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Background Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Methods Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. Results We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Conclusions Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.

Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs (rRNA) and in eukaryotic genomes are typically present as one or more arrays of tandem repeats. Species have characteristic rDNA copy numbers, ranging from tens to thousands of copies, with the number thought to be redundant for rRNA production. However, the tandem rDNA repeats are prone to recombination-mediated changes in copy number, resulting in substantial intra-species copy number variation. There is growing evidence that these copy number differences can have phenotypic consequences. However, we lack a comprehensive understanding of what determines rDNA copy number, how it evolves, and what the consequences are, in part because of difficulties in quantifying copy number. Here, we developed a genomic sequence read approach that estimates rDNA copy number from the modal coverage of the rDNA and whole genome to help overcome limitations in quantifying copy number with existing mean coverage-based approaches. We validated our method using strains of the yeast Saccharomyces cerevisiae with previously-determined rDNA copy numbers, and then applied our pipeline to investigate rDNA copy number in a global sample of 788 yeast isolates. We found that wild yeast have a mean copy number of 92, consistent with what is reported for other fungi but much lower than in laboratory strains. We also show that different populations have different rDNA copy numbers. These differences can partially be explained by phylogeny, but other factors such as environment are also likely to contribute to population differences in copy number. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.Author summaryThe ribosomal RNA gene repeats (rDNA) form large tandem repeat arrays in most eukaryote genomes. Their tandem arrangement makes the rDNA prone to copy number variation, and there is increasing evidence that this copy number variation has phenotypic consequences. However, difficulties in measuring rDNA copy number hamper investigation into rDNA copy number dynamics and their significance. Here we developed a novel bioinformatics method for measuring rDNA copy number from whole genome sequence data that is based on the modal sequence read coverage. We established parameters for optimal performance of the method and validated it using yeast strains of known rDNA copy numbers. We then applied the method to a dataset of almost 800 global yeast isolates and demonstrate that yeast populations have different rDNA copy numbers that partially correlate with phylogeny. Our work provides a simple and accurate method for determining rDNA copy number that leverages the growing number of whole genome datasets, and highlights the dynamic nature of rDNA copy number.

The nucleolar surveillance pathway (NSP) monitors nucleolar fidelity and responds to nucleolar stresses (i.e., inactivation of ribosome biogenesis) by mediating the inhibitory binding of ribosomal proteins (RPs) to mouse double minute 2 homolog (MDM2), a nuclear-localised E3 ubiquitin ligase, which results in p53 accumulation. Inappropriate activation of the NSP has been implicated in the pathogenesis of collection of human diseases termed “ribosomopathies”, while drugs that selectively activate the NSP are now in trials for cancer. Despite the clinical significance, the precise molecular mechanism(s) regulating the NSP remain poorly understood. Using genome-wide loss of function screens, we demonstrate the ribosome biogenesis (RiBi) axis as the most potent class of genes whose disruption stabilises p53. Furthermore, we identified a novel suite of genes critical for the NSP, including a novel mammalian protein implicated in 5S ribonucleoprotein particle (5S-RNP) biogenesis, HEATR3. By selectively disabling the NSP, we unexpectedly demonstrate that a functional NSP is required for the ability of all nuclear acting stresses tested, including DNA damage, to robustly induce p53 accumulation. Together, our data demonstrates that the NSP has evolved as the dominant central integrator of stresses that regulate nuclear p53 abundance, thus ensuring RiBi is hardwired to cellular proliferative capacity.

The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA., Jeannine Diesch et al. report the changes in rDNA chromatin state associated with cell transition into malignancy. They show that a specific transcription factor regulates this transition by altering rDNA chromatin, resulting in the reorganization of contacts between rDNA and the genome.

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21 and STAG2 and screened for synthetic lethality with 3,009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunit rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin mutant cancers.

Here, we report novel tumour suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, our data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth. This article has an associated ‘The people behind the papers’ interview.

Psi, the sole FUSE Binding Protein (FUBP) family single stranded DNA/RNA binding protein inDrosophila, is essential for proper cell and tissue growth, however its mechanism of function remains unclear. Here we use Targeted DamID combined with RNA-sequencing to generate the first genome-wide binding and expression profiles for Psi. Surprisingly, we demonstrate Psi drives growth in theDrosophilawing through transcriptional repression of key developmental pathways (e.g. Wnt, Notch and TGFβ). Thus, Psi patterns tissue growth by directly repressing transcription of developmental growth suppressors. Analysis of direct Psi targets identified novel growth inhibitors, includingTolkin(Zinc metallopeptidase implicated in TGFβ signalling),Ephexin(Rho-GEF) andemp(CD36 scavenger receptor-related protein). Their depletion not only suppressed impaired growth associated with Psi knockdown, but alone was sufficient to drive wing overgrowth. Thus, Psi drives wing growth twofold, through direct activation ofMycand through transcriptional repression of growth inhibitors comprising core developmental pathways.

Here we report novel tumour suppressor activity for the Drosophila Argonaute family RNA binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion, and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1 depleted animals requires RNA Pol II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA Pol II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together our data show AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.

Exquisite regulation of PI3K/AKT/mTORC1 signaling is essential for homeostatic control of cell growth, proliferation, and survival. Aberrant activation of this signaling network is an early driver of many sporadic human cancers. Paradoxically, sustained hyperactivation of the PI3K/AKT/mTORC1 pathway in nontransformed cells results in cellular senescence, which is a tumor-suppressive mechanism that must be overcome to promote malignant transformation. While oncogene-induced senescence (OIS) driven by excessive RAS/ERK signaling has been well studied, little is known about the mechanisms underpinning the AKT-induced senescence (AIS) response. Here, we utilize a combination of transcriptome and metabolic profiling to identify key signatures required to maintain AIS. We also employ a whole protein-coding genome RNAi screen for AIS escape, validating a subset of novel mediators and demonstrating their preferential specificity for AIS as compared with OIS. As proof of concept of the potential to exploit the AIS network, we show that neurofibromin 1 (NF1) is upregulated during AIS and its ability to suppress RAS/ERK signaling facilitates AIS maintenance. Furthermore, depletion of NF1 enhances transformation of p53-mutant epithelial cells expressing activated AKT, while its overexpression blocks transformation by inducing a senescent-like phenotype. Together, our findings reveal novel mechanistic insights into the control of AIS and identify putative senescence regulators that can potentially be targeted, with implications for new therapeutic options to treat PI3K/AKT/mTORC1-driven cancers.

Transcription of the ribosomal RNA genes (rDNA) that encode the three largest ribosomal RNAs (rRNA), is mediated by RNA Polymerase I (Pol I) and is a key regulatory step for ribosomal biogenesis. Although it has been reported over a century ago that the number and size of nucleoli, the site of ribosome biogenesis, are increased in cancer cells, the significance of this observation for cancer etiology was not understood. The realization that the increase in rRNA expression has an active role in cancer progression, not only through increased protein synthesis and thus proliferative capacity but also through control of cellular check points and chromatin structure, has opened up new therapeutic avenues for the treatment of cancer through direct targeting of Pol I transcription. In this review, we discuss the rational of targeting Pol I transcription for the treatment of cancer; review the current cancer therapeutics that target Pol I transcription and discuss the development of novel Pol I-specific inhibitors, their therapeutic potential, challenges and future prospects.

Elevated ribosome biogenesis in oncogene‐driven cancers is commonly targeted by DNA‐damaging cytotoxic drugs. Our previous first‐in‐human trial of CX‐5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single‐agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX‐5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP‐dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I‐directed therapies., The elevated metabolic activity and activation of cAMP‐dependent pro‐survival mechanisms that lead to resistance of hematological cancers towards rRNA synthesis inhibition also poses a potentially targetable vulnerability.

The nucleolus is a dynamic subnuclear compartment that has a number of different functions, but its primary role is to coordinate the production and assembly of ribosomes. For well over 100 years, pathologists have used changes in nucleolar number and size to stage diseases such as cancer. New information about the nucleolus' broader role within the cell is leading to the development of drugs which directly target its structure as therapies for disease. Traditionally, it has been difficult to develop high-throughput image analysis pipelines to measure nucleolar changes due to the broad range of morphologies observed. In this study, we describe a simple high-content image analysis algorithm using Harmony software (PerkinElmer), with a PhenoLOGIC™ machine-learning component, that can measure and classify three different nucleolar morphologies based on nucleolin and fibrillarin staining ("normal," "peri-nucleolar rings" and "dispersed"). We have utilized this algorithm to determine the changes in these classes of nucleolar morphologies over time with drugs known to alter nucleolar structure. This approach could be further adapted to include other parameters required for the identification of new therapies that directly target the nucleolus.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA Polymerase I (Pol I) transcription, revealed single agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in thein vivoefficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here we show that this improved efficacy is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this co-treatment is driven by translational re-wiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies identify the molecular mechanisms underpinning the response of blood cancers to selective ribosome biogenesis inhibitors and identify metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

Limited effective therapeutic options are available for patients with recurrent high-grade serous carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. We have shown efficacy in poly-ADP ribose polymerase (PARP) inhibitor-resistant HGSC for the RNA Polymerase I (Pol I) transcription inhibitor CX-5461 through its ability to activate a nucleolar-associated DNA damage response (DDR). Here, we screen the protein-coding genome to identify potential targets whose inhibition enhances the efficacy of CX-5461. We identify a network of cooperating inhibitory interactions, including components of homologous recombination (HR) DNA repair and DNA topoisomerase 1 (TOP1). We highlight that CX-5461 combined with topotecan, a TOP1 inhibitor used as salvage therapy in HGSC, induces robust cell cycle arrest and cell death in a panel of HR-proficient HGSC cell lines. The combination potentiates a nucleolar-associated DDR via recruitment of phosphorylated replication protein A (RPA) and ataxia telangiectasia and Rad3 related protein (ATR). CX-5461 plus low-dose topotecan cooperate to potently inhibit xenograft tumour growth, indicating the potential for this strategy to improve salvage therapeutic regimens to treat HGSC.

The majority of patients with neuroblastoma due to MYCN oncogene amplification and consequent N-Myc oncoprotein over-expression die of the disease. Here our analyses of RNA sequencing data identify the long noncoding RNA lncNB1 as one of the transcripts most over-expressed in MYCN-amplified, compared with MYCN-non-amplified, human neuroblastoma cells and also the most over-expressed in neuroblastoma compared with all other cancers. lncNB1 binds to the ribosomal protein RPL35 to enhance E2F1 protein synthesis, leading to DEPDC1B gene transcription. The GTPase-activating protein DEPDC1B induces ERK protein phosphorylation and N-Myc protein stabilization. Importantly, lncNB1 knockdown abolishes neuroblastoma cell clonogenic capacity in vitro and leads to neuroblastoma tumor regression in mice, while high levels of lncNB1 and RPL35 in human neuroblastoma tissues predict poor patient prognosis. This study therefore identifies lncNB1 and its binding protein RPL35 as key factors for promoting E2F1 protein synthesis, N-Myc protein stability and N-Myc-driven oncogenesis, and as therapeutic targets., MYCN amplification is common in neuroblastomas. Here, the authors identify a long noncoding RNA, lncNB1 in these cancers and show that it promotes tumorigenesis by binding to ribosomal protein, RPL35 to enhance E2F1 and DEPDC1B protein synthesis, which phosphorylates ERK to stabilise N-Myc.

Nucleoli form around actively transcribed ribosomal RNA (rRNA) genes (rDNA), and the morphology and location of nucleolus-associated genomic domains (NADs) are linked to the RNA Polymerase I (Pol I) transcription status. The number of rDNA repeats (and the proportion of actively transcribed rRNA genes) is variable between cell types, individuals and disease state. Substantial changes in nucleolar morphology and size accompanied by concomitant changes in the Pol I transcription rate have long been documented during normal cell cycle progression, development and malignant transformation. This demonstrates how dynamic the nucleolar structure can be. Here, we will discuss how the structure of the rDNA loci, the nucleolus and the rate of Pol I transcription are important for dynamic regulation of global gene expression and genome stability, e.g., through the modulation of long-range genomic interactions with the suppressive NAD environment. These observations support an emerging paradigm whereby the rDNA repeats and the nucleolus play a key regulatory role in cellular homeostasis during normal development as well as disease, independent of their role in determining ribosome capacity and cellular growth rates.

Purpose: The MYC oncogene is frequently overexpressed in prostate cancer. Upregulation of ribosome biogenesis and function is characteristic of MYC-driven tumors. In addition, PIM kinases activate MYC signaling and mRNA translation in prostate cancer and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer. Experimental Design:The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective, and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription, has been successfully exploited therapeutically but only in models of hematologic malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in prostate cancer cell lines, in Hi-MYC- and PTEN-deficient mouse models and in patient-derived xenografts (PDX) of metastatic tissue obtained from a patient with castration-resistant prostate cancer. Results: CX-5461 inhibited anchorage-independent growth and induced cell-cycle arrest in prostate cancer cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced TP53 expression and activity and reduced proliferation (MKI67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN-null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of prostate cancer. Conclusions: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of prostate cancer. Clin Cancer Res; 22(22); 5539–52. ©2016 AACR.

Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets. High-throughput screening provides a powerful platform for target discovery. Here, we describe an approach to use RNAi transfection of a pre-established AKT-induced senescent cell population and subsequent high-content imaging to screen for senescence regulators. We have incorporated multiparametric readouts, including cell number, proliferation, and senescence-associated beta-galactosidase (SA-βGal) staining. Using machine learning and automated image analysis, we also describe methods to classify distinct phenotypes of cells with SA-βGal staining. These methods can be readily adaptable to high-throughput functional screens interrogating the mechanisms that maintain and prevent senescence in various contexts.

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.

Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I–mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K–AKT–mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eμ-Myc lymphoma–bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. Significance: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eμ-Myc lymphoma–bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer. Cancer Discov; 6(1); 59–70. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 1

Summary Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5′TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5′ UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes., Graphical Abstract, Highlights • Selective TCP-seq dissects translation mechanisms in mammals and yeast • 40S-bound eIF2 and eIF3 traverse 5′ UTRs and sequentially dissociate at AUG codons • AUG recognition involves four conformational intermediates of the 48S PIC • Initiation factors can assemble co-translationally into higher-order complexes, Changing environmental conditions require reprogramming of gene expression for cells to adapt and survive. Translational control is central to this complex response. Wagner et al. developed assays capturing footprints of mRNA-bound ribosomal complexes associated with selected translation-promoting factors to uncover mechanisms underlying translation in the context of its regulatory potential.

The cohesin complex comprises four core subunits: RAD21, SMC3, SMC1A, and STAG2. Cohesin is required for many biological processes, including sister chromatid cohesion, DNA damage response, regulation of gene expression, and ribosomal biogenesis. Germline mutations of cohesin subunits or their regulators give rise to human developmental disorders, while somatic mutations in cohesin subunits are found in a wide range of human cancers, including acute myeloid leukemia (AML) (12%), Ewing sarcoma (15%) and glioblastoma (19%). In particular, cohesin subunit STAG2, has been identified as one of only 12 genes that are significantly mutated in more than 4 human cancers. Currently, there is no targeted therapeutic strategy for cancer patients with cohesin mutations. Synthetic lethality has emerged as a promising approach for targeted therapy. While synthetic lethal interactions of STAG2 have been demonstrated with its paralog, STAG1, and with the DNA damage repair pathway, synthetic lethal interactions with other pathways have not been explored. To identify synthetic lethal drug activity with cohesin mutations, we generated three isogenic cohesin-deficient MCF10A cell lines (RAD21+/-, SMC3+/-, and STAG2-/-) using CRISPR/Cas9. Cohesin-deficient MCF10A cell lines are sufficiently similar to their parental line concerning growth rate and chromosome stability. However, these cell lines exhibit alterations in nucleolar morphology and increased nuclear location of γH2AX and p53 (except in STAG2-/-). RNA-sequencing analysis of the cell lines showed that cohesin subunit mutation led to downregulation of genes involved in regulation of ribosome biogenesis and RNA processing. Furthermore, ribosomal stress mediated by Actinomycin D led to severe nucleolar fragmentation in cohesin-deficient MCF10A, but not MCF10A parental, suggesting that cohesin mutation might confer vulnerability to perturbation of ribosome biogenesis. Using MCF10A parental and isogenic cohesin-deficient MCF10A cell lines, we performed a quantitative high-throughput drug screen against 3,009 compounds of FDA-approved drugs, kinase, and epigenetic inhibitors. Several classes of inhibitors targeting mTOR signaling, Wnt signaling and chromatin modification were found to selectively inhibit the growth of cohesin-deficient MCF10A cells by 30% or more. WAY-600 (mTORC1/mTORC2 inhibitor) and I-BET-762 (Bromodomain inhibitor) were among the top hits. Mechanistic studies on how these drugs preferentially kill cohesin-deficient cells are under way. In summary, we have identified potential synthetic lethal compounds that may provide the basis for development of targeted therapies of cancers with cohesin deficiency or mutation. Citation Format: Chue Vin Chin, Jisha Antony, Amee J George, JinShu He, Kate Parson, Ross D Hannan, Julia A Horsfield. A synthetic lethal drug screen identifies exploitable vulnerabilities in cohesin-deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A12. doi:10.1158/1535-7163.TARG-19-LB-A12

Significance We demonstrate that ATRX maintains ribosomal DNA (rDNA) heterochromatin formation and stability. ATRX-depleted cells suffer extensive rDNA copy loss, resulting in a reduced ribosomal RNA transcription output and an increased sensitivity to RNA polymerase I (Pol I) inhibition. Supporting these data, we have also detected reduced rDNA copy in ATRX mutated ALT-positive human primary tumor samples and increased sensitivity of human ALT cancer cell lines to RNA Pol I transcription inhibitor. Our study highlights the therapeutic potential of Pol I transcription inhibitors for the treatment of ATRX-mutated cancers., ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Diamond-Blackfan anaemia (DBA) is a rare congenital erythroid hypoplastic anaemia characterised by severely reduced numbers of erythroid precursors in the bone marrow. This reduction of erythroid progenitors is partly due to p53-induced apoptosis during erythroid differentiation in response to nucleolar stress. Nucleolar stress can be caused by mutations in ribosomal proteins. Mutations in ribosomal proteins are found in over 50% of DBA patients and mutations in RPS19 are found in ∼25% of DBA patients. We hypothesise that preventing p53-induced apoptosis in DBA erythroid cells would lead to the survival and differentiation of erythroid progenitors. We performed a genome-wide, gain-of-function screen to identify factors that can modulate levels of p53 in RPS19 knockdown cells. For screening, we generated a human cell line with doxycycline-inducible shRNA-mediated RPS19 knockdown. Induction leads to a decrease in RPS19 protein and robust induction of p53. Using a lentiviral human ORF expression library containing ∼17000 genes in a high-throughput overexpression assay, we confirmed 36 high-confidence candidates that can modulate levels of p53 in the presence of perturbed ribosome biogenesis. To determine if overexpression of genes that led to a reduction of p53 (p53low hits) can regulate p53 within erythroid cells and thereby rescue the DBA phenotype, we utilised a doxycycline-inducible RPS19 knock-down mouse model. This mouse displays many characteristics of DBA such as bone marrow failure which can be rescued by RPS19 gene transfer or loss of Trp53. We tested if expression of p53low hits can rescue the differentiation and proliferation of shRPS19 erythroid progenitors from foetal livers of 14.5dpc embryos in vitro and bone marrow in vivo. By identifying factors that regulate p53, we aim to identify pathways involved in the disease and targets for novel DBA therapies.

RNA Polymerase I (PolI)-mediated transcription of ribosomal RNAs is the limiting factor of ribosomal biogenesis, is often dysregulated in cancer 1 and is a new target for cancer therapy 2 , 3 . Decreased levels of PolI transcription is also associated with cell differentiation 4 suggesting that high levels of PolI transcription are required for stemness. Mammalian cells contain over 200 copies of the ribosomal genes (rDNA) clustered in specific chromosomal regions that aggregate in the nucleolus, where only a proportion acquires an active chromatic configuration allowing PolI transcription to occur. Malignant transformation is associated with an increase in the portion of active rDNA loci in the nucleolus. We showed that during malignant progression of B-cell lymphoma, the rDNA chromatin acquires an open state and rDNA-genome contacts are reorganized, which correlate with the repression of genes involved in B-cell differentiation 5 . These data support the intriguing hypothesis that dynamic changes in rDNA chromatin and long-range rDNA-genomic interactions regulate distinct programs of gene expression required for stemness, differentiation and malignant transformation. To address this hypothesis we are; (i) examining the molecular mechanisms by which nucleolar-associated chromatin domains dynamically regulate spatial organisation of the genome; (ii) testing whether the concomitant changes in distinct patterns of gene expression actively drive developmental programs such as differentiation and contribute to malignant transformations; and (iii) examining whether the therapeutic efficacy of Pol I inhibitors is mediated in part through the disruption of long-range rDNA genomic interaction.

Survival rates for pediatric patients suffering from mixed lineage leukemia (MLL)-rearranged leukemia remain below 50% and more targeted, less toxic therapies are urgently needed. A screening method optimized to discover cytotoxic compounds selective for MLL-rearranged leukemia identified CCI-006 as a novel inhibitor of MLL-rearranged and CALM-AF10 translocated leukemias that share common leukemogenic pathways. CCI-006 inhibited mitochondrial respiration and induced mitochondrial membrane depolarization and apoptosis in a subset (7/11, 64%) of MLL-rearranged leukemia cell lines within a few hours of treatment. The unresponsive MLL-rearranged leukemia cells did not undergo mitochondrial membrane depolarization or apoptosis despite a similar attenuation of mitochondrial respiration by the compound. In comparison to the sensitive cells, the unresponsive MLL-rearranged leukemia cells were characterized by a more glycolytic metabolic phenotype, exemplified by a more pronounced sensitivity to glycolysis inhibitors and elevated HIF1α expression. Silencing of HIF1α expression sensitized an intrinsically unresponsive MLL-rearranged leukemia cell to CCI-006, indicating that this pathway plays a role in determining sensitivity to the compound. In addition, unresponsive MLL-rearranged leukemia cells expressed increased levels of MEIS1, an important leukemogenic MLL target gene that plays a role in regulating metabolic phenotype through HIF1α. MEIS1 expression was also variable in a pediatric MLL-rearranged ALL patient dataset, highlighting the existence of a previously undescribed metabolic variability in MLL-rearranged leukemia that may contribute to the heterogeneity of the disease. This study thus identified a novel small molecule that rapidly kills MLL-rearranged leukemia cells by targeting a metabolic vulnerability in a subset of low HIF1α/low MEIS1-expressing MLL-rearranged leukemia cells.

Ribosme biogenesis can consume up to 80% of the cellular energy in rapidly proliferating cells and thus cells have involved various mechanisms/checkpoints to monitor the integrity of this process, of which the best understood is the

Exquisite control of ribosome biogenesis is fundamental for the maintenance of cellular growth and proliferation. Importantly, synthesis of ribosomal RNA by RNA polymerase I is a key regulatory step in ribosome biogenesis and a major biosynthetic and energy consuming process. Consequently, ribosomal RNA gene transcription is tightly coupled to the availability of growth factors, nutrients and energy. Thus cells have developed an intricate sensing network to monitor the cellular environment and modulate ribosomal DNA transcription accordingly. Critical controllers in these sensing networks, which mediate growth factor activation of ribosomal DNA transcription, include the PI3K/AKT/mTORC1, RAS/RAF/ERK pathways and MYC transcription factor. mTORC1 also responds to amino acids and energy status, making it a key hub linking all three stimuli to the regulation of ribosomal DNA transcription, although this is achieved via overlapping and distinct mechanisms. This review outlines the current knowledge of how cells respond to environmental cues to control ribosomal RNA synthesis. We also highlight the critical points within this network that are providing new therapeutic opportunities for treating cancers through modulation of RNA polymerase I activity and potential novel imaging strategies.

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). Refereed/Peer-reviewed

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naive, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

Huntington's disease is caused by polyglutamine (polyQ)-expansion mutations in the CAG tandem repeat of the Huntingtin gene. The central feature of Huntington's disease pathology is the aggregation of mutant Huntingtin (Htt) protein into micrometer-sized inclusion bodies. Soluble mutant Htt states are most proteotoxic and trigger an enhanced risk of death whereas inclusions confer different changes to cellular health, and may even provide adaptive responses to stress. Yet the molecular mechanisms underpinning these changes remain unclear. Using the flow cytometry method of pulse-shape analysis (Pu1SA) to sort neuroblastoma (Neuro2a) cells enriched with mutant or wild-type Htt into different aggregation states, we clarified which transcriptional signatures were specifically attributable to cells before versus after inclusion assembly. Dampened CREB signalling was the most striking change overall and invoked specifically by soluble mutant Httexl states. Toxicity could be rescued by stimulation of CREB signalling. Other biological processes mapped to different changes before and after aggregation included NF-kB signalling, autophagy, SUMOylation, transcription regulation by histone deacetylases and BRD4, NAD + biosynthesis, ribosome biogenesis and altered HIF-1 signalling. These findings open the path for therapeutic strategies targeting key molecular changes invoked prior to, and subsequently to, Httexl aggregation.

Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 μM, 6.89 μM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis., Author summary Trypanosoma brucei is protected by an essential Variant Surface Glycoprotein (VSG) coat in the mammalian bloodstream. The active VSG gene is transcribed by RNA polymerase I (Pol I), which typically only transcribes rDNA. Pol I transcription inhibitors are under clinical trials for cancer chemotherapy. As T. brucei relies on Pol I for VSG transcription, we investigated its susceptibility to these drugs. We show that quarfloxin (CX-3543), CX-5461, and BMH-21 are effective against T. brucei at nanomolar concentrations. T. brucei death was due to rapid and specific inhibition of Pol I transcription. Incubation with Pol I transcription inhibitors also resulted in disappearance of Pol I subnuclear structures like the nucleolus and the VSG expression site body (ESB). Rapid ESB loss followed the Pol I transcription block, arguing that the ESB is nucleated by Pol I transcription. Pol I transcription inhibitors could therefore potentially function as novel drugs against trypanosomiasis.

The transcript encoding the proto-oncogene MYC is commonly overexpressed in prostate cancer (PC). MYC protein abundance is also increased in the majority of cases of advanced and metastatic castrate-resistant PC (mCRPC). Accordingly, the MYC-directed transcriptional program directly contributes to PC by upregulating the expression of a number of pro-tumorigenic factors involved in cell growth and proliferation. A key cellular process downstream of MYC activity is the regulation of ribosome biogenesis which sustains tumor growth. MYC activity also cooperates with the dysregulation of the phosphoinositol-3-kinase (PI3K)/AKT/mTOR pathway to promote PC cell survival. Recent advances in the understanding of these interactions through the use of animal models have provided significant insight into the therapeutic efficacy of targeting MYC activity by interfering with its transcriptional program, and indirectly by targeting downstream cellular events linked to MYC transformation potential.

Overall survival for patients with ovarian cancer (OC) has shown little improvement for decades meaning new therapeutic options are critical. OC comprises multiple histological subtypes, of which the most common and aggressive subtype is high-grade serous ovarian cancer (HGSOC). HGSOC is characterized by genomic structural variations with relatively few recurrent somatic mutations or dominantly acting oncogenes that can be targeted for the development of novel therapies. However, deregulation of pathways controlling homologous recombination (HR) and ribosome biogenesis has been observed in a high proportion of HGSOC, raising the possibility that targeting these basic cellular processes may provide improved patient outcomes. The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib has been approved to treat women with defects in HR due to germline BRCA mutations. Recent evidence demonstrated the efficacy of targeting ribosome biogenesis with the specific inhibitor of ribosomal RNA synthesis, CX-5461 in v-myc avian myelocytomatosis viral oncogene homolog (MYC)-driven haematological and prostate cancers. CX-5461 has now progressed to a phase I clinical trial in patients with haematological malignancies and phase I/II trial in breast cancer. Here we review the currently available targeted therapies for HGSOC and discuss the potential of targeting ribosome biogenesis as a novel therapeutic approach against HGSOC.

The contribution of the nucleolus to cancer is well established with respect to its traditional role in facilitating ribosome biogenesis and proliferative capacity. More contemporary studies however, infer that nucleoli contribute a much broader role in malignant transformation. Specifically, extra-ribosomal functions of the nucleolus position it as a central integrator of cellular proliferation and stress signaling, and are emerging as important mechanisms for modulating how oncogenes and tumor suppressors operate in normal and malignant cells. The dependence of certain tumor cells to co-opt nucleolar processes to maintain their cancer phenotypes has now clearly been demonstrated by the application of small molecule inhibitors of RNA Polymerase I to block ribosomal DNA transcription and disrupt nucleolar function (Bywater et al., 2012 [1]). These drugs, which selectively kill tumor cells in vivo while sparing normal cells, have now progressed to clinical trials. It is likely that we have only just begun to scratch the surface of the potential of the nucleolus as a new target for cancer therapy, with “suppression of nucleolar stress” representing an emerging “hallmark” of cancer. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

The tumor suppressor protein p53 plays a crucial part in the cellular defense against malignancies. DNA-damaging chemotherapeutics rely on the activation of p53 for their anticancer activity at the expense of genotoxicity. Nongenotoxic approaches for p53 activation have been extensively investigated validating p53 as a therapeutic target. However, their development has been hampered by low efficacy and a narrow therapeutic window. An alternate nongenotoxic approach for cancer-specific activation of wild-type p53 has been recently identified. It relies on the activation of a cellular checkpoint mechanism termed 'nucleolar stress', which can be triggered by acute inhibition of rRNA biogenesis. CX5461, the first selective inhibitor of rRNA biogenesis, and thus a potent activator of nucleolar stress, is poised to enter clinical development.

Revertant somatic mosaicism (RM) has been reported in genetic diseases including skin disorders and bone marrow failure (BMF) syndromes. We present examples of RM of haematopoietic cells in the context of inherited BMF syndromes with dramatically different clinical outcomes. Diamond Blackfan Anaemia (DBA) is an inherited BMF disorder caused by mutations/deletions in ribosomal proteins. We have a 9 year old male who required regular transfusions until he became transfusion independent at age 4. We identified a 184kb deletion on chromosome 12 encompassing 11 genes including RPS26 (known DBA gene). Significantly, we observed multiple independent copy neutral loss of heterozygosity (CNLOH) events on chromosome 12, correcting the 184kb deletion in a subset of blood cells, but not in hair. We propose RM as a mechanism for correction of the germline deletion resulting in spontaneous remission in the patient. We also recently identified germline mutations in SAMD9L, in a family with thrombocytopaenia, macrocytosis and ALL. In this family, we see examples for somatic mosaicism leading to a severe blood phenotype as well as CNLOH RM causing milder phenotypes in different family members. RM needs to be considered when selecting tissue source for diagnosis in carriers with mild phenotype/remission, and use of patient-derived cell lines for drug screens with multiple rounds of cell replication. We are currently trying to understand and manipulate revertant clone selection and hope this will open windows for rational correction and selection protocols for effective therapeutic intervention. Experimental manipulations to select clones that improve clinical phenotype have substantive implications on efficiencies of gene manipulations, manufacture and quality control requirements of autologous cellular therapies.

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

A safe and efficient therapy for patients with the bone marrow failure syndrome Diamond-Blackfan Anemia (DBA) is urgently needed. To identify novel drug candidates for DBA, a small molecule screen was performed using c-Kit positive E14.5 fetal liver cells from a DBA mouse model in which the DBA phenotype is induced upon doxycycline (DOX)-inducible silencing of Rps19, the most frequently mutated gene in DBA (Jaako et al. PMID: 21989989). Test compounds were added 24 hours after cell seeding and DOX addition. After four days the number of live metabolically active cells in each well was estimated based on quantitation of intracellular ATP. Fifteen commercial annotated small-molecule libraries (3 800 molecules) and 10 500 selected compounds from a diverse compound library were screened. Between the screens we identified 20 molecules that reproducibly increased proliferation of rps19-deficient erythroid progenitors 4-8 fold in a concentration-dependent manner. Hits from annotated libraries included inhibitors of TGFb receptor, DYRK and Casein kinases. The most potent hits however were compounds in a series of thienopyridines, with an unknown target profile, but with a core structure suggesting kinase inhibition activity. Database searches revealed that the structure-activity relationships (SAR) of 12 active and 10 inactive analogues were similar to that of a series of thienopyridines previously reported as bone anabolic agents by unknown mechanism (Saito et al. PMID: 23453217). The most potent analogues described by Saito et al. as bone anabolic agents were synthesized, and the compounds rescue also RPS19-deficient erythroid cell proliferation in a potent manner (EC50= 20-50 nM). In an attempt to identify the molecular target of these compounds, six actives including thienopyridines 15k and 15w were subjected to kinase profiling against 468 kinases (DiscoverX). Three of the compounds targeted cyclin- dependent kinases CDK8 and its paralog CDK19, and in particular the most potent molecule (15w) is a highly selective CDK8 inhibitor. To confirm whether CDK8 inhibition underlies the rescue of the DBA phenotype, structurally unrelated and potent CDK8 inhibitors including CCT-251545, Senexin A, Senexin B, and Sel120-34A were also evaluated. All tested CDK8-inhibitors rescue proliferation and erythroid maturation of c-kit+ cells from the DBA mouse in a concentration-dependent manner. To further investigate the potential of CDK8 as a therapeutic target in DBA several CDK8 inhibitors were evaluated in erythroid cultures of primary DBA patient cells. CDK8 inhibitors 15w, Senexin B and Sel120-34A increase erythroid progenitor proliferation 5-10 fold of CD34+ peripheral blood cells from three DBA patients (RPS19, RPS26, RPL35a mutations), and increase the fraction of transferrin receptor (CD71) and glycophorin A positive cells in culture. Healthy CD34+ peripheral blood treated with CDK8 inhibitors show no increase in proliferation. Finally, we show that bone marrow failure and anemia in the DBA mouse model is partially rescued (RBC and Hemoglobin levels, p Disclosures Rzymski: Selvita S.A.: Employment, Equity Ownership. Johansson:LU Holding: Patents & Royalties. Lundbäck:LU Holding: Patents & Royalties. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

The upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. We recently reported in Sanij et al. (2014) [1] that the two isoforms of UBF (UBF1/2) are enriched at Pol II-transcribed genes throughout the mouse and human genomes. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) of UBF1/2, Pol I, Pol II, H3K9me3, H3K4me4, H3K9ac and H4 hyperacetylation, we reported a correlation of UBF1/2 binding with enrichments in Pol II and markers of active chromatin. In addition, we examined a functional role for UBF1/2 in mediating Pol II transcription by performing expression array analysis in control and UBF1/2 depleted NIH3T3 cells. Our data demonstrate that UBF1/2 bind highly active Pol II-transcribed genes and mediate their expression without recruiting Pol I. Furthermore, we reported ChIP-sequencing analysis of UBF1/2 in immortalized human epithelial cells and their isogenically matched transformed counterparts. Here we report the experimental design and the description of the ChIP-sequencing and microarray expression datasets uploaded to NCBI Sequence Research Archive (SRA) and Gene Expression Omnibus (GEO)., Highlights • ChIP-seq analysis of UBF binding in NIH3T3, HMEC and HMLER cell lines • Correlation analysis of UBF binding with Pol I, Pol lI and histone modifications in NIH3T3 cells • Identifications of a novel biological function for the Pol I transcription factor UBF

To influence physiology and pathophysiology, G protein-coupled receptors (GPCRs) have evolved to appropriate additional signalling modalities, such as activation of adjacent membrane receptors. Epidermal growth factor receptors (EGFRs) mediate growth and remodelling actions of GPCRs, although the precise network of gene products and molecular cascades linking GPCRs to EGFRs (termed EGFR transactivation) remains incomplete. In this review, we describe the current view of GPCR-EGFR transactivation, identifying the established models of receptor cross-talk. We consider the limitations in our current knowledge, and propose that recent advances in molecular and cell biology technology, including functional genomics approaches, will allow a renewed focus of efforts to understand the mechanism underlying EGFR transactivation. Using an unbiased approach for identification of the molecules required for GPCR-mediated EGFR transactivation will provide a contemporary and more complete representation from which to extrapolate therapeutic control in diseases from cardiovascular remodelling to cancer. Cross-talk between G protein-coupled receptors (GPCRs) and the epidermal growth factor receptor (EGFR) facilitate many important cellular activities. However, the molecular mechanism still remains poorly understood. In this review, we discuss the current view of GPCR-EGFR transactivation, and propose that functional genomics approaches will allow a renewed concentration of our efforts to further investigate the mechanism underlying this process.

The nucleoli are the site of the production of ribosomes, the protein synthetic apparatus of the cell. The presence of enlarged nucleoli, reflecting increased ribosomal gene transcription, has long been used by pathologists as an indicator of aggressive tumors. However, over the last 10 years a growing body of evidence has revealed that the nucleolus contains a dynamic cohort of over 4500 proteins, the majority of which have no function in ribosome production. The activity of some of these proteins is modulated by their regulated sequestration and release from the nucleolus. In particular, the nucleolus plays a central role in sensing cellular stress to modulate the abundance of the critical tumor suppressor protein p53. The finding that p53 activity is dysregulated in up to 50% of all human cancers highlights the importance of the nucleolar stress response in limiting malignant transformation. The development of drugs to selectively inhibit transcription of the ribosomal RNA genes in the nucleolus has paved the way for a new therapeutic approach to hijack nucleolar stress to selectively and non-genotoxically activate p53 in tumor cells. Here, we describe the potential application of this exciting new class of drugs for the treatment of human cancer.

Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.

Transcription of the ribosomal RNA genes by the dedicated RNA polymerase I enzyme and subsequent processing of the ribosomal RNA are fundamental control steps in the synthesis of functional ribosomes. Dysregulation of Pol I transcription and ribosome biogenesis is linked to the etiology of a broad range of human diseases. Diseases caused by loss of function mutations in the molecular constituents of the ribosome, or factors intimately associated with RNA polymerase I transcription and processing are collectively termed ribosomopathies. Ribosomopathies are generally rare and treatment options are extremely limited tending to be more palliative than curative. Other more common diseases are associated with profound changes in cellular growth such as cardiac hypertrophy, atrophy or cancer. In contrast to ribosomopathies, altered RNA polymerase I transcriptional activity in these diseases largely results from dysregulated upstream oncogenic pathways or by direct modulation by oncogenes or tumor suppressors at the level of the RNA polymerase I transcription apparatus itself. Ribosomopathies associated with mutations in ribosomal proteins and ribosomal RNA processing or assembly factors have been covered by recent excellent reviews. In contrast, here we review our current knowledge of human diseases specifically associated with dysregulation of RNA polymerase I transcription and its associated regulatory apparatus, including some cases where this dysregulation is directly causative in disease. We will also provide insight into and discussion of possible therapeutic approaches to treat patients with dysregulated RNA polymerase I transcription. This article is part of a Special Issue entitled: Transcription by Odd Pols.

The dysregulation of PI3K/AKT/mTORC1 signalling and/or hyperactivation of MYC are observed in a high proportion of human cancers, and together they form a 'super signalling' network mediating malignancy. A fundamental downstream action of this signalling network is up-regulation of ribosome biogenesis and subsequent alterations in the patterns of translation and increased protein synthesis, which are thought to be critical for AKT/MYC-driven oncogenesis. We have demonstrated that AKT and MYC cooperate to drive ribosomal DNA (rDNA) transcription and ribosome biogenesis, with AKT being essential for rDNA transcription and in vitro survival of lymphoma cells isolated from a MYC-driven model of B-cell lymphoma (Eμ-Myc) [Chan JC et al., (2011) Science Signalling 4, ra56]. Here we show that the allosteric AKT inhibitor MK-2206 rapidly and potently antagonizes rDNA transcription in Eμ-Myc B-cell lymphomas in vivo, and this is associated with a rapid reduction in indicators of disease burden, including spleen weight and the abundance of tumour cells in both the circulation and lymph nodes. Extended treatment of tumour-bearing mice with MK-2206 resulted in a significant delay in disease progression, associated with increased B-cell lymphoma apoptosis. Our findings suggest that malignant diseases characterized by unrestrained ribosome biogenesis may be vulnerable to therapeutic strategies that target the PI3K/AKT/mTORC1/MYC growth control network.

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs. Refereed/Peer-reviewed