Viral reprogramming of host transcription initiation.

Viruses are master remodelers of the host cell environment in support of infection and virus production. For example, viruses typically regulate cell gene expression through modulating canonical cell promoter activity. Here, we show that Epstein Barr virus (EBV) replication causes 'de novo' transcription initiation at 29674 new transcription start sites throughout the cell genome. De novo transcription initiation is facilitated in part by the unique properties of the viral pre-initiation complex (vPIC) that binds a TATT[T/A]AA, TATA box-like sequence and activates transcription with minimal support by additional transcription factors. Other de novo promoters are driven by the viral transcription factors, Zta and Rta and are influenced by directional proximity to existing canonical cell promoters, a configuration that fosters transcription through existing promoters and transcriptional interference. These studies reveal a new way that viruses interact with the host transcriptome to inhibit host gene expression and they shed light on primal features driving eukaryotic promoter function.

Comparative Analysis of Gammaherpesvirus Circular RNA Repertoires: Conserved and Unique Viral Circular RNAs.

Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) human DNA tumor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expression was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV latency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for viral circRNA functions in latency and viral replication.IMPORTANCE Infection with oncogenic gammaherpesviruses leads to long-term viral persistence through a dynamic interplay between the virus and the host immune system. Critical for remodeling of the host cell environment after the immune responses are viral noncoding RNAs that modulate host signaling pathways without attracting adaptive immune recognition. Despite the importance of noncoding RNAs in persistent infection, the circRNA class of noncoding RNAs has only recently been identified in gammaherpesviruses. Accordingly, their roles in virus infection and associated oncogenesis are unknown. Here we report evolutionary conservation of EBV-encoded circRNAs determined by assessing the circRNAome in rLCV-infected lymphomas from an SIV-infected rhesus macaque, and we report latent and lytic circRNAs from KSHV and MHV68. These experiments demonstrate utilization of the circular RNA class of RNAs across 4 members of the gammaherpesvirus subfamily, and they identify orthologues and potential homoplastic circRNAs, implying conserved circRNA functions in virus biology and associated malignancies.

Identification of novel allosteric inhibitors of mutant isocitrate dehydrogenase 1 by cross docking-based virtual screening.

IDH1 mutation (mIDH1) occurs in 20-30% of gliomas and is a promising target for the cancer therapy. In this article, a cross docking-based virtual screening was employed to identify seven small molecules for the allosteric site of mIDH1. Compounds ZX01, ZX05 and ZX06 exhibited the potent inhibitory activity and the high selectivity against WT-IDH1, providing a good starting point for the further development of highly selective mIDH1 inhibitors. Importantly, the parallel artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) identified ZX06 with a good ability to penetrate BBB. These findings indicate that ZX06 deserves further optimization as a lead compound for the treatment of patients with IDH1 mutated brain cancers.

Design, synthesis and biological activity of 3-pyrazine-2-yl-oxazolidin-2-ones as novel, potent and selective inhibitors of mutant isocitrate dehydrogenase 1.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG) generating carbon dioxide and NADPH/NADH. Evidence suggests that the specific mutations in IDH1 are critical to the growth and reproduction of some tumor cells such as gliomas and acute myeloid leukemia, emerging as an attractive antitumor target. In order to discovery potent new mutant IDH1 inhibitors, we designed, synthesized and evaluated a series of allosteric mIDH1 inhibitors harboring the scaffold of 3-pyrazine-2-yl-oxazolidin-2-ones. All tested compounds effectively suppress the D-2-hydroxyglutarate (D-2-HG) production in cells transfected with IDH1-R132H and IDH1-R132C mutations at 10 µM and 50 µM. Importantly, compound 3g owns the similar inhibitory activity to the positive agent NI-1 and shows no significant toxicity at the two concentrations. The parallel artificial membrane permeation assay of the blood-brain barrier (PAMPA-BBB) identified 3g with a good ability to penetrate the blood-brain barrier (BBB). These findings indicate that 3g deserves further optimization as a lead compound for the treatment of patients with IDH1 mutated brain cancers.

Design, synthesis and biological activity of 3-oxoamino-benzenesulfonamides as selective and reversible LSD1 inhibitors.

Lysine specific demethylase 1 (LSD1) is a flavin-dependent amine oxidase that selectively removes one or two methyl groups from H3 at Lys4 and is recognized as a promising therapeutic target for cancer and other diseases. Here, a series of 3-oxoamino-benzenesulfonamides were synthesized and evaluated for their inhibitory activity against LSD1. Compounds 7b and 7h showed the most potent inhibition with the IC50 values of 9.5 and 6.9µM, respectively. Furthermore, the LSD1 inhibition of 7b and 7h were reversible and selective. Docking study presented the possible binding mode between 7b, 7h and the LSD1 active site. Taken together, 3-oxoamino-benzenesulfonamides may represent a new class of reversible LSD1 inhibitors and 7b and 7h were two hit compounds deserved further structural optimization.

Discovery of Novel Dual Inhibitors Targeting Mutant IDH1 and NAMPT for the Treatment of Glioma with IDH1Mutation.

The targeting of cancer cell intrinsic metabolism has emerged as a promising strategy for antitumor intervention. In the study, we identified the first-in-class small molecules that effectively inhibit both mutant isocitrate dehydrogenase 1 (mIDH1) and nicotinamide phosphoribosyltransferase (NAMPT), two crucial targets in cancer metabolism, through structure-based drug design. Notably, compound 23h exhibits excellent and balanced inhibitory activities against both mIDH1 (IC50 = 14.93 nM) and NAMPT (IC50 = 12.56 nM), leading to significant suppression of IDH1-mutated glioma cell (U87 MG-IDH1R132H) proliferation. Significantly, compound 23h has the ability to cross the blood-brain barrier (B/P ratio, 0.76) and demonstrates remarkable in vivo antitumor efficacy (20 mg/kg) in the U87 MG-IDH1R132H orthotopic transplantation mouse models without any notable toxicity. This proof-of-concept investigation substantiates the viability of discovering small molecules that concurrently target mIDH1 and NAMPT, providing valuable leads for the treatment of glioma and an efficient approach for the discovery of multitarget antitumor drugs.

Loss of feedback regulation between FAM3B and androgen receptor driving prostate cancer progression.

BACKGROUND: Although the fusion of the transmembrane serine protease 2 gene (TMPRSS2) with the erythroblast transformation-specific-related gene (ERG), or TMPRSS2-ERG, occurs frequently in prostate cancer, its impact on clinical outcomes remains controversial. Roughly half of TMPRSS2-ERG fusions occur through intrachromosomal deletion of interstitial genes and the remainder via insertional chromosomal rearrangements. Because prostate cancers with deletion-derived TMPRSS2-ERG fusions are more aggressive than those with insertional fusions, we investigated the impact of interstitial gene loss on prostate cancer progression. METHODS: We conducted an unbiased analysis of transcriptome data from large collections of prostate cancer samples and employed diverse in vitro and in vivo models combined with genetic approaches to characterize the interstitial gene loss that imposes the most important impact on clinical outcome. RESULTS: This analysis identified FAM3B as the top-ranked interstitial gene whose loss is associated with a poor prognosis. The association between FAM3B loss and poor clinical outcome extended to fusion-negative prostate cancers where FAM3B downregulation occurred through epigenetic imprinting. Importantly, FAM3B loss drives disease progression in prostate cancer. FAM3B acts as an intermediator of a self-governing androgen receptor feedback loop. Specifically, androgen receptor upregulates FAM3B expression by binding to an intronic enhancer to induce an enhancer RNA and facilitate enhancer-promoter looping. FAM3B, in turn, attenuates androgen receptor signaling. CONCLUSION: Loss of FAM3B in prostate cancer, whether through the TMPRSS2-ERG translocation or epigenetic imprinting, causes an exit from this autoregulatory loop to unleash androgen receptor activity and prostate cancer progression. These findings establish FAM3B loss as a new driver of prostate cancer progression and support the utility of FAM3B loss as a biomarker to better define aggressive prostate cancer.

Data of relative mRNA and protein abundances of androgen receptor splice variants in castration-resistant prostate cancer.

These data include secondary analysis of publicly available RNA-seq data from castration-resistant prostate cancer (CRPC) patients as well as RT-qPCR and Western blotting analyses of patient-derived xenograft models and a CRPC cell line. We applied Spearman correlation analysis to assess the relationship between canonical androgen receptor (AR) splicing and alternative AR splicing. We also assessed the ratio of AR splice variants (AR-Vs) to the full-length AR (AR-FL) at the RNA and protein levels by absolute RT-qPCR and Western blotting, respectively. These data are critical for studying the mechanisms underlying upregulated expression of AR-Vs after AR-directed therapies and the importance of AR-Vs to castration-resistant progression of prostate cancer. Data presented here are related to the research article by Ma et al., "Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy", Cancer Lett. In Press [1].

Increased transcription and high translation efficiency lead to accumulation of androgen receptor splice variant after androgen deprivation therapy.

Upregulation of androgen receptor splice variants (AR-Vs), especially AR-V7, is associated with castration resistance of prostate cancer. At the RNA level, AR-V7 upregulation is generally coupled with increased full-length AR (AR-FL); consequently, AR-V7 and AR-Vs collectively constitute a minority of the AR population. However, Western blotting showed that the relative abundance of AR-V proteins is much higher in many castration-resistant prostate cancers (CRPCs). To address the mechanism underlying this discrepancy, we analyzed RNA-seq data from ~350 CRPC samples and found a positive correlation between all canonical and alternative AR splicing. This indicates that increased alternative splicing is not at the expense of canonical splicing. Instead, androgen deprivation releases AR-FL from repressing the transcription of the AR gene to induce coordinated increase of AR-FL and AR-V mRNAs. At the protein level, however, androgen deprivation induces AR-FL, but not AR-V, degradation. Moreover, AR-V7 is translated much faster than AR-FL. Thus, androgen-deprivation-induced AR-gene transcription and AR-FL protein decay, together with efficient AR-V7 translation, explain the discrepancy between the relative AR-V mRNA and protein abundances in many CRPCs, highlighting the inevitability of AR-V induction after endocrine therapy.

A detailed characterization of stepwise activation of the androgen receptor variant 7 in prostate cancer cells.

Expression of the androgen receptor splice variant 7 (AR-V7) is frequently detected in castrate resistant prostate cancer and associated with resistance to AR-targeted therapies. While we have previously noted that homodimerization is required for the transcriptional activity of AR-V7 and that AR-V7 can also form heterodimers with the full-length AR (AR-FL), there are still many gaps of knowledge in AR-V7 stepwise activation. In the present study, we show that neither AR-V7 homodimerization nor AR-V7/AR-FL heterodimerization requires cofactors or DNA binding. AR-V7 can enter the nucleus as a monomer and drive a transcriptional program and DNA-damage repair as a homodimer. While forming a heterodimer with AR-FL to induce nuclear localization of unliganded AR-FL, AR-V7 does not need to interact with AR-FL to drive gene transcription or DNA-damage repair in prostate cancer cells that co-express AR-V7 and AR-FL. These data indicate that AR-V7 can function independently of its interaction with AR-FL in the true castrate state or "absence of ligand", providing support for the utility of targeting AR-V7 in improving outcomes of patients with castrate resistant prostate cancer.

Two crossed axonal projections contribute to binaural unmasking of frequency-following responses in rat inferior colliculus.

Frequency-following responses (FFRs) are sustained potentials based on phase-locked neural activities elicited by low- to medium-frequency periodical sound waveforms. Human brainstem FFRs, which are able to encode some critical acoustic features of speech, can be unmasked by binaural processing. However, the underlying unmasking mechanisms have not previously been reported. In rats, most neurons in the inferior colliculus (IC) exhibit binaural responses which are affected by axonal projections from both the contralateral dorsal nucleus of the lateral lemniscus (DNLL) and the contralateral IC. The present study investigated whether the contralateral DNLL and the contralateral IC modulate binaural unmasking of FFRs recorded in the rat IC. The results show that IC FFRs to the rat pain call (chatter) were enhanced by local injection of the excitatory glutamate receptor antagonist kynurenic acid (KYNA) into the contralateral DNLL but were reduced by KYNA injection into the contralateral IC. Introducing a disparity between the interaural time difference (ITD) of the FFR-eliciting chatter and the ITD of the masking noise enhanced IC FFRs. Moreover, the ITD-disparity-induced FFR enhancement was weakened by injection of KYNA into either the contralateral DNLL or the contralateral IC when the ipsilateral chatter preceded the contralateral chatter. Thus, binaural hearing can improve IC FFRs against noise masking. More importantly, both inhibitory projections from the contralateral DNLL and excitatory projections from the contralateral IC modulate IC FFRs and play a role in forming binaural unmasking of IC FFRs.

I-8, a novel inhibitor of mutant IDH1, inhibits cancer progression in vitro and in vivo.

Isocitrate dehydrogenase 1 mutations have been discovered in an array of hematologic malignancies and solid tumors. These mutations could cause the production of high levels of 2-hydroxyglutarate, which in turn implicated in epigenetic changes and impaired cell differentiation. Here, we described the characterization of compound I-8, a novel mutant IDH1 inhibitor, both in vitro and in vivo. Compound I-8 specifically inhibited 2-HG production, reduced histone methylation levels, induced differentiation and depleted stem characteristics in engineered and endogenous IDH1 mutant cells. In addition, oral administration of I-8 also significantly suppressed 2-HG production and histone methylation with dose of 150 mg/kg. And I-8 treatment also could induce differentiation and attenuate stem characteristics in tumor tissue. Together, these studies indicated that compound I-8 has clinical potential in tumor therapies as a effective mutant IDH1 inhibitor, and provided scientific guidance for the development of mutant IDH1 inhibitor in the future.

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

Recent advances in inhibitors of sirtuin1/2: an update and perspective.

Sirtuins (SIRT) are coenzyme NAD+-dependent histone deacetylases for the transfer of modified acetyl groups. Sirtuins are widely involved in various physiological processes and therefore associated with cardiovascular disease, diabetes, Parkinson's disease, cancer and beyond. Consequently, the development of modulators for  sirtuins has considerable clinical value. To date, a variety of SIRT1/2 inhibitors have been reported and none has been approved for the market. This review summarizes the recent progress in the discovery and development of SIRT1/2 inhibitors including their inhibitory potency, structure-activity relationship and binding mode analysis as well as discusses the perspective for the future development of SIRT1/2 inhibitors.

Inhibitors of Mutant Isocitrate Dehydrogenases 1 and 2 (mIDH1/2): An Update and Perspective.

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are homodimeric enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid cycle. However, mutant IDH1/2 (mIDH1/2) reduces α-KG to the oncometabolite 2-hydroxyglutarate (2-HG). High levels of 2-HG competitively inhibit the α-KG-dependent dioxygenases involved in histone and DNA demethylation, thereby impairing normal cellular differentiation and promoting tumor development. Thus, small molecules that inhibit these mutant enzymes may be therapeutically beneficial. Recently, an increasing number of mIDH1/2 inhibitors have been reported. In this review, we summarize the molecular basis of mIDH1/2 and the activity, binding modes, and progress in clinical application of mIDH1/2 inhibitors. We note important future research directions for mIDH1/2 inhibitors and discuss potential therapeutic strategies for the development of mIDH1/2 inhibitors to treat IDH1/2 mutated tumors.

Interplay between Cytoplasmic and Nuclear Androgen Receptor Splice Variants Mediates Castration Resistance.

Androgen receptor splice variants (AR-V) are implicated in resistance of prostate cancer to androgen-directed therapies. When expressed alone in cells, some AR-Vs (e.g., AR-V7) localize primarily to the nucleus, whereas others (e.g., AR-V1, AR-V4, and AR-V6) localize mainly to the cytoplasm. Significantly, the latter are often coexpressed with the nucleus-predominant AR-Vs and the full-length AR (AR-FL). An important question to be addressed is whether the cytoplasmic-localized AR-Vs play a role in castration-resistant prostate cancer (CRPC) through interaction with the nucleus-predominant AR-Vs and AR-FL. Here, it is demonstrated that AR-V1, -V4, and -V6 can dimerize with both AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V1, -V4, and -V6, and these variants, in turn, mitigated the ability of the antiandrogen enzalutamide to inhibit androgen-induced AR-FL nuclear localization. Interestingly, the impact of nuclear localization of AR-V4 and -V6 on AR transactivation differs from that of AR-V1. Nuclear localization leads to an increased ability of AR-V4 and -V6 to transactivate both canonical AR targets and AR-V-specific targets and to confer castration-resistant cell growth. However, although AR-V1, which lacks inherent transcriptional activity, appears to activate AR-FL in an androgen-independent manner, it significantly antagonizes AR-V7 transactivation. Together, these data demonstrate that the complex interactions among different AR-Vs and AR-FL play a significant role in castration-resistant disease. IMPLICATIONS: This study suggests important consequences for clinical castration resistance due to simultaneous expression of AR-FL and AR-Vs in patient tumors and suggests that dissecting these interactions should help develop effective strategies to disrupt AR-V signaling. Mol Cancer Res; 15(1); 59-68. ©2016 AACR.