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The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

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Background: Serum biomarkers for diagnosis and risk stratification of childhood solid tumors would improve the accuracy/timeliness of diagnosis and reduce the need for invasive biopsies. We hypothesized that differential expression and/or release of microRNAs (miRNAs) by such tumors may be detected as altered serum miRNA profiles.Methods: We undertook global quantitative reverse transcription PCR (qRT-PCR) miRNA profiling (n = 741) on RNA from 53 serum samples, representing 33 diagnostic cases of common childhood cancers plus 20 controls. Technical confirmation was performed in a subset of 21 cases, plus four independent samples.Results: We incorporated robust quality control steps for RNA extraction, qRT-PCR efficiency and hemolysis quantification. We evaluated multiple methods to normalize global profiling data and identified the ‘global mean’ approach as optimal. We generated a panel of six miRNAs that were most stable in pediatric serum samples and therefore most suitable for normalization of targeted miRNA qRT-PCR data. Tumor-specific serum miRNA profiles were identified for each tumor type and selected miRNAs underwent confirmatory testing. We identified a panel of miRNAs (miR-124-3p/miR-9-3p/miR-218-5p/miR-490-5p/miR-1538) of potential importance in the clinical management of neuroblastoma, as they were consistently highly overexpressed in MYCN-amplified high-risk cases (MYCN-NB). We also derived candidate miRNA panels for noninvasive differential diagnosis of a liver mass (hepatoblastoma vs. combined MYCN-NB/NB), an abdominal mass (Wilms tumor vs. combined MYCN-NB/NB), and sarcoma subtypes.Conclusions: This study describes a pipeline for robust diagnostic serum miRNA profiling in childhood solid tumors, and has identified candidate miRNA profiles for prospective testing.Impact: We propose a new noninvasive method with the potential to diagnose childhood solid tumors. Cancer Epidemiol Biomarkers Prev; 24(2); 350–60. ©2014 AACR.

Supplementary Materials, Tables S1 and S2, Figures S1-S10. Supplementary Table S1. Number of over-expressed serum microRNAs in solid tumor types of childhood. Supplementary Table S2. Ten over-expressed serum microRNAs selected for the technical confirmation qRT-PCR study. Supplementary Table and Figure Legends, Supplementary References. Supplementary Figure S1. Quality control (QC) assessments in the initial qRT-PCR run. Supplementary Figure S2. Confirmation of suitability of the four serum QC housekeeping microRNAs for screening samples for the full discovery-phase qRT-PCR run. Supplementary Figure S3. QC assessments in the full discovery-phase qRT-PCR run. Supplementary Figure S4. Hemolysis assessment of the 53 serum samples in the initial QC and full discovery-phase qRT-PCR run. Supplementary Figure S5. Further QC assessment of the 53 serum samples in the full discovery-phase qRT-PCR run. Supplementary Figure S6. Top-ranking overlapping housekeeping microRNAs identified by NormFinder and geNorm algorithms, suitable for targeted serum qRT-PCR studies. Supplementary Figure S7. Comparison of expression of miR-122-3p and miR-122-5p in the 53 serum samples in the full discovery-phase qRT-PCR run. Supplementary Figure S8. Comparison of the average raw Ct values in the 21 serum samples common to both the full discovery-phase and technical confirmation qRT-PCR run. Supplementary Figure S9. QC assessment in the technical confirmation qRT-PCR run for all 25 serum samples used. Supplementary Figure S10. Serum microRNA expression in osteosarcoma.

Supplementary Tables S3 to S13. Over-expressed serum microRNAs in individual types of solid tumor of childhood. The Tables list the over-expressed microRNAs in individual types of solid tumor of childhood compared with the 'other tumor' group and control group, their fold-changes, coefficient of variation and robust rank aggregate (RRA) score. Key: FC =fold-change; CV = coefficient of variation. Table S3: MYCN-amplified INRG high-risk neuroblastoma (MYCN-NB). Table S4: non-MYCN-amplified INRG low-risk neuroblastoma (NB). Table S5: mhepatoblastoma(HB). Table S6: Wilms tumor (WT). Table S7 = rhabdomyosarcoma (RMS). Table S8: Ewings sarcoma (ES). Table S9: osteosarcoma (OS). Table S10: B-cell non-Hodgkin's lymphoma (BNHL). Table S11: Hodgkin's disease (HD). Table S12: pleuropulmonary blastoma (PPB) Table S13: Central nervous system glioma (GL).

PDF - 62K, Supplementary Table 1: Clinico-pathological data for sample set-3. Supplementary Table 2: Primers used for mRNA qRT-PCR. Supplementary Table 3: Expression of let-7 family microRNAs in pediatric malignant-GCTs. Supplementary Table 4: Let-7 mRNA targets identified in combined microarray analysis of pediatric and adult malignant-GCTs. Supplementary Table 5: The six let-7 mRNA targets selected following microarray and qRT-PCR analysis.

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Supplementary Figures 1-9, Tables 1-10 from Malignant Germ Cell Tumors Display Common MicroRNA Profiles Resulting in Global Changes in Expression of Messenger RNA Targets

Supplementary Results, Figure and Table Legends from Malignant Germ Cell Tumors Display Common MicroRNA Profiles Resulting in Global Changes in Expression of Messenger RNA Targets

PDF - 492K, Supplementary Figure 1: Levels of the nine members of the let-7 family in pediatric malignant-GCTs. Supplementary Figure 2: PCR quantification of let-7e, LIN28 and LIN28B in malignant-GCTs.Supplementary Figure 3: Sylamer analysis of up-regulated genes in pediatric and adult malignant-GCTs. Supplementary Figure 4: Correlations between levels of let-7 and mRNA targets. Supplementary Figure 5: PCR quantification of HMGA2 in pediatric malignant-GCTs. Supplementary Figure 6: PCR validation of expression of selected let-7 mRNA targets in pediatric malignant-GCTs. Supplementary Figure 7: Effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 8: Independent confirmation of specific effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 9: Effects of let-7e mimic in malignant-GCT cells. Supplementary Figure 10: Relationships between MYCN and C-MYC versus LIN28 and LIN28B in malignant-GCTs. Supplementary Figure 11: MYCN depletion in malignant-GCTs (S11). Supplementary Figure 12: Schematic of the LIN28/let-7 axis in malignant-GCTs.

Tolinapant (ASTX660) is a potent, nonpeptidomimetic antagonist of cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) and X-linked IAP, which is currently being evaluated in a phase 2 study in T-cell lymphoma (TCL) patients. Tolinapant has demonstrated evidence of single-agent clinical activity in relapsed/refractory peripheral TCL and cutaneous TCL. To investigate the mechanism of action underlying the single-agent activity observed in the clinic, we have used a comprehensive translational approach integrating in vitro and in vivo models of TCL confirmed by data from human tumor biopsies. Here, we show that tolinapant acts as an efficacious immunomodulatory molecule capable of inducing complete tumor regression in a syngeneic model of TCL exclusively in the presence of an intact immune system. These findings were confirmed in samples from our ongoing clinical study showing that tolinapant treatment can induce changes in gene expression and cytokine profile consistent with immune modulation. Mechanistically, we show that tolinapant can activate both the adaptive and the innate arms of the immune system through the induction of immunogenic forms of cell death. In summary, we describe a novel role for IAP antagonists as immunomodulatory molecules capable of promoting a robust antitumor immune response in TCL.

Analyses of small non-coding RNA (ncRNA) expression in malignant germ cell tumours (GCTs) have focused on microRNAs (miRNAs). As GCTs all arise from primordial germ cells, and piwi-interacting RNAs (piRNAs) have important roles in maintaining germline integrity via transposon silencing, we hypothesised that malignant GCTs are characterised by fundamental piRNA dysregulation.We undertook global small ncRNA sequencing in malignant GCTs, in order to describe small ncRNA expression changes for both miRNAs and piRNAs.We performed small ncRNA next generation sequencing on a representative panel of 47 samples, comprising malignant GCT (n = 31) and control (n = 16) tissues/cell lines. Following quality control and normalisation, filtered count reads were used for differential miRNA and piRNA expression analyses via DESeq2. Predicted mRNA targets for piRNAs were identified and utilised for pathway enrichment analyses.Overall, miRNAs and piRNAs comprised 21.9% and 43.0% of small ncRNA species, respectively. There were 749 differentially expressed miRNAs in malignant GCTs, of which 536 (72%) were over-expressed and 213 (28%) under-expressed. The top-ranking over-expressed miRNAs were exclusively from the miR-371∼373 and miR-302/367 clusters. The most significantly under-expressed miRNAs were miR-100-5p, miR-214-3p, miR-125b-5p and let-7 family members, including miR-202-3p. There were 1,121 differentially expressed piRNAs in malignant GCTs, of which 167 (15%) were over-expressed and 954 (85%) under-expressed. Of note, of the top-20 differentially expressed piRNAs, 16 were over-expressed, of which piR-hsa-2506793 was both top-ranking and most abundant. Mobile element (ME; i.e., transposon)-associated piRNAs comprised 166 (15%) of the 1,121 differentially expressed piRNAs, of which 165 (99%) were down-regulated. The remaining 955 (85%) non-ME-associated piRNAs may have wider cellular roles. To explore this, predicted mRNA targets of differentially expressed piRNAs identified putative involvement in cancer-associated pathways.This study confirms previous miRNA observations, giving credence to our novel demonstration of global piRNA dysregulation in gonadal malignant GCTs, through both ME and non-ME-associated pathways, which likely contributes to GCT pathogenesis.

Summary Sequestosome-1 (SQSTM1/p62) is involved in cellular processes such as autophagy and metabolic reprogramming. Mutations resulting in the loss of function of SQSTM1 lead to neurodegenerative diseases including frontotemporal dementia. The pathogenic mechanism that contributes to SQSTM1-related neurodegeneration has been linked to its role as an autophagy adaptor, but this is poorly understood, and its precise role in mitochondrial function and clearance remains to be clarified. Here, we assessed the importance of SQSTM1 in human induced pluripotent stem cell (iPSC)-derived cortical neurons through the knockout of SQSTM1. We show that SQSTM1 depletion causes altered mitochondrial gene expression and functionality, as well as autophagy flux, in iPSC-derived neurons. However, SQSTM1 is not essential for mitophagy despite having a significant impact on early PINK1-dependent mitophagy processes including PINK1 recruitment and phosphorylation of ubiquitin on depolarized mitochondria. These findings suggest that SQSTM1 is important for mitochondrial function rather than clearance., Highlights • SQSTM1 is dispensable for cortical neuron differentiation, modeled with human iPSCs • Expression of bioenergetic genes is altered in human cortical neurons lacking SQSTM1 • Loss of SQSTM1 causes aberration in mitochondrial functionality • SQSTM1 affects mitophagic processes but is not required for mitochondrial clearance, Sequestosome-1 (SQSTM1/p62) deficiency leads to several neurodegenerative diseases wherein mitochondrial dysfunction is observed in neurons. Whether SQSTM1 contributes to pathogenesis by playing a role in mitochondrial function and clearance remains controversial. Poon and colleagues generated cortical neurons depleted of SQSTM1 from human induced pluripotent stem cells and demonstrated that SQSTM1 is a regulator of mitochondrial respiratory capacity but is dispensable for neuronal mitophagy.

RNA-Seq is a powerful transcriptome profiling technology enabling transcript discovery and quantification. Whilst most commonly used for gene-level quantification, the data can be used for the analysis of transcript isoforms. However, when the underlying transcript assemblies are complex, current visualization approaches can be limiting, with splicing events a challenge to interpret. Here, we report on the development of a graph-based visualization method as a complementary approach to understanding transcript diversity from short-read RNA-Seq data. Following the mapping of reads to a reference genome, a read-to-read comparison is performed on all reads mapping to a given gene, producing a weighted similarity matrix between reads. This is used to produce an RNA assembly graph, where nodes represent reads and edges similarity scores between them. The resulting graphs are visualized in 3D space to better appreciate their sometimes large and complex topology, with other information being overlaid on to nodes, e.g. transcript models. Here we demonstrate the utility of this approach, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity.

There is growing evidence for the importance of 3’ untranslated region (3’UTR) dependent regulatory processes. However, our current human 3’UTR catalogue is incomplete. Here, we developed a machine learning-based framework, leveraging both genomic and tissue-specific transcriptomic features to predict previously unannotated 3’UTRs. We identify unannotated 3’UTRs associated with 1,513 genes across 39 human tissues, with the greatest abundance found in brain. These unannotated 3’UTRs were significantly enriched for RNA binding protein (RBP) motifs and exhibited high human lineage-specificity. We found that brain-specific unannotated 3’UTRs were enriched for the binding motifs of important neuronal RBPs such as TARDBP and RBFOX1, and their associated genes were involved in synaptic function and brain- related disorders. Our data is shared through an online resource F3UTER (https://astx.shinyapps.io/F3UTER/). Overall, our data improves 3’UTR annotation and provides novel insights into the mRNA-RBP interactome in the human brain, with implications for our understanding of neurological and neurodevelopmental diseases.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL). Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand). Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays. Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability. Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo. By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro. Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds. Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. Disclosures Sims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.

Whilst mitogen-activated protein kinase (MAPK) pathway inhibitors are approved therapies in mutant-BRAF driven cancers and have demonstrated a high response rate in the clinic, the duration of response is often short-lived. Approaches targeting the immune system have elicited durable responses and led to the approval of checkpoint inhibitors such as the anti-PD1 therapy, pembrolizumab, in indications where MAPK pathway activation is often observed, such as melanoma. Activation of the MAPK pathway has been associated with an immunosuppressive tumor microenvironment (TME). Further, preclinical studies have demonstrated that in addition to inhibiting MAPK activity in tumor cells, MAPK pathway inhibitors such as the BRAFV600E inhibitor dabrafenib or MEK inhibitor trametinib have promoted a more proinflammatory TME leading to upregulated antigen presentation on tumor cells, increased CD8+ T cell infiltration and tumor cell killing. Similar preclinical results were recently reported for a KRASG12C inhibitor, AMG 510, where treatment of in vivo models led to an increase in tumor infiltrating lymphocytes, macrophages, dendritic cells and an increase in active immune gene signatures leading to tumor immunity. We have recently described the discovery of ASTX029, which is currently undergoing clinical development in advanced solid tumors (NCT03520075). ASTX029 is a dual-mechanism ERK1/2 (ERK) inhibitor, inhibiting both the catalytic activity and phosphorylation of ERK, and shows potent inhibition of MAPK-activated tumor growth in preclinical models. Previous studies have demonstrated a difference in regulation of genes involved in response to type I interferon following treatment with dual-mechanism compared to catalytic ERK inhibitors. We investigated whether treatment with ASTX029 modulates antigen presentation and the TME in MAPK-activated tumors. Following treatment of cell lines in vitro, we observed ASTX029-dependent changes consistent with increased antigen presentation, including an increase in cell surface expression of MHC class I and the increase in gene expression of tumor-specific antigens gp100 and MART-1 following treatment of melanoma cells. We have previously demonstrated that ASTX029 has good bioavailability following oral dosing in mice. To further investigate the effect of ASTX029 on the TME, syngeneic tumors grown in immunocompetent mice dosed with ASTX029 were characterized in terms of immune cell composition of the TME and gene expression. Our results were consistent with increased immune activation, including increased interferon signaling and a change in the immune cell composition of the TME. These data demonstrate that treatment with ASTX029 leads to modulation of the TME and we hypothesize that to optimize therapeutic activity, ASTX029 could be partnered either with an immunomodulatory or tumor-directed agent. Citation Format: Chris Hindley, Andrea Biondo, Kim-Hien Dao, Matthew Davis, Lynsey Fazal, Roberta Ferraldeschi, Keisha Hearn, Vanessa Martins, Harpreet Saini, Martin Sims, Nicola Wallis, Nicola Wilsher, Harold Keer, John Lyons, Joanne Munck. Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1661.

RNA-sequencing (RNA-Seq) is a powerful transcriptome profiling technology enabling transcript discovery and quantification. RNA-Seq data are large, and most commonly used as a source of genelevel quantification measurements, whilst the underlying assemblies of reads, if inspected, are usually viewed as sequence reads mapped on to a reference genome. Whilst sufficient for many needs, when the underlying transcript assemblies are complex, this visualisation approach can be limiting; errors in assembly can be difficult to spot and interpretation of splicing events is challenging.Here we report on the development of a graph-based visualisation method as a complementary approach to understanding transcript diversity and read assembly from short-read RNA-Seq data. Following the mapping of reads to the reference genome, read-to-read comparison is performed on all reads mapping to a given gene, producing a matrix of weighted similarity scores between reads. This is used to produce an RNA assembly graph where nodes represent reads derived from a cDNA and edges similarity scores between reads, above a defined threshold. Visualisation of resulting graphs is performed using Graphia Professional. This tool can render the often large and complex graph topologies that result from DNA/RNA sequence assembly in 3D space and supports info rmatio no verlay on to nodes, e.g. transcript models. We have also implemented an analysis pipeline for the creation of RNA assembly graphs with both a command-line and web-based interface that allows users to create and visualise these data. Here we demonstrate the utility of this approach on RNA-Seq data, including the unusual structure of these graphs and how they can be used to identify issues in assembly, repetitive sequences within transcripts and splice variants. We believe this approach has the potential to significantly improve our understanding of transcript complexity.

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non‐coding transcripts (lncRNA). This process occurs post‐mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon‐driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non‐coding genes. Accordingly, we show that a small fraction of these novel ERV‐driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue‐specific context. In summary, we demonstrate that LTRs can act as tissue‐specific promoters and contribute to post‐mitotic spermatogenic transcriptome diversity.

Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain C − O in a β-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the N − H component of hydrogen bond can be replaced by a C(γ) -H group in the side chain, culminating in a nonconventional C − H···O interaction. Because of its shape this β-turn mimic is designated as ω-turn, which is found to occur ∼ three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C − H···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ'1(i + 2) (using the interacting C(γ) atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. C(β) -branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two β-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

BACKGROUND: When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs. It is assumed that the higher the number of predicted transcriptional OTEs, the greater the size of the actual OTE signature and the more detrimental the phenotypic consequences in target-negative cells. METHODS: We tested this general assumption by investigating the OTEs of potential therapeutic siRNAs targeting the human papillomavirus (HPV) type-16 E7 oncogene. We studied HPV-negative squamous epithelial cells, from normal cervix (NCx) and skin (HaCaT), which would be vulnerable to 'bystander' OTEs following transfection in vivo. RESULTS: We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted). The unpredicted OTEs included stimulation of innate immune pathways, as well as indirect (downstream) effects of other OTEs, which affected important cancer-associated pathways. Although most significant OTEs observed were seen in both NCx and HaCaT cells, only 0-5.9% of differentially expressed genes overlapped between the two cell types. CONCLUSION: These data do not support the assumption that actual OTEs correlate well with predicted OTEs.

Background: Tyrosine kinase inhibitors, such as crizotinib and erlotinib, are widely used to treat non-small-cell lung cancer, but after initial response, relapse is common because of the emergence of resistance through multiple mechanisms. Here, we investigated whether a frontline combination with an HSP90 inhibitor could delay the emergence of resistance to these inhibitors in preclinical lung cancer models. Methods: The HSP90 inhibitor, onalespib, was combined with either crizotinib or erlotinib in ALK- or EGFR-activated xenograft models respectively (H2228, HCC827). Results: In both models, after initial response to the monotherapy kinase inhibitors, tumour relapse was observed. In contrast, tumour growth remained inhibited when treated with an onalespib/kinase inhibitor combination. Analysis of H2228 tumours, which had relapsed on crizotinib monotherapy, identified a number of clinically relevant crizotinib resistance mechanisms, suggesting that HSP90 inhibitor treatment was capable of suppressing multiple mechanisms of resistance. Resistant cell lines, derived from these tumours, retained sensitivity to onalespib (proliferation and signalling pathways were inhibited), indicating that, despite their resistance to crizotinib, they were still sensitive to HSP90 inhibition. Conclusions: Together, these preclinical data suggest that frontline combination with an HSP90 inhibitor may be a method for delaying the emergence of resistance to targeted therapies.

Embryonic stem (ES) cells can be induced to differentiate into embryoid bodies (EBs) in a synchronised manner when plated at a fixed density in hanging drops. This differentiation procedure mimics post-implantation development in mouse embryos and also serves as the starting point of protocols used in differentiation of stem cells into various lineages. Currently, little is known about the potential influence of microRNAs (miRNAs) on mRNA expression patterns during EB formation. We have measured mRNA and miRNA expression in developing EBs plated in hanging drops until day 3, when discrete structural changes occur involving their differentiation into three germ layers. We observe significant alterations in mRNA and miRNA expression profiles during this early developmental time frame, in particular of genes involved in germ layer formation, stem cell pluripotency and nervous system development. Computational target prediction using Pictar, TargetScan and miRBase Targets reveals an enrichment of binding sites corresponding to differentially and highly expressed miRNAs in stem cell pluripotency genes and a neuroectodermal marker, Nes. We also find that members of let-7 family are significantly down-regulated at day 3 and the corresponding up-regulated genes are enriched in let-7 seed sequences. These results depict how miRNA expression changes may affect the expression of mRNAs involved in EB formation on a genome-wide scale. Understanding the regulatory effects of miRNAs during EB formation may enable more efficient derivation of different cell types in culture.

Although gain of chromosome 5p is one of the most frequent DNA copy-number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study, we showed that the microRNA processor Drosha (located on chromosome 5p) demonstrates frequent copy-number gain and overexpression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/overexpression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in 18 cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of 14 that were differentially expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha-overexpressing samples/cell lines was the highest-ranked change (by adjusted p value) in both analyses, an observation validated by northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.

In recent years there has been an explosion in cancer genomic data that has enabled understanding of the incidence and patterns of mutations in cancer patients. The challenge is to translate this vast resource of genomic research and data into precision medicines that target specific mutations in patient populations and improve clinical outcomes. Delivering personalised cancer medicines requires greater understanding of how cancer mutations impact the structure, function, and druggability of known and potential protein drug targets. In this study we annotate each missense mutation in COSMIC, via COSMIC-3D (http://cancer.sanger.ac.uk/cosmic3d), with protein structural features derived from the wwPDB, and rich functional features derived from the UniProt protein database. The structural features include small molecule binding sites (including drug binding sites), protein-protein interaction interfaces, protein-nucleic acid interactions, solvent inaccessible protein “core” versus solvent exposed areas, and predicted small-molecule druggability. The UniProt-derived functional features represent high quality functional site annotations, including post-translational modification sites, functional domains and motifs, and regions of biological interest. In addition, we explore the relationship between mutation occurrence in protein structural and functional sites, and recurrence in cancer. We found that recurrent missense mutations are significantly enriched in specific structural and functional environments, including small-molecule binding sites and predicted druggable pockets. These results provide promising hypotheses that recurrent, cancer driving mutations may be targeted by small-molecule drugs. We posit that further characterisation of the structure-function implications of each mutation in COSMIC-3D will increasingly aid mutation-guided target discovery and drug design. Citation Format: Harry C. Jubb, Harpreet K. Saini, Marcel L. Verdonk, Simon A. Forbes. COSMIC-3D: Impacts of cancer mutations on protein structure, function, and druggability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3285.

miRBase is the central online repository for microRNA (miRNA) nomenclature, sequence data, annotation and target prediction. The current release (10.0) contains 5071 miRNA loci from 58 species, expressing 5922 distinct mature miRNA sequences: a growth of over 2000 sequences in the past 2 years. miRBase provides a range of data to facilitate studies of miRNA genomics: all miRNAs are mapped to their genomic coordinates. Clusters of miRNA sequences in the genome are highlighted, and can be defined and retrieved with any inter-miRNA distance. The overlap of miRNA sequences with annotated transcripts, both protein- and non-coding, are described. Finally, graphical views of the locations of a wide range of genomic features in model organisms allow for the first time the prediction of the likely boundaries of many miRNA primary transcripts. miRBase is available at http://microrna.sanger.ac.uk/.

Recently a number of computational approaches have been developed for the prediction of protein-protein interactions. Complete genome sequencing projects have provided the vast amount of information needed for these analyses. These methods utilize the structural, genomic, and biological context of proteins and genes in complete genomes to predict protein interaction networks and functional linkages between proteins. Given that experimental techniques remain expensive, time-consuming, and labor-intensive, these methods represent an important advance in proteomics. Some of these approaches utilize sequence data alone to predict interactions, while others combine multiple computational and experimental datasets to accurately build protein interaction maps for complete genomes. These methods represent a complementary approach to current high-throughput projects whose aim is to delineate protein interaction maps in complete genomes. We will describe a number of computational protocols for protein interaction prediction based on the structural, genomic, and biological context of proteins in complete genomes, and detail methods for protein interaction network visualization and analysis.

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

The RAS-RAF-MEK-ERK signalling cascade is activated through mutations in RAS or RAF in over 30% of cancers. The successful development of inhibitors of BRAF and MEK kinases has led to effective treatment particularly of melanomas whose tumor growth is driven by activating mutations in BRAF such as V600E. Despite these successes, resistance emerges after several months, leading to increased signaling through ERK1/2. This has prompted the development of direct inhibitors of ERK1/2, several of which are in early clinical trials. The majority of clinical ERK1/2 inhibitors are ATP competitive, blocking ERK1/2 catalytic phosphorylation of downstream substrates such as RSK, but do not modulate phosphorylation of ERK1/2 by MEK. Crystal structural studies performed by us and others on the pERK1/2 modulating inhibitor SCH772984 suggested that it induces a conformational change in the glycine-rich loop of ERK2, which leads to Tyr36 becoming tucked under the loop and creating a new binding pocket. We hypothesized that this binding mode might underlie the ability of SCH772984 to block the phosphorylation of ERK1/2, and initiated a fragment-based approach to develop novel, orally bioavailable inhibitors that elicit a similar conformational change and also modulate the phosphorylation of ERK1/2. Using screening methods including high-throughput X-ray crystallography and biophysical assays, we identified fragments binding to both the hinge and the inducible pocket of ERK2. Progressive rounds of structure-guided fragment optimization and growing led to an understanding of inhibitor structure determinants required to induce the conformational change in ERK2. These efforts, together with iterative optimization in a screening cascade including measurement of pRSK and pERK levels and antiproliferative activity in RAS and BRAF mutant cells, led to the discovery of a novel series of pERK modulating ERK1/2 inhibitors. The lead compound shows low nanomolar potency in biochemical ERK1/2 assays and an excellent kinome selectivity profile. In BRAF and RAS mutant cell lines, the lead shows low nanomolar cell proliferation IC50 values, while sparing cell lines not driven by the MAPK pathway. The lead exhibits robust antitumor activity upon oral dosing in a range of subcutaneous xenograft models including the mutant BRAF colorectal line Colo205, providing a promising basis for further optimization towards clinical pERK1/2 modulating ERK1/2 inhibitors. Citation Format: Tom D. Heightman, Valerio Berdini, Hannah Braithwaite, Ildiko Buck, Megan Cassidy, Juan Castro, Aurélie Courtin, James Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte Griffiths-Jones, John Lyons, Vanessa Martins, Sandra Muench, Joanne Munck, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David Rees, Sharna Rich, Caroline Richardson, Harpreet Saini, Neil Thompson, Nicola Wallis, Hugh Walton, Nicola Wilsher, Alison Woolford, Chris Murray. Fragment-based discovery of a highly potent, orally bioavailable ERK1/2 inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B161.

The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF, RAS and the upregulation of RTKs. The therapeutic potential of MAPK pathway inhibition has been demonstrated by the clinical efficacy of RAF and MEK1/2 (MEK) inhibitors in the treatment of BRAF-mutant melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms, which in the majority of cases result in the reactivation of ERK1/2 (ERK) signalling. Therefore, the direct targeting of ERK is an attractive therapeutic approach to overcoming the limitations of RAF or MEK inhibitors. Here, we describe a novel, potent, and selective ERK inhibitor, which inhibits both ERK catalytic activity and also the phosphorylation of ERK by MEK. Using fragment-based drug discovery we have developed a selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nM IC50 value. This lead compound has strong antiproliferative effects in a wide range of MAPK-activated cell lines, including the BRAF-mutant cell lines A375 (melanoma) and Colo205 (colorectal), the KRAS-mutant cell lines HCT116 (colorectal), Calu6 (lung) and Panc05.04 (pancreatic), and the NRAS-mutant cell line Ma-mel-27 (melanoma). The lead compound potently inhibits ERK cell signalling. The potent (nM) inhibition of RSK phosphorylation (a direct ERK substrate) was confirmed in A375 (BRAF-mutant melanoma) cells, using MSD analysis. In addition to inhibiting downstream ERK signalling, we demonstrated by ELISA and Western blotting that the lead compound confers a decrease in phospho-ERK levels in both BRAF-mutant and KRAS-mutant cell lines. We investigated the biochemical mechanism of the modulation of ERK phosphorylation in vitro and demonstrated that the compound prevents the phosphorylation of ERK by MEK (at key ERK activation loop residues, T202/Y204), without directly inhibiting MEK activity. The compound was profiled in a range of subcutaneous xenograft models including A375 (BRAF-mutant melanoma) and Calu-6 (KRAS-mutant lung). Once-daily oral dosing of the lead compound conferred significant antitumor activity in a range of in vivo efficacy studies. The compound potently inhibited the phosphorylation of downstream ERK substrates (including RSK) in tumor xenograft tissue. There was a clear relationship between in vivo compound concentrations and the modulation of ERK substrate phosphorylation. Furthermore, as was demonstrated in vitro, we confirmed that in addition to inhibiting ERK catalytic activity the compound potently inhibited the phosphorylation of ERK itself, in both KRAS and BRAF-mutant tumor xenografts. Here, we characterize a novel, highly potent, selective ERK inhibitor, which inhibits both ERK catalytic activity and also the upstream phosphorylation of ERK by MEK. These data support the further optimization of this series of compounds for clinical development. Citation Format: Joanne M. Munck, Valerio Berdini, Luke D. Bevan, Hannah Braithwaite, Ildiko M. Buck, Megan Cassidy, Juan Castro, Aurelie Courtin, James E. Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte M. Griffiths-Jones, Tom D. Heightman, Chris J. Hindley, Birikiti Kidane, Justyna Kucia-Tran, John F. Lyons, Vanessa Martins, Sandra Muench, Chris W. Murray, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David C. Rees, Sharna J. Rich, Caroline J. Richardson, Harpreet K. Saini, Alpesh Shah, Lukas Stanczuk, Neil T. Thompson, Hugh Walton, Nicola E. Wilsher, Alison J. Woolford, Nicola G. Wallis. Characterization of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B154.

Explosions in the availability of cancer genomic data and protein structure data give us the potential to explore cancer molecular biology at an unprecedented scale, and in atomic detail. We present COSMIC-3D, which combines COSMIC, the most comprehensive cancer mutation database available (http://cancer.sanger.ac.uk), with the wealth of publicly available 3D protein structure data, to create a resource with which the protein-structural nature of cancer can be probed. COSMIC-3D will help us understand which cancer mutations drive the progression of cancers, by identifying which are in functional sites that have an effect on driving cell growth and proliferation, and where they are clustered in protein structures across the proteome, for specific cancer types. Through understanding the effects of cancer mutations on known and predicted drug binding sites, we aim to predict potential new drug targets in cancers, and improve the specificity and efficacy of new or existing drugs, by using protein structure with cancer mutation data to guide mutation-specific drug design. COSMIC-3D is available as a web interface at http://cancer.sanger.ac.uk/cosmic3d, and enables interactive exploration of the cancer mutome in a 3D peptide environment, showing all forms of exonic point mutation. Individual mutant locations can be highlighted as molecular surfaces, while recurrence across nonsynonymous substitutions is visualized as 3D heat-maps. Cancer mutations can also be visualised in combination with precalculated small-molecule “druggable” binding sites, providing a powerful visual approach for development of hypotheses across structural, functional, and drug-resistance impacts of cancer variants. COSMIC-3D shows, for example, the steric occlusion of the binding site of ATP-competitive small-molecule inhibitors in the EGFR kinase domain by the mutation of L858 to arginine; these kinds of insights can be applied to novel cancer targets for any protein-structural mutation of interest. The human cancer structural proteome comprising COSMIC-3D extends to nearly 8,500 human genes; 1/3rd of genes in COSMIC. Over 30,000 human protein structures are available across these genes, to which over 345,000 cancer mutations are mapped. From structure based druggability prediction, over 6,500 proteins in COSMIC are predicted to have small-molecule druggable binding sites, making COSMIC-3D a powerful and exciting resource for the exploration and guidance of drug design and discovery in oncology. Citation Format: Harry C. Jubb, Harpreet Saini, Marcel Verdonk, Simon Forbes. COSMIC-3D: exploring cancer mutations in three dimensions for drug design and discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2601. doi:10.1158/1538-7445.AM2017-2601

Background: Serum biomarkers for diagnosis and risk stratification of childhood solid tumors would improve the accuracy/timeliness of diagnosis and reduce the need for invasive biopsies. We hypothesized that differential expression and/or release of microRNAs (miRNAs) by such tumors may be detected as altered serum miRNA profiles. Methods: We undertook global quantitative reverse transcription PCR (qRT-PCR) miRNA profiling (n = 741) on RNA from 53 serum samples, representing 33 diagnostic cases of common childhood cancers plus 20 controls. Technical confirmation was performed in a subset of 21 cases, plus four independent samples. Results: We incorporated robust quality control steps for RNA extraction, qRT-PCR efficiency and hemolysis quantification. We evaluated multiple methods to normalize global profiling data and identified the ‘global mean’ approach as optimal. We generated a panel of six miRNAs that were most stable in pediatric serum samples and therefore most suitable for normalization of targeted miRNA qRT-PCR data. Tumor-specific serum miRNA profiles were identified for each tumor type and selected miRNAs underwent confirmatory testing. We identified a panel of miRNAs (miR-124-3p/miR-9-3p/miR-218-5p/miR-490-5p/miR-1538) of potential importance in the clinical management of neuroblastoma, as they were consistently highly overexpressed in MYCN-amplified high-risk cases (MYCN-NB). We also derived candidate miRNA panels for noninvasive differential diagnosis of a liver mass (hepatoblastoma vs. combined MYCN-NB/NB), an abdominal mass (Wilms tumor vs. combined MYCN-NB/NB), and sarcoma subtypes. Conclusions: This study describes a pipeline for robust diagnostic serum miRNA profiling in childhood solid tumors, and has identified candidate miRNA profiles for prospective testing. Impact: We propose a new noninvasive method with the potential to diagnose childhood solid tumors. Cancer Epidemiol Biomarkers Prev; 24(2); 350–60. ©2014 AACR.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed “GAGGUA” was enriched in the 3′ untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention. Cancer Res; 73(15); 4872–84. ©2013 AACR.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

Transposons present an acute challenge to the germline, and mechanisms that repress their activity are essential for transgenerational genomic integrity. LINE1 (L1) is the most successful retrotransposon and is epigenetically repressed by CpG DNA methylation. Here, we identify two additional important mechanisms by which L1 is repressed during spermatogenesis. We demonstrate that the Piwi protein Mili and the piRNA pathway are required to posttranscriptionally silence L1 in meiotic pachytene cells even in the presence of normal L1 DNA methylation. Strikingly, in the absence of both a functional piRNA pathway and DNA methylation, L1 elements are normally repressed in mitotic stages of spermatogenesis. Accordingly, we find that the euchromatic repressive histone H3 dimethylated lysine 9 modification cosuppresses L1 expression therein. We demonstrate the existence of multiple epigenetic mechanisms that in conjunction with the piRNA pathway sequentially enforce L1 silencing and genomic stability during mitotic and meiotic stages of adult spermatogenesis.

Mast cells have essential effector and immunoregulatory functions in IgE-associated allergic disorders and certain innate and adaptive immune responses, but the role of miRNAs in regulating mast cell functions is almost completely unexplored. To examine the role of the activation-induced miRNA miR-221 in mouse mast cells, we developed robust lentiviral systems for miRNA overexpression and depletion. While miR-221 favored mast cell adhesion and migration towards SCF or antigen in trans-well migration assays, as well as cytokine production and degranulation in response to IgE-antigen complexes, neither miR-221 overexpression, nor its ablation, interfered with mast cell differentiation. Transcriptional profiling of miR-221-overexpressing mast cells revealed modulation of many transcripts, including several associated with the cytoskeleton; indeed, miR-221 overexpression was associated with reproducible increases in cortical actin in mast cells, and with altered cellular shape and cell cycle in murine fibroblasts. Our bioinformatics analysis showed that this effect was likely mediated by the composite effect of miR-221 on many primary and secondary targets in resting cells. Indeed, miR- 221-induced cellular alterations could not be recapitulated by knockdown of one of the major targets of miR-221. We propose a model in which miR-221 has two different roles in mast cells: in resting cells, basal levels of miR-221 contribute to the regulation of the cell cycle and cytoskeleton, a general mechanism probably common to other miR-221- expressing cell types, such as fibroblasts. Vice versa, upon induction in response to mast cell stimulation, miR-221 effects are mast cell-specific and activation-dependent, contributing to the regulation of degranulation, cytokine production and cell adherence. Our studies provide new insights into the roles of miR-221 in mast cell biology, and identify novel mechanisms that may contribute to mast cell-related pathological conditions, such as asthma, allergy and mastocytosis.

Background We hypothesised that differences in microRNA expression profiles contribute to the contrasting natural history and clinical outcome of the two most common types of malignant germ cell tumour (GCT), yolk sac tumours (YSTs) and germinomas. Results By direct comparison, using microarray data for paediatric GCT samples and published qRT-PCR data for adult samples, we identified microRNAs significantly up-regulated in YSTs (n = 29 paediatric, 26 adult, 11 overlapping) or germinomas (n = 37 paediatric). By Taqman qRT-PCR we confirmed differential expression of 15 of 16 selected microRNAs and further validated six of these (miR-302b, miR-375, miR-200b, miR-200c, miR-122, miR-205) in an independent sample set. Interestingly, the miR-302 cluster, which is over-expressed in all malignant GCTs, showed further over-expression in YSTs versus germinomas, representing six of the top eight microRNAs over-expressed in paediatric YSTs and seven of the top 11 in adult YSTs. To explain this observation, we used mRNA expression profiles of paediatric and adult malignant GCTs to identify 10 transcription factors (TFs) consistently over-expressed in YSTs versus germinomas, followed by linear regression to confirm associations between TF and miR-302 cluster expression levels. Using the sequence motif analysis environment iMotifs, we identified predicted binding sites for four of the 10 TFs (GATA6, GATA3, TCF7L2 and MAF) in the miR-302 cluster promoter region. Finally, we showed that miR-302 family over-expression in YST is likely to be functionally significant, as mRNAs down-regulated in YSTs were enriched for 3' untranslated region sequences complementary to the common seed of miR-302a~miR-302d. Such mRNAs included mediators of key cancer-associated processes, including tumour suppressor genes, apoptosis regulators and TFs. Conclusions Differential microRNA expression is likely to contribute to the relatively aggressive behaviour of YSTs and may enable future improvements in clinical diagnosis and/or treatment.

Despite their extensive clinical and pathologic heterogeneity, all malignant germ cell tumors (GCT) are thought to originate from primordial germ cells. However, no common biological abnormalities have been identified to date. We profiled 615 microRNAs (miRNA) in pediatric malignant GCTs, controls, and GCT cell lines (48 samples in total) and re-analyzed available miRNA expression data in adult gonadal malignant GCTs. We applied the bioinformatic algorithm Sylamer to identify miRNAs that are of biological importance by inducing global shifts in mRNA levels. The most significant differentially expressed miRNAs in malignant GCTs were all from the miR-371–373 and miR-302 clusters (adjusted P < 0.00005), which were overexpressed regardless of histologic subtype [yolk sac tumor (YST)/seminoma/embryonal carcinoma (EC)], site (gonadal/extragonadal), or patient age (pediatric/adult). Sylamer revealed that the hexamer GCACTT, complementary to the 2- to 7-nucleotide miRNA seed AAGUGC shared by six members of the miR-371–373 and miR-302 clusters, was the only sequence significantly enriched in the 3′-untranslated region of mRNAs downregulated in pediatric malignant GCTs (as a group), YSTs and ECs, and in adult YSTs (all versus nonmalignant tissue controls; P < 0.05). For the pediatric samples, downregulated genes containing the 3′-untranslated region GCACTT showed significant overrepresentation of Gene Ontology terms related to cancer-associated processes, whereas for downregulated genes lacking GCACTT, Gene Ontology terms generally represented metabolic processes only, with few genes per term (adjusted P < 0.05). We conclude that the miR-371–373 and miR-302 clusters are universally overexpressed in malignant GCTs and coordinately downregulate mRNAs involved in biologically significant pathways. Cancer Res; 70(7); 2911–23

Background MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in development, differentiation and pathogenesis. Hundreds of miRNAs have been discovered in mammalian genomes. Approximately 50% of mammalian miRNAs are expressed from introns of protein-coding genes; the primary transcript (pri-miRNA) is therefore assumed to be the host transcript. However, very little is known about the structure of pri-miRNAs expressed from intergenic regions. Here we annotate transcript boundaries of miRNAs in human, mouse and rat genomes using various transcription features. The 5' end of the pri-miRNA is predicted from transcription start sites, CpG islands and 5' CAGE tags mapped in the upstream flanking region surrounding the precursor miRNA (pre-miRNA). The 3' end of the pri-miRNA is predicted based on the mapping of polyA signals, and supported by cDNA/EST and ditags data. The predicted pri-miRNAs are also analyzed for promoter and insulator-associated regulatory regions. Results We define sets of conserved and non-conserved human, mouse and rat pre-miRNAs using bidirectional BLAST and synteny analysis. Transcription features in their flanking regions are used to demarcate the 5' and 3' boundaries of the pri-miRNAs. The lengths and boundaries of primary transcripts are highly conserved between orthologous miRNAs. A significant fraction of pri-miRNAs have lengths between 1 and 10 kb, with very few introns. We annotate a total of 59 pri-miRNA structures, which include 82 pre-miRNAs. 36 pri-miRNAs are conserved in all 3 species. In total, 18 of the confidently annotated transcripts express more than one pre-miRNA. The upstream regions of 54% of the predicted pri-miRNAs are found to be associated with promoter and insulator regulatory sequences. Conclusion Little is known about the primary transcripts of intergenic miRNAs. Using comparative data, we are able to identify the boundaries of a significant proportion of human, mouse and rat pri-miRNAs. We confidently predict the transcripts including a total of 77, 58 and 47 human, mouse and rat pre-miRNAs respectively. Our computational annotations provide a basis for subsequent experimental validation of predicted pri-miRNAs.

MicroRNAs (miRNAs) are important genetic regulators of development, differentiation, growth, and metabolism. The mammalian genome encodes ≈500 known miRNA genes. Approximately 50% are expressed from non-protein-coding transcripts, whereas the rest are located mostly in the introns of coding genes. Intronic miRNAs are generally transcribed coincidentally with their host genes. However, the nature of the primary transcript of intergenic miRNAs is largely unknown. We have performed a large-scale analysis of transcription start sites, polyadenylation signals, CpG islands, EST data, transcription factor-binding sites, and expression ditag data surrounding intergenic miRNAs in the human genome to improve our understanding of the structure of their primary transcripts. We show that a significant fraction of primary transcripts of intergenic miRNAs are 3–4 kb in length, with clearly defined 5′ and 3′ boundaries. We provide strong evidence for the complete transcript structure of a small number of human miRNAs.

The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system.

Background As malignant germ cell tumors (MGCTs) are thought to have a common origin from primordial germ cells, they may share fundamental biological abnormalities despite their clinical and histopathological heterogeneity. We tested the hypothesis that MGCTs are characterized by common patterns of microRNA (miRNA) expression that are functionally significant by affecting levels of mRNA targets. We further investigated relationships between miRNA profiles and transcription factor expression, in order to identify potential regulators of miRNA transcription in MGCTs. Methods We used the miRCURY microarray (Exiqon) to quantify global microRNA expression levels in 28 gonadal and extragonadal GCTs and 14 non-malignant samples (eight controls, six teratomas) from pediatric subjects. For 21 of these samples (17 MGCT, four non-malignant) global mRNA expression profiles had been obtained using the U133A GeneChip (Affymetrix). We compared our findings with a re-analysis of published qRT-PCR miRNA and U133A GeneChip mRNA profiling in adult gonadal MGCTs. Data were analysed using R and Bioconductor. We used the novel bioinformatic algorithm Sylamer to investigate enrichment and depletion of miRNA seed complementary regions (SCRs) in 3’UTRs of mRNAs ranked according to expression levels in MGCTs. For the pediatric MGCT samples we compared miRNA profiles with transcription factor expression using linear regression plots. Results miR-302 and miR-371∼373 clusters were universally over-expressed in MGCTs regardless of patient age (pediatric or adult), tumor histological type [yolk sac tumors (YSTs), seminomas or embryonal carcinomas] or anatomical site (gonadal or extragonadal). Sylamer demonstrated that the most significantly over-represented SCR in the 3’UTRs of down-regulated mRNAs was GCACTT, corresponding to the AAGUGC seed common to the six members of the miR-302a∼d and miR-372∼373 family. Gene Ontology (GO) analysis revealed that the down-regulated mRNAs containing this common SCR in pediatric MGCTs were members of major cancer-associated cellular pathways. The transcription factor SOX17 was highly significantly over-expressed in pediatric and adult MGCTs (adjusted p-value=3.0×10−4 and 1.4×10−11, respectively), and showed a strong positive correlation in matched pediatric samples with combined median expression values for miR-302a∼d and miR-372∼3 (p-value=4.9×10−4). Conclusion Our data suggests a fundamental role for miR-302 and miR-371∼373 clusters in the biology of MGCTs and identifies SOX17 as a candidate regulator of these clusters. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3424.