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Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039. Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified. Publisher PDF

Many viruses that cause serious diseases in humans and animals, including the betacoronaviruses (beta-CoVs), such as SARS-CoV, MERS-CoV, and the recently identified SARS-CoV-2, have natural reservoirs in bats. Because these viruses rely entirely on the host cellular machinery for survival, their evolution is likely to be guided by the link between the codon usage of the virus and that of its host. As a result, specific cellular microenvironments of the diverse hosts and/or host tissues imprint peculiar molecular signatures in virus genomes. Our study is aimed at deciphering some of these signatures. Using a variety of genetic methods we demonstrated that trends in codon usage across chiroptera-hosted CoVs are collaboratively driven by geographically different host-species and temporal-spatial distribution. We not only found that chiroptera-hosted CoVs are the ancestors of SARS-CoV-2, but we also revealed that SARS-CoV-2 has the codon usage characteristics similar to those seen in CoVs infecting the Rhinolophus sp. Surprisingly, the envelope gene of beta-CoVs infecting Rhinolophus sp., including SARS-CoV-2, had extremely high CpG levels, which appears to be an evolutionarily conserved trait. The dissection of the furin cleavage site of various CoVs infecting hosts revealed host-specific preferences for arginine codons, however, arginine is encoded by a wider variety of synonymous codons in the murine CoV (MHV-A59) furin cleavage site. Our findings also highlight the latent diversity of CoVs in mammals that has yet to be fully explored.

Background Hepatocellular carcinoma is the second most deadly cancer with late presentation and limited treatment options, highlighting an urgent need to better understand HCC to facilitate the identification of early-stage biomarkers and uncover therapeutic targets for the development of novel therapies for HCC. Methods Deep transcriptome sequencing of tumor and paired non-tumor liver tissues was performed to comprehensively evaluate the profiles of both the host and HBV transcripts in HCC patients. Differential gene expression patterns and the dys-regulated genes associated with clinical outcomes were analyzed. Somatic mutations were identified from the sequencing data and the deleterious mutations were predicted. Lastly, human-HBV chimeric transcripts were identified, and their distribution, potential function and expression association were analyzed. Results Expression profiling identified the significantly upregulated TP73 as a nodal molecule modulating expression of apoptotic genes. Approximately 2.5% of dysregulated genes significantly correlated with HCC clinical characteristics. Of the 110 identified genes, those involved in post-translational modification, cell division and/or transcriptional regulation were upregulated, while those involved in redox reactions were downregulated in tumors of patients with poor prognosis. Mutation signature analysis identified that somatic mutations in HCC tumors were mainly non-synonymous, frequently affecting genes in the micro-environment and cancer pathways. Recurrent mutations occur mainly in ribosomal genes. The most frequently mutated genes were generally associated with a poorer clinical prognosis. Lastly, transcriptome sequencing suggest that HBV replication in the tumors of HCC patients is rare. HBV-human fusion transcripts are a common observation, with favored HBV and host insertion sites being the HBx C-terminus and gene introns (in tumors) and introns/intergenic-regions (in non-tumors), respectively. HBV-fused genes in tumors were mainly involved in RNA binding while those in non-tumors tissues varied widely. These observations suggest that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts may occur during tumorigenesis. Conclusions Transcriptome sequencing of HCC patients reveals key cancer molecules and clinically relevant pathways deregulated/mutated in HCC patients and suggests that while HBV may integrate randomly during chronic infection, selective expression of functional chimeric transcripts likely occur during the process of tumorigenesis.

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥ 4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

The transition from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is characterized by reprogramming of the CML transcriptome (Radich et al. PNAS 2006), and shortened survival. Current models propose genomic instability as causal in BC transformation with enhanced DNA damage and impaired DNA repair inducing genetic mutations (ranging from large chromosomal aberrations to point mutations), altered gene function, and eventually BC transformation (Perrotti et al. JCI 2010). Consistent with this model are the phenomena of BC clonal evolution, and the increased frequency of ABL kinase domain mutations found in BC. Because different mutational processes are associated with distinct cancer-specific mutation signatures (Alexandrov et al. Nature 2013), this model also predicts the existence of a CML-specific mutation signature. In addition, recent work has highlighted the importance of epigenetic alterations in hematologic malignancies (Shih et al., Nat. Rev. Cancer, 2012). However, we lack a complete understanding of the type or frequency of genetic alterations in BC, and the relative contribution of genetic vs. epigenetic events in reprogramming the BC transcriptome. To address these knowledge gaps, we analyzed the CML progression genome, epigenome, and transcriptome in 12 CP/BC sample pairs. Whole-genome sequencing revealed the CML genome to be relatively stable with respect to structural variations, indels, and somatic single nucleotide variants. The average number of nonsynonymous coding mutations per BC genome was 5, placing the BC coding genome in the same mutation frequency range as AML and ALL genomes (Alexandrov et al. Nature 2013). In addition, we identified a novel mutation signature in all CML samples suggesting a CML-specific mutational process. 1175 genes were 'hit' by genomic, mostly copy number, alterations in >1 sample, and included TCR genes and Ikaros (IKZF1) among lymphoid BC pairs. Only 21 recurrently altered genes were affected by somatic SNVs or indels, with resistance-associated ABL1 mutations being commonest. We next used DNA methylation arrays to assess the BC epigenome, and found 20,651 CpG sites (out of 455,187) to be hyper-methylated, and 3225 to be hypo-methylated in BC compared to CP. Combined methylome and transcriptome analysis demonstrated an inverse relationship between methylation and expression changes at a subset of CpG sites enriched at promoters. Genes with increased methylation/decreased expression or decreased methylation/increased expression included those involved in cell cycle control/heme biosynthesis, and molecular mechanisms of cancer/G-protein coupled receptor signaling/MAPK signaling respectively. Unsupervised methylation-based clustering segregated samples into CP, lymphoid BC and myeloid BC groups, recapitulating expression-based clustering, and further supporting a functional role for DNA methylation in BC transcriptional reprogramming. We next performed an integrative analysis by combining the genome, methylome, and transcriptome datasets, and included data from 34 additional CML samples. Top ranking candidate genes included epigenetic modifiers, and hematopoetic differentiation- and stem cell-related genes. Functional analysis of candidate genes and epigenetic processes using genetic and epigenetic drug-based approaches are ongoing. In summary, we conclude that: 1. The genomic and epigenomic landscapes in BC are characterized by a modest number of recurring events in the former, but consistent and striking differences in the latter, 2. The BC methylome is functionally associated with the robust gene expression changes found in BC, and 3. Epigenetic modifier drugs may be of use in reversing the gene expression changes characteristic of BC. Disclosures Chuah: Children International: Honoraria; Novartis: Honoraria; Bristol Meyers Squibb: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau.

Current models of CML blast crisis (BC) propose that expression of BCR-ABL results in genomic instability and the acquisition of genetic alterations that affect cell proliferation and survival, self-renewal and differentiation. To characterize the molecular events that underlie progression, we performed whole genome sequencing of paired samples of the same patient at CP and at BC (n = 12), as well as expression and methylation arrays of these samples and a larger validation cohort of unpaired CD34-selected samples (n = 38). Contrary to expectations, we found that the CML BC genome is relatively quiescent with regards to SNVs, indels and structural variations. In contrast, we observed widespread hyper-methylation in BC that was associated with distinct changes in expression and was independent of lineage/differentiation state. These findings suggest that in addition to genetic alterations, epigenomic events are likely to contribute substantively to BC progression. To understand the functional effects of the dysregulated transcriptome and epigenome in BC CML, we employed both pharmacologic and genetic methods to target candidate genes of interest identified in our earlier studies. To induce de-methylation of the BC genome, we treated primary samples with low doses of decitabine, a DNMT inhibitor. We found that decitabine impaired colony formation ability of BC CD34+ progenitors and concomitantly activated regulators of myeloid differentiation that were both hyper-methylated and down-regulated in BC CD34+ progenitors, such as MPO and KLF1. These results suggest that hyper-methylation does contribute to BC CD34+ progenitor function, and support the use of epigenetic therapies as a rational approach to targeting BC. The genetic approach we chose was a CRISPR-based in vitro pooled screen. We created a custom library targeting 200 genes, with an average of 5 sgRNAs per gene, and 50 non-targeting controls. We transduced K562 with the library and harvested samples at different time-points post-transduction/selection - Day 0, 7 and 21 - for deep sequencing. As expected, sgRNAs targeting essential genes such as MYC and MCM 2-7 were recurrently depleted in the population over time. More importantly, enriched sgRNAs targeted genes including TET2, which has been previously reported to be inactivated in myeloid malignancies, as well as novel candidates including RREB1, a transcription factor that binds to RAS-responsive elements (RREs) and may be involved in MAPK signaling. We will validate these targets by knocking them out individually and assessing their effect on the ability of CP cells to serially replate and/or engraft immune-deficient mice. Disclosures Chuah: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern International: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Astellas: Speakers Bureau. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.