Sara Napoli

Promoter-specific transcriptional interference and c-myc gene silencing by siRNAs in human cells

Small interfering RNAs (siRNAs) directed to gene promoters can silence genes at the transcriptional level. siRNA‐directed transcriptional silencing (RdTS) was first described in plants and yeasts and more recently in mammalian cells. RdTS has been associated with the induction of epigenetic changes and the formation of complexes containing RNA interference and chromatin‐remodelling factors. Here, we show that a promoter‐targeted siRNA inhibits transcription of the c‐myc gene. Transcriptional silencing of c‐myc did not involve changes of known epigenetic marks. Instead, the c‐myc promoter‐targeted siRNA interfered with transcription initiation blocking the assembly of the pre‐initiation complex. Transcriptional interference depended on Argonaute 2 and a noncoding promoter‐associated RNA initiated upstream and overlapping the transcription start site. Silencing of c‐myc led to growth arrest, reduced clonogenic potential and senescence of c‐myc over‐expressing prostate cancer cells with minimal effect on normal cells. RNA‐directed transcriptional interference may be a natural mechanism of transcriptional control and siRNAs targeting noncoding RNAs participating in this regulatory pathway could be valuable tools to control expression of deregulated genes in human diseases.

RNAi-mediated silencing of Myc transcription inhibits stem-like cell maintenance and tumorigenicity in prostate cancer

Several studies link disease progression, recurrence, and treatment failures to the cancer stem-like cell (CSC) subpopulation within the heterogeneous tumor cell population. Myc is a transcription factor having a central function in stem cell biology and in human cancers. Hence, Myc represents an attractive target to develop CSC-specific therapies. Recent findings suggest that Myc transcription can be silenced using an RNA interference (RNAi)-based strategy that targets noncoding promoter-associated RNA (paRNA) overlapping the transcription start site. In this study, we investigated the effects of silencing Myc transcription on prostate CSC in cell culture and xenograft models of human prostate cancer. Treatment with an effective promoter-targeting siRNA reduced the fraction of CSCs, leading to reduced self-renewal, tumor-initiating, and metastatic capability. Combined analysis of stem-like cells and senescence markers indicated that Myc silencing triggered a phenotypic shift and senescence in the CSC subpopulation. Notably, systemic delivery of the promoter-targeting siRNA in the xenograft model produced a striking suppression in the development of prostate tumors. Our results support a pivotal role for Myc in CSC maintenance and show that Myc targeting via RNAi-based transcriptional silencing can trigger CSC senescence and loss of their tumor-initiating capability. More generally, our findings demonstrate the efficacy of RNAi-based transcriptional strategies and the potential to target regulatory noncoding paRNAs for therapeutic applications.

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer–associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP–CUL3 substrates that are preferentially degraded by endometrial cancer–associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer–specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

Mechanisms of triplex DNA-mediated inhibition of transcription initiation in cells.

Triplex-forming oligonucleotides (TFOs) are attractive tools to control gene expression at the transcriptional level. This anti-gene approach has proven to be successful in various experimental settings. However, the mechanisms leading to transcriptional repression in cells have not been fully investigated yet. Here, we examined the consequence of triplex DNA formation on the binding of transcriptional activators, co-activators and RNA Polymerase II to the ets2 gene promoter using chromatin immunoprecipitation assays. The triplex target sequence was located approximately 40-bp upstream of the transcription start site (TSS) and overlapped an Sp1 binding site relevant for ets2 transcription. We found that the ets2-TFO prevented binding of Sp1, TAF(II)130 and TAF(II)250 to the ets2 promoter, while binding of RNA polymerase II and TBP were not affected. The effects were both sequence and target specific, since the TFO had no effect on the c-myc promoter and a mutated ets2 promoter construct. Thus, triplex DNA formation near a TSS leads to formation of a non-functional pre-initiation complex (PIC) by blocking binding of transcriptional activators and co-activator molecules. This is the first direct demonstration of interference with PIC assembly at the TSS by oligonucleotide-triplex DNA formation in cells.

Growth inhibition and apoptosis induced by daunomycin-conjugated triplex-forming oligonucleotides targeting the c-myc gene in prostate cancer cells

Covalent attachment of intercalating agents to triplex-forming oligonucleotides (TFOs) is a promising strategy to enhance triplex stability and biological activity. We have explored the possibility to use the anticancer drug daunomycin as triplex stabilizing agent. Daunomycin-conjugated TFOs (dauno-TFOs) bind with high affinity and maintain the sequence-specificity required for targeting individual genes in the human genome. Here, we examined the effects of two dauno-TFOs targeting the c-myc gene on gene expression, cell proliferation and survival. The dauno-TFOs were directed to sequences immediately upstream (dauno-GT11A) and downstream (dauno-GT11B) the major transcriptional start site in the c-myc gene. Both dauno-TFOs were able to down-regulate promoter activity and transcription of the endogenous gene. Myc-targeted dauno-TFOs inhibited growth and induced apoptosis of prostate cancer cells constitutively expressing the gene. Daunomycin-conjugated control oligonucleotides with similar sequences had only minimal effects, confirming that the activity of dauno-TFOs was sequence-specific and triplex-mediated. To test the selectivity of dauno-TFOs, we examined their effects on growth of normal human fibroblasts, which express low levels of c-myc. Despite their ability to inhibit c-myc transcription, both dauno-TFOs failed to inhibit growth of normal fibroblasts at concentrations that inhibited growth of prostate cancer cells. In contrast, daunomycin inhibited equally fibroblasts and prostate cancer cells. Thus, daunomycin per se did not contribute to the antiproliferative activity of dauno-TFOs, although it greatly enhanced their ability to form stable triplexes at the target sites and down-regulate c-myc. Our data indicate that dauno-TFOs are attractive gene-targeting agents for development of new cancer therapeutics.

Small RNA-directed transcriptional control: New insights into mechanisms and therapeutic applications

The discovery of RNA interference (RNAi) has opened new avenues in biology and medicine. In addition to post-transcriptional gene silencing, new findings are expanding the range of action of small duplex RNAs and broadening the spectrum of the potential applications of RNAi-based therapeutics. In recent years a complex and heterogeneous network of non-protein coding RNAs (ncRNAs) with potential regulatory functions has come into the spotlight providing an unexpected perspective on the mechanisms of transcriptional and epigenetic control of gene expression in human cells. The spread and complexity of these RNA-based transcriptional regulatory networks are still to be explored. However, they are likely to be important mechanisms controlling gene expression in human cells. As we will learn more about these processes, endogenous small RNAs and ncRNAs participating in these transcriptional regulatory networks might become valuable targets to modulate expression of genes involved in human diseases. Thus, understanding these basic processes of gene regulation might be translated in the near future into innovative therapeutic strategies to treat human diseases.

A promoter-proximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers

Long noncoding RNAs are emerging players in the epigenetic machinery with key roles in development and diseases. Here we uncover a complex network comprising a promoter-associated noncoding RNA (paRNA), microRNA and epigenetic regulators that controls transcription of the tumour suppressor E-cadherin in epithelial cancers. E-cadherin silencing relies on the formation of a complex between the paRNA and microRNA-guided Argonaute 1 that, together, recruit SUV39H1 and induce repressive chromatin modifications in the gene promoter. A single nucleotide polymorphism (rs16260) linked to increased cancer risk alters the secondary structure of the paRNA, with the risk allele facilitating the assembly of the microRNA-guided Argonaute 1 complex and gene silencing. Collectively, these data demonstrate the role of a paRNA in E-cadherin regulation and the impact of a noncoding genetic variant on its function. Deregulation of paRNA-based epigenetic networks may contribute to cancer and other diseases making them promising targets for drug discovery.

Natural antisense transcripts drive a regulatory cascade controlling c-MYC transcription

ABSTRACT Cis-natural antisense transcripts (cis-NATs) are long noncoding RNAs transcribed from the opposite strand and overlapping coding and noncoding genes on the sense strand. cis-NATs are widely present in the human genome and can be involved in multiple mechanisms of gene regulation. Here, we describe the presence of cis-NATs in the 3′ distal region of the c-MYC locus and investigate their impact on transcriptional regulation of this key oncogene in human cancers. We found that cis-NATs are produced as consequence of the activation of cryptic transcription initiation sites in the 3′ distal region downstream of the c-MYC 3′UTR. The process is tightly regulated and leads to the formation of two main transcripts, NAT6531 and NAT6558, which differ in their ability to fold into stem-loop secondary structures. NAT6531 acts as a substrate for DICER and as a source of small RNAs capable of modulating c-MYC transcription. This complex system, based on the interplay between cis-NATs and NAT-derived small RNAs, may represent an important layer of epigenetic regulation of the expression of c-MYC and other genes in human cells.

Abstract 5179: The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma

Background. ETS transcription factors, such as FLI1 and SPIB, are recurrently deregulated in human lymphomas (Bonetti et al, Blood 2013; Lenz et al, PNAS 2008). The small molecule YK-4-279 inhibits binding of EWS1-FLI1 fusion protein to RHA resulting in growth arrest and apoptosis in Ewing sarcoma cells (Erkizan et al, Nat Med 2009) and we previously showed that YK-4-279 has in vitro anti-lymphoma activity (Chung et al, AACR 2015). TK-216 is a YK-4-279 clinical derivative that is in phase 1 for patients with relapsed or refractory Ewing sarcoma (NCT02657005). Here, we present extensive preclinical results obtained with TK-216 in lymphoma models. Methods. 56 cell lines [27 diffuse large B cell lymphoma (DLBCL); 10 mantle cell lymphoma; 6 marginal zone lymphoma; 5 anaplastic large T-cell lymphoma; 8 others] were exposed to TK-216 increasing doses for 72h using a Tecan D300e Digital Dispenser and 384well plates; cell proliferation was measured with MTT. In vivo studies were performed in NOD-SCID mice and treatments started with approximately sc 60mm3 tumor volumes. Results. TK-216 displayed high activity: median IC50 was 449 nM (95%CI: 367-506). Sensitivity was not affected by the lymphoma cell of origin [B vs T; activated B cell type (ABC) vs germinal center type DLBCL] or MYC and TP53 status. There was a non-statistically significant trend for lower sensitivity in cell lines bearing BCL2 chromosomal translocation (P=0.07, DLBCL only; P=0.06, all cell lines). Anti-tumor activity was mainly cytotoxic as confirmed by performing cell cycle analysis and Annexin V staining in 6 DLBCL cell lines (TMD8, U2932, HBL1, OCI-LY-18, WSU-DLCL2, DOHH2 for 24, 48, 72h), in which a time-dependent apoptosis was preceded by G2/M arrest. Antitumor activity was confirmed in DLBCL TMD8 xenografts. Compared with control group (n=10), mice treated with TK-216 (100 mg/Kg, BID; n=9) clearly presented a reduction in tumor growth, already evident at day 3 and becoming much stronger with time (D3, D5, D8, D11: P TK-216 was tested in combination with other targeted agents in DLBCL cell lines. A benefit was observed with the combination of TK-216 with the immunomodulator lenalidomide (synergism in 2/2 ABC DLBCL), with the BET inhibitor OTX015 (MK-8628) (synergism in 2/4 cells and additive effect in 1/4), the anti-CD20 monoclonal antibody rituximab (synergism in 2/3 cells) and the BCL2 inhibitor venetoclax (synergism in 3/4 cells). The latter synergism could be linked to the previously mentioned negative trend between TK-216 IC50 values and the presence of BCL2 translocation. Conclusions. The novel small molecule TK-216 presented strong preclinical anti-lymphoma activity, which provides evidence for further preclinical and clinical development as single agent and in combination. Citation Format: Filippo Spriano, Chiara Tarantelli, Eugenio Gaudio, Elaine YL Chung, Alberto J. Arribas, Luciano Cascione, Sara Napoli, Ivo Kwee, Andrea Rinaldi, Davide Rossi, Emanuele Zucca, Anastasios Stathis, Katti Jessen, Brian Lannutti, Jeffrey Toretsky, Francesco Bertoni. The first in class FLI1 inhibitor TK-216 presents both in vitro and in vivo anti-tumor activity in lymphoma [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 5179. doi:10.1158/1538-7445.AM2017-5179

Abstract 2349: An RNA-based epigenetic network controls the expression of E-cadherin in epithelial normal and cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Epigenetic mechanisms play important roles in many human diseases. However, what drives epigenetic regulators to specific genomic locations is still an open question. Promoter-associated long non-coding RNAs (paRNAs) have been identified in many genes and have been proposed to act as docking elements for recruitment of epigenetic regulators to gene promoters, although the underlying mechanisms are still poorly characterized. In this study we investigated the role of paRNAs in transcriptional regulation of E-cadherin (CDH1), a trans-membrane glycoprotein and an important determinant of epithelial cell differentiation. Transcriptional silencing of E-cadherin is frequent in epithelial cancers and loss of E-cadherin expression triggers epithelial-to-mesenchymal transition (EMT) and acquisition of tumor-initiating properties. We found that bidirectional transcription occurred from independent initiation sites in the E-cadherin promoter and generated sense (S) and antisense (AS) paRNAs that coordinated the transcriptional activity of the gene. S and AS paRNAs had distinct expression patterns in normal and cancer cell lines and human prostate tumors. Both in cancer cell lines and human tumors the prevalence of S paRNAs and low AS/S paRNA ratio were associated with low E-cadherin expression. We found that the S paRNA bound Argonaute 1 (AGO1) and coordinated silencing of the gene by recruiting, along with AGO1, the histone methyltransferase SUV39H1 to the promoter. Consistently, knockdown of either S paRNA or AGO1 reduced SUV39H1 promoter occupancy and reactivated E-cadherin transcription in low expressing cells. Using promoter reporter and expression constructs we showed that the S paRNA and AGO1 acted in cis to control promoter activity and that the interaction with AGO1 required specific element of the S paRNA. Furthermore, recruitment of AGO1 to the S paRNA and CDH1 promoter depended on an isomiR derived through alternative processing and editing of pre-miR-4534. Accordingly, mutations that disrupted the isomiR binding sequence in the S paRNA reduced AGO1 binding and increased promoter activity, while depletion of the isomiR induced CDH1 expression. Notably, the novel isomiR was more abundant in transformed epithelial cells and cancer cell lines than normal prostate epithelial cells, accumulated preferentially in nuclei and was specifically associated with S paRNA and chromatin-bound AGO1 in low CDH1 expressing cancer cells. This study reveals a complex RNA-based epigenetic network that relies on sequence-specific interactions between a paRNA, a small RNA and AGO1 and coordinates transcriptional silencing of a critical gene involved in tumor development and progression. Our findings give also a new prospective and mechanistic insights on the interplay between epigenetic regulatory factors indentifying paRNAs as relevant elements in these processes and potential targets for gene modulation strategies. Citation Format: Sara Napoli, Giuseppina Pisignano, Ramon Garcia-Escudero, Giuseppina Carbone, Carlo V. Catapano. An RNA-based epigenetic network controls the expression of E-cadherin in epithelial normal and cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2349. doi:10.1158/1538-7445.AM2014-2349