Functional Annotation of MicroRNAs Using Existing Resources.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs that are involved in most biological signaling pathways, including the cell cycle, apoptosis, proliferation, immune response, metabolism as well as in biological processes including organ development and in human diseases like cancers. During the past two decades, high-throughput transcriptomic profiling using next generation sequencing and microarrays have been extensively utilized to identify differentially expressed miRNAs across different conditions and diseases. A natural extension of miRNA identification is to the process of functionally annotating known or predicted gene targets of those miRNAs and, by inference, revealing their potential influences on diverse biological pathways and functions. In this chapter, we provide a stepwise guideline on how to perform functional enrichment analyses on miRNAs of interest using publicly available resources such as miRWalk2.0.

Methyltransferase inhibitors restore SATB1 protective activity against cutaneous T cell lymphoma in mice.

Cutaneous T cell lymphoma (CTCL) has a poorly understood etiology and no known cure. Using conditional knockout mice, we found that ablation of the genomic organizer special AT-rich sequence-binding protein 1 (Satb1) caused malignant transformation of mature, skin-homing, Notch-activated CD4+ and CD8+ T cells into progressively fatal lymphoma. Mechanistically, Satb1 restrained Stat5 phosphorylation and the expression of skin-homing chemokine receptors in mature T cells. Notably, methyltransferase-dependent epigenetic repression of SATB1 was universally found in human Sézary syndrome, but not in other peripheral T cell malignancies. H3K27 and H3K9 trimethylation occluded the SATB1 promoter in Sézary cells, while inhibition of SUV39H1/2 methyltransferases (unlike EZH2 inhibition) restored protective SATB1 expression and selectively abrogated the growth of primary Sézary cells more effectively than romidepsin. Therefore, inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides/Sézary syndrome, a set of incurable diseases.

Epigenetic specifications of host chromosome docking sites for latent Epstein-Barr virus.

Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.

Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization.

Senescence is induced by various stimuli such as oncogene expression and telomere shortening, referred to as oncogene-induced senescence (OIS) and replicative senescence (RS), respectively, and accompanied by global transcriptional alterations and 3D genome reorganization. Here, we demonstrate that the human condensin II complex participates in senescence via gene regulation and reorganization of euchromatic A and heterochromatic B compartments. Both OIS and RS are accompanied by A-to-B and B-to-A compartmental transitions, the latter of which occur more frequently and are undergone by 14% (430 Mb) of the human genome. Mechanistically, condensin is enriched in A compartments and implicated in B-to-A transitions. The full activation of senescence genes (SASP genes and p53 targets) requires condensin; its depletion impairs senescence markers. This study describes that condensin reinforces euchromatic A compartments and promotes B-to-A transitions, both of which are coupled to optimal expression of senescence genes, thereby allowing condensin to contribute to senescent processes.

N6-Methylation of Adenosine of FZD10 mRNA Contributes to PARP Inhibitor Resistance.

Despite the high initial response rates to PARP inhibitors (PARPi) in BRCA-mutated epithelial ovarian cancers (EOC), PARPi resistance remains a major challenge. Chemical modifications of RNAs have emerged as a new layer of epigenetic gene regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of mRNA, yet the role of m6A modification in PARPi resistance has not previously been explored. Here, we show that m6A modification of FZD10 mRNA contributes to PARPi resistance by upregulating the Wnt/ß-catenin pathway in BRCA-mutated EOC cells. Global m6A profile revealed a significant increase in m6A modification in FZD10 mRNA, which correlated with increased FZD10 mRNA stability and an upregulation of the Wnt/ß-catenin pathway. Depletion of FZD10 or inhibition of the Wnt/ß-catenin sensitizes resistant cells to PARPi. Mechanistically, downregulation of m6A demethylases FTO and ALKBH5 was sufficient to increase FZD10 mRNA m6A modification and reduce PARPi sensitivity, which correlated with an increase in homologous recombination activity. Moreover, combined inhibition of PARP and Wnt/ß-catenin showed synergistic suppression of PARPi-resistant cells in vitro and in vivo in a xenograft EOC mouse model. Overall, our results show that m6A contributes to PARPi resistance in BRCA-deficient EOC cells by upregulating the Wnt/ß-catenin pathway via stabilization of FZD10. They also suggest that inhibition of the Wnt/ß-catenin pathway represents a potential strategy to overcome PARPi resistance. SIGNIFICANCE: These findings elucidate a novel regulatory mechanism of PARPi resistance in EOC by showing that m6A modification of FZD10 mRNA contributes to PARPi resistance in BRCA-deficient EOC cells via upregulation of Wnt/ß-catenin pathway.

A Gene Expression Classifier from Whole Blood Distinguishes Benign from Malignant Lung Nodules Detected by Low-Dose CT.

Low-dose CT (LDCT) is widely accepted as the preferred method for detecting pulmonary nodules. However, the determination of whether a nodule is benign or malignant involves either repeated scans or invasive procedures that sample the lung tissue. Noninvasive methods to assess these nodules are needed to reduce unnecessary invasive tests. In this study, we have developed a pulmonary nodule classifier (PNC) using RNA from whole blood collected in RNA-stabilizing PAXgene tubes that addresses this need. Samples were prospectively collected from high-risk and incidental subjects with a positive lung CT scan. A total of 821 samples from 5 clinical sites were analyzed. Malignant samples were predominantly stage 1 by pathologic diagnosis and 97% of the benign samples were confirmed by 4 years of follow-up. A panel of diagnostic biomarkers was selected from a subset of the samples assayed on Illumina microarrays that achieved a ROC-AUC of 0.847 on independent validation. The microarray data were then used to design a biomarker panel of 559 gene probes to be validated on the clinically tested NanoString nCounter platform. RNA from 583 patients was used to assess and refine the NanoString PNC (nPNC), which was then validated on 158 independent samples (ROC-AUC = 0.825). The nPNC outperformed three clinical algorithms in discriminating malignant from benign pulmonary nodules ranging from 6-20 mm using just 41 diagnostic biomarkers. Overall, this platform provides an accurate, noninvasive method for the diagnosis of pulmonary nodules in patients with non-small cell lung cancer. SIGNIFICANCE: These findings describe a minimally invasive and clinically practical pulmonary nodule classifier that has good diagnostic ability at distinguishing benign from malignant pulmonary nodules.

Evidence for Persistent Monocyte and Immune Dysregulation After Prolonged Viral Suppression Despite Normalization of Monocyte Subsets, sCD14 and sCD163 in HIV-Infected Individuals.

BACKGROUND: People living with HIV on antiretroviral therapy (HIV/ART) experience excess non-AIDS comorbidities, and also remain at increased risk for certain infections and viral malignancies. Monocytes/macrophages are central to many of these comorbidities, and elevated plasma cytokines and immune activation during untreated infection are often incompletely reversed by ART and are also associated with comorbidities. METHODS: We investigated monocyte surface markers, gene expression, and plasma cytokines in 11 HIV-infected older individuals (median 53 years) who started therapy with low CD4 counts (median 129 cells/µl), with elevated hsCRP (≥ 2mg/L) despite long-term ART (median 7.4 years), along with matched controls. RESULTS: Frequency of monocyte subsets (based on CD14/CD16/CD163), were not different from controls, but surface expression of CD163 was increased (P = 0.021) while PD1 was decreased (P = 0.013) along with a trend for higher tissue factor (P = 0.096). As a group, HIV/ART participants had elevated plasma CCL2 (MCP-1; P = 0.0001), CXCL9 (MIG; P = 0.04), and sIL2R (P = 0.015), which were correlated, while sCD14 was not elevated. Principal component analysis of soluble markers revealed that 6/11 HIV/ART participants clustered with controls, while 5 formed a distinct group, driven by IL-10, CCL11, CXCL10, CCL2, CXCL9, and sIL2R. These individuals were significantly older than those clustering with controls. Transcriptomic analysis revealed multiple genes linked to immune functions including inflammation, immune cell development, and cell-cell signaling that were downregulated in HIV/ART monocytes and distinct from patterns in untreated subjects. CONCLUSIONS: Long-term ART-treated individuals normalize monocyte subsets but exhibit immune dysregulation involving both aberrant inflammation and monocyte dysfunction, as well as inter-individual heterogeneity, suggesting complex mechanisms linking monocytes and HIV/ART comorbidities.

ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5-MAVS-IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6-p38-MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3'-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3'-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.

Structural and functional analysis of the human POT1-TPP1 telomeric complex.

POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1-TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1-TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancer-associated mutations, partially disrupt the POT1-TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1-TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer.

BET Inhibitors Suppress ALDH Activity by Targeting ALDH1A1 Super-Enhancer in Ovarian Cancer.

The emergence of tumor cells with certain stem-like characteristics, such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression, contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to chemotherapy efficacy. Here, we show that bromodomain and extraterminal (BET) inhibitors suppress ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. The clinically applicable small-molecule BET inhibitor JQ1 suppressed the outgrowth of cisplatin-treated ovarian cancer cells both in vitro and in vivo Combination of JQ1 and cisplatin improved the survival of ovarian cancer-bearing mice in an orthotopic model. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and other stem-related genes in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using clinically applicable small-molecule inhibitors, such as JQ1, is a promising strategy for targeting ALDH activity in epithelial ovarian cancer. Cancer Res; 76(21); 6320-30. ©2016 AACR.

Transgenic Expression of the Mitochondrial Chaperone TNFR-associated Protein 1 (TRAP1) Accelerates Prostate Cancer Development.

Protein homeostasis, or proteostasis, is required for mitochondrial function, but its role in cancer is controversial. Here we show that transgenic mice expressing the mitochondrial chaperone TNFR-associated protein 1 (TRAP1) in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten+/- background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten+/- mice without affecting hyperplasia or prostatic intraepithelial neoplasia. Global profiling of Pten+/--TRAP1 transgenic mice by RNA sequencing and reverse phase protein array reveals modulation of oncogenic networks of cell proliferation, apoptosis, cell motility, and DNA damage. Mechanistically, reconstitution of Pten+/- prostatic epithelial cells with TRAP1 increases cell proliferation, reduces apoptosis, and promotes cell invasion without changes in mitochondrial bioenergetics. Therefore, TRAP1 is a driver of prostate cancer in vivo and an "actionable" therapeutic target.

The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis.

Metastasis is a critical event affecting breast cancer patient survival. To identify molecules contributing to the metastatic process, we analysed The Cancer Genome Atlas (TCGA) breast cancer data and identified 41 genes whose expression is inversely correlated with survival. Here we show that GABAA receptor alpha3 (Gabra3), normally exclusively expressed in adult brain, is also expressed in breast cancer, with high expression of Gabra3 being inversely correlated with breast cancer survival. We demonstrate that Gabra3 activates the AKT pathway to promote breast cancer cell migration, invasion and metastasis. Importantly, we find an A-to-I RNA-edited form of Gabra3 only in non-invasive breast cancers and show that edited Gabra3 suppresses breast cancer cell invasion and metastasis. A-to-I-edited Gabra3 has reduced cell surface expression and suppresses the activation of AKT required for cell migration and invasion. Our study demonstrates a significant role for mRNA-edited Gabra3 in breast cancer metastasis.

Telomeric repeat-containing RNA (TERRA) constitutes a nucleoprotein component of extracellular inflammatory exosomes.

Telomeric repeat-containing RNA (TERRA) has been identified as a telomere-associated regulator of chromosome end protection. Here, we report that TERRA can also be found in extracellular fractions that stimulate innate immune signaling. We identified extracellular forms of TERRA in mouse tumor and embryonic brain tissue, as well as in human tissue culture cell lines using RNA in situ hybridization. RNA-seq analyses revealed TERRA to be among the most highly represented transcripts in extracellular fractions derived from both normal and cancer patient blood plasma. Cell-free TERRA (cfTERRA) could be isolated from the exosome fractions derived from human lymphoblastoid cell line (LCL) culture media. cfTERRA is a shorter form (∼200 nt) of cellular TERRA and copurifies with CD63- and CD83-positive exosome vesicles that could be visualized by cyro-electron microscopy. These fractions were also enriched for histone proteins that physically associate with TERRA in extracellular ChIP assays. Incubation of cfTERRA-containing exosomes with peripheral blood mononuclear cells stimulated transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) Exosomes engineered with elevated TERRA or liposomes with synthetic TERRA further stimulated inflammatory cytokines, suggesting that exosome-associated TERRA augments innate immune signaling. These findings imply a previously unidentified extrinsic function for TERRA and a mechanism of communication between telomeres and innate immune signals in tissue and tumor microenvironments.

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Diamond Blackfan Anemia (DBA) is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal protein haploinsufficiency or changes in the bone marrow microenvironment that leads to red cell aplasia in DBA patients.

Interaction between TBP and Condensin Drives the Organization and Faithful Segregation of Mitotic Chromosomes.

Genome/chromosome organization is highly ordered and controls various nuclear events, although the molecular mechanisms underlying the functional organization remain largely unknown. Here, we show that the TATA box-binding protein (TBP) interacts with the Cnd2 kleisin subunit of condensin to mediate interphase and mitotic chromosomal organization in fission yeast. TBP recruits condensin onto RNA polymerase III-transcribed (Pol III) genes and highly transcribed Pol II genes; condensin in turn associates these genes with centromeres. Inhibition of the Cnd2-TBP interaction disrupts condensin localization across the genome and the proper assembly of mitotic chromosomes, leading to severe defects in chromosome segregation and eventually causing cellular lethality. We propose that the Cnd2-TBP interaction coordinates transcription with chromosomal architecture by linking dispersed gene loci with centromeres. This chromosome arrangement can contribute to the efficient transmission of physical force at the kinetochore to chromosomal arms, thereby supporting the fidelity of chromosome segregation.

AKAP4 is a circulating biomarker for non-small cell lung cancer.

Cancer testis antigens (CTAs) are widely expressed in tumor tissues, circulating tumor cells (CTCs) and in cancer derived exosomes that are frequently engulfed by lymphoid cells. To determine whether tumor derived CTA mRNAs could be detected in RNA from purified peripheral blood mononuclear cells (PBMC) of non-small cell lung cancer (NSCLC) patients, we assayed for the expression of 116 CTAs in PBMC RNA in a discovery set and identified AKAP4 as a potential NSCLC biomarker. We validated AKAP4 as a highly accurate biomarker in a cohort of 264 NSCLCs and 135 controls from 2 different sites including a subset of controls with high risk lung nodules. When all (264) lung cancers were compared with all (135) controls the area under the ROC curve (AUC) was 0.9714. When 136 stage I NSCLC lung cancers are compared with all controls the AUC is 0.9795 and when all lung cancer patients were compared to 27 controls with histologically confirmed benign lung nodules, a comparison of significant clinical importance, the AUC was 0.9825. AKAP4 expression increases significantly with tumor stage, but independent of age, gender, smoking history or cancer subtype. Follow-up studies in a small number of resected NSCLC patients revealed a decrease of AKAP4 expression post-surgical resection that remained low in patients in remission and increased with tumor recurrence. AKAP4 is a highly accurate biomarker for the detection of early stage lung cancer.

Romidepsin in peripheral and cutaneous T-cell lymphoma: mechanistic implications from clinical and correlative data.

Romidepsin is an epigenetic agent approved for the treatment of patients with cutaneous or peripheral T-cell lymphoma (CTCL and PTCL). Here we report data in all patients treated on the National Cancer Institute 1312 trial, demonstrating long-term disease control and the ability to retreat patients relapsing off-therapy. In all, 84 patients with CTCL and 47 with PTCL were enrolled. Responses occurred early, were clinically meaningful and of very long duration in some cases. Notably, patients with PTCL receiving romidepsin as third-line therapy or later had a comparable response rate (32%) of similar duration as the total population (38%). Eight patients had treatment breaks of 3.5 months to 10 years; in four of six patients, re-initiation of treatment led to clear benefit. Safety data show slightly greater haematological and constitutional toxicity in PTCL. cDNA microarray studies show unique individual gene expression profiles, minimal overlap between patients, and both induction and repression of gene expression that reversed within 24 h. These data argue against cell death occurring as a result of an epigenetics-mediated gene induction programme. Together this work supports the safety and activity of romidepsin in T-cell lymphoma, but suggests a complex mechanism of action.

Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes.

Molecular stratification of tumors is essential for developing personalized therapies. Although patient stratification strategies have been successful; computational methods to accurately translate the gene-signature from high-throughput platform to a clinically adaptable low-dimensional platform are currently lacking. Here, we describe PIGExClass (platform-independent isoform-level gene-expression based classification-system), a novel computational approach to derive and then transfer gene-signatures from one analytical platform to another. We applied PIGExClass to design a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) based molecular-subtyping assay for glioblastoma multiforme (GBM), the most aggressive primary brain tumors. Unsupervised clustering of TCGA (the Cancer Genome Altas Consortium) GBM samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 19% of the samples, resulting in significant (P = 0.0103) survival differences among the refined subgroups. PIGExClass derived four-class classifier, which requires only 121 transcript-variants, assigns GBM patients' molecular subtype with 92% accuracy. This classifier was translated to an RT-qPCR assay and validated in an independent cohort of 206 GBM samples. Our results demonstrate the efficacy of PIGExClass in the design of clinically adaptable molecular subtyping assay and have implications for developing robust diagnostic assays for cancer patient stratification.

CD164 and FCRL3 are highly expressed on CD4+CD26- T cells in Sézary syndrome patients.

Sézary syndrome (SS) cells express cell surface molecules also found on normal activated CD4 T cells. In an effort to find a more specific surface marker for malignant SS cells, a microarray analysis of gene expression was performed. Results showed significantly increased levels of mRNA for CD164, a sialomucin found on human CD34+ hematopoietic stem cells, and FCRL3, a molecule present on a subset of human natural T regulatory cells. Both markers were increased in CD4 T cells from SS patients compared with healthy donors (HD). Flow cytometry studies confirmed the increased expression of CD164 and FCRL3 primarily on CD4+CD26- T cells of SS patients. Importantly, a statistically significant correlation was found between an elevated percentage of CD4+CD164+ T cells and an elevated percentage of CD4+CD26- T cells in all tested SS patients but not in patients with mycosis fungoides and atopic dermatitis or HD. FCRL3 expression was significantly increased only in patients with high tumor burden. CD4+CD164+ cells displayed cerebriform morphology and their loss correlated with clinical improvement in treated patients. Our results suggest that CD164 can serve as a marker for diagnosis and for monitoring progression of cutaneous T-cell lymphoma (CTCL)/SS and that FCRL3 expression correlates with a high circulating tumor burden.