimental model system was used to study changes over time in the expression of miRNA associated with the loss of CD28 expression in monoclonal T cell populations at a lower or higher number of population doublings (PDs). This approach allows identification of age-associated miRNA expression changes in a longitudinal model. Results were validated in ex vivo samples. The cumulative number of PDs but not the age of the donor of the T cell clone was correlated with decreased expression of CD28. Principal component analysis of 252 expressed miRNAs showed clustering based on low and high PDs, irrespective of the age of the clone donor. Increased expression of miR-9-5p and miR-34a-5p was seen in clones at higher PDs, and miR-9-5p expression inversely correlated with CD28 expression in ex vivo sorted T-cells from healthy subjects. We then examined the involvement of miR-9-5p, miR-34a-5p, and the members of the miR-23a~24-2 cluster, in which all are predicted to bind to the 3′UTR of CD28, in the IL-15-induced loss of CD28 in T cells. Culture of fresh naive CD28+ T cells in the presence of IL-15 resulted in a gradual loss of CD28 expression, while the expression of miR-9-5p, miR-34a-5p, and members of the miR-23a~24-2 cluster increased. Binding of miR-9-5p, miR-34a-5p, miR-24-3p, and miR-27- 3p to the 3′UTR of CD28 was studied using luciferase reporter constructs. Functional binding to the 3′UTR was shown for miR-24-3p and miR-27a-3p. Our results indicate involvement of defined miRNAs in T cells in relation to specific characteristics of T cell aging, i.e., PD and CD28 expression.

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

Whole exome sequencing reveals a distinct mutation pattern in metastatic small cell lung cancer compared to non-small cell lung cancer

Purpose: Lung cancer is divided into two main groups, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter subdivided in squamous, large cell and adenocarcinoma. Many studies have shown intra-tumor heterogeneity in lung cancer with multiple minor clones characterized by their own specific mutational landscape. These minor clones can become predominant under selective pressure caused by therapy. Aim of our study is to investigate the relation between primary lung cancer and multiple distant metastases using whole exome sequencing. Materials and methods: Normal tissue, primary lung tumor, and multiple metastases were obtained from 2 SCLC and 3 NSCLC patients resulting in a total of 29 samples (Table 1). DNA was isolated from total, macro-dissected, or laser micro-dissected tissue to obtain a purity of at least 80% tumor cells. Library preparation was based on the SureSelect All Exon V5 bait (Agilent) and paired-end sequencing was done using Illumina® HiSeq2000. Genome analysis toolkit (GATK) was used to analyze data and the 1000-Genome database was used to remove single nucleotide polymorphisms. Results: In four patients more than 70% of the observed variants were true somatic mutations. In the adenocarcinoma patient, only 32% of the variants were called as true somatic mutations. After excluding personal variants, 79 to 446 true somatic mutations were observed in any of the tumor samples of the five lung cancer patients (Table 1). Comparison of the mutations observed in the primary and metastatic samples revealed that around 50% of the mutations were shared between all tumor samples of the three NSCLC patients, whereas more than 95% of mutations were shared in all samples of the two SCLC patients. Conclusion: We found a high percentage of metastasis specific mutations in NSCLC patients. In contrast, SCLC patients show a limited number of metastasis specific mutations, consistent with the aggressiveness of these tumor and the very poor prognosis. Citation Format: Ali Saber, Klaas Kok, Martijn M. Terpstra, Wim Timens, Sijmen Aukema, T. Jeroen Hiltermann, Harry J. M. Groen, Anke van den Berg. Whole exome sequencing reveals a distinct mutation pattern in metastatic small cell lung cancer compared to non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2015-4757

Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients

Purpose: A small proportion of lung cancer patients have an inversion involving the ALK gene on chromosome 2. These ALK+ patients initially respond to crizotinib, but later develop progressive disease. This resistance can be due to gatekeeper mutations in the kinase domain of the ALK gene like L1196M and G1269A, copy number gain of the EML4-ALK fusion gene, and mutations in EGFR and KRAS. We aimed at identifying fusion genes that may explain the resistance mechanism in patients progressing on crizotinib. Materials and Methods: Frozen tissue of the resistant tumor samples was available for four EML4-ALK positive patients (Table 1). Paired end RNA sequencing was performed using the Illumina TruSeq RNA platform. DeFuse (v.0.6.1) was used to identify fusion products. Candidate fusion genes were validated by RT-PCR. In addition, we used the IGV browser to identify mutations in resistance-associated genes in the RNA-seq data. Results: Thirteen fusions genes were identified with high confidence (Table 1). In three of the four patients we detected an EML4-ALK fusion product. Four novel fusion products with a functional ORF were confirmed by RT-PCR. Three of these fusion products were present in patient 1 (Table 1). The fourth fusion involving CLIP4-VSNL1 was detected in patient 3. Interestingly, three of the 4 fusion products observed in patient 3 involved 5 genes that are all close to the ALK locus, i.e. EML4, ALK, CLIP4, VSNL1 and MCFD2 (Table 1). In addition, we found two resistance-related mutations in the tyrosine kinase domain of the ALK gene, i.e. p.C1156Y and p.G1269A, in patients 2 and 3, respectively. Conclusion: We found four new fusion products with a functional ORF using RNA sequencing of crizotinib resistant tumor samples. Further analysis of the primary tumor samples is ongoing and will reveal if these fusion genes might have been induced by the crizotinib treatment. Two of the four patients have a known resistance-inducing mutation in the ALK gene. Citation Format: Ali Saber, Anthonie van der Wekken, Klaas Kok, Martijn M. Terpstra, Mirjam F. Mastik, Wim Timens, Ed M.D. Schuuring, T. Jeroen Hiltermann, Harry J.M. Groen, Anke van den Berg. Detection of fusion genes in lung cancer biopsies of crizotinib resistant patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2015-4245