Publications

A novel role for Top2α as a mediator of resistance to ribosomal DNA transcription inhibition

Changes in long-range rDNA-genomic interactions associate with altered RNA polymerase II gene programs during malignant transformation

Abstract 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.

Amino acid-dependent signaling <i>via</i> S6K1 and MYC is essential for regulation of rDNA transcription

// Jian Kang 1 , Eric P. Kusnadi 1 , Allison J. Ogden 1 , Rodney J. Hicks 2,3 , Lukas Bammert 4 , Ulrike Kutay 4 , Sandy Hung 5 , Elaine Sanij 1 , Ross D. Hannan 1,2,6,7,8,9 , Katherine M. Hannan 6,9 and Richard B. Pearson 1,2,6,7 1 Oncogenic Signaling and Growth Control Program, Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia 2 Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria, Australia 3 Molecular Imaging and Targeted Therapeutics Laboratory, Cancer Therapeutics Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia 4 Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland 5 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital &amp; Department of Ophthalmology, University of Melbourne, East Melbourne, Victoria, Australia 6 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia 7 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 8 School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia 9 Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberaa, ACT, Australia Correspondence to: Richard B. Pearson, email: // Katherine M. Hannan, email: // Keywords : amino acids, rDNA transcription, S6K1, MYC, Gerotarget Received : June 08, 2016 Accepted : June 15, 2016 Published : June 30, 2016 Abstract Dysregulation of RNA polymerase I (Pol I)-dependent ribosomal DNA (rDNA) transcription is a consistent feature of malignant transformation that can be targeted to treat cancer. Understanding how rDNA transcription is coupled to the availability of growth factors and nutrients will provide insight into how ribosome biogenesis is maintained in a tumour environment characterised by limiting nutrients. We demonstrate that modulation of rDNA transcription initiation, elongation and rRNA processing is an immediate, co-regulated response to altered amino acid abundance, dependent on both mTORC1 activation of S6K1 and MYC activity. Growth factors regulate rDNA transcription initiation while amino acids modulate growth factor-dependent rDNA transcription by primarily regulating S6K1-dependent rDNA transcription elongation and processing. Thus, we show for the first time amino acids regulate rRNA synthesis by a distinct, post-initiation mechanism, providing a novel model for integrated control of ribosome biogenesis that has implications for understanding how this process is dysregulated in cancer.

Ets-2 Is Induced by Oxidative Stress and Sensitizes Cells to H2O2-Induced Apoptosis: Implications for Down's Syndrome

Cystathionine-β-synthase is essential for AKT-induced senescence and suppresses the development of gastric cancers with PI3K/AKT activation

Hyperactivation of oncogenic pathways downstream of RAS and PI3K/AKT in normal cells induces a senescence-like phenotype that acts as a tumor-suppressive mechanism that must be overcome during transformation. We previously demonstrated that AKT-induced senescence (AIS) is associated with profound transcriptional and metabolic changes. Here, we demonstrate that human fibroblasts undergoing AIS display upregulated cystathionine-β-synthase (CBS) expression and enhanced uptake of exogenous cysteine, which lead to increased hydrogen sulfide (H

Exploiting dysregulated iron homeostasis to eradicate persistent high-grade serous ovarian cancer

Treatments for high-grade serous ovarian cancer (HGSOC) are initially effective but most invariably fail. Although they can successfully suppress the bulk of the tumour cell population, residual cancer cells can enter alternative therapy-resistant cell fates highlighted by proliferative arrest. Understanding the nature of these fates and how cells may resume uncontrolled proliferation will lead to the development of new treatments for HGSOC. In this study, we examine the response of HGSOC cells to standard of care cisplatin chemotherapy and to the RNA Polymerase I transcription inhibitor CX-5461/Pidnarulex, two drugs that elicit a potent DNA damage response and growth arrest. Here, we identify that HGSOC cells exposed to these therapies show multiple hallmarks of therapy-induced senescence (TIS) and derive a core TIS gene expression signature irrespective of genetic background or senescence trigger. Given that TIS is a potentially escapable state, we have performed a focussed drug screen to identify drugs that eradicate senescent HGSOC cells. We identify that therapy-induced senescent HGSOC cells, including those with decreased sensitivity to senolytic drugs that inhibit the pro-survival protein BCL-XL, can be eliminated using drugs that induce ferroptosis, an iron-dependent form of cell death. Mechanistically, we demonstrate that senescent HGSOC cells have altered expression of regulators of iron metabolism leading to intracellular iron overload that underpins this targetable vulnerability. Together, we highlight elevated levels of iron as a TIS biomarker in HGSOC and the potential of inducing ferroptosis to eradicate residual HGSOC cells following initial therapy.

c-MYC coordinately regulates ribosomal gene chromatin remodeling and Pol I availability during granulocyte differentiation

Loss of c-MYC is required for downregulation of ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) during granulocyte differentiation. Here, we demonstrate a robust reduction of Pol I loading onto rDNA that along with a depletion of the MYC target gene upstream binding factor (UBF) and a switch from epigenetically active to silent rDNA accompanies this MYC reduction. We hypothesized that MYC may coordinate these mechanisms via direct regulation of multiple components of the Pol I transcription apparatus. Using gene expression arrays we identified a ‘regulon’ of Pol I factors that are both downregulated during differentiation and reinduced in differentiated granulocytes upon activation of the MYC-ER transgene. This regulon includes the novel c-MYC target genes RRN3 and POLR1B. Although enforced MYC expression during granulocyte differentiation was sufficient to increase the number of active rRNA genes, its activation in terminally differentiated cells did not alter the active to inactive gene ratio despite increased rDNA transcription. Thus, c-MYC dynamically controls rDNA transcription during granulocytic differentiation through the orchestrated transcriptional regulation of core Pol I factors and epigenetic modulation of number of active rRNA genes.

Supplementary Methods, Tables S1 - S3, Figure Legends from Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma

&lt;p&gt;This document contains additional materials and methods details, tables of antibody and primer details and supplementary data figure legends.&lt;/p&gt;

Genome wide mapping of UBF binding-sites in mouse and human cell lines

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).

A functional genetic screen defines the AKT-induced senescence signaling network

Abstract 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.

Supplementary Figure S2 from Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma

&lt;p&gt;This figure shows supplementary data pertinent to Figure 2 including cell death and tumour cell analysis data.&lt;/p&gt;

Targeting the ribosome to treat multiple myeloma

<title>Abstract</title> The high rates of protein synthesis and processing render multiple myeloma (MM) cells vulnerable to perturbations in protein homeostasis. The induction of proteotoxic stress by targeting protein degradation with proteasome inhibitors (PI) has revolutionized the treatment of MM. However, resistance to PI is inevitable and represents an ongoing clinical challenge. Our first-in-human study of the selective inhibitor of RNA polymerase I transcription of ribosomal RNA genes, CX-5461 has demonstrated a potential signal for anti-tumor activity in three of six heavily pre-treated MM patients. Here we show that CX-5461 has potent antimyeloma activity in PI-resistant MM preclinical models in vitro and in vivo. In addition to inhibiting ribosome biogenesis, CX-5461 causes topoisomerase II trapping and replication-dependent DNA damage, leading to G2/M cell cycle arrest and apoptotic cell death. Surprisingly, the addition of PI does not enhance the therapeutic benefit of CX-5461. In contrast, CX-5461 shows synergistic interaction with the histone deacetylase inhibitor panobinostat in both the Vk*MYC and the 5T33-KaLwRij mouse models of MM by targeting ribosome biogenesis and protein synthesis through distinct mechanisms. Our findings thus provide strong evidence to facilitate the clinical development of targeting the ribosome to treat relapsed and refractory MM.

Data from Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma

&lt;div&gt;Abstract&lt;p&gt;Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene &lt;i&gt;c-MYC&lt;/i&gt; 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-&lt;i&gt;Myc&lt;/i&gt; 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.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; 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-&lt;i&gt;Myc&lt;/i&gt; lymphoma–bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer. &lt;i&gt;Cancer Discov; 6(1); 59–70. ©2015 AACR&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 1&lt;/p&gt;&lt;/div&gt;

Investigating Ribosome-Targeting Therapies for the Treatment of Relapsed/Refractory Multiple Myeloma

The nucleolus: an emerging target for cancer therapy

CX-5461 can destabilize replication forks in PARP inhibitor-resistant models of ovarian cancer

Acquired drug resistance leads to poor clinical outcome in high grade serous ovarian cancer (HGSOC). We have demonstrated the efficacy of the novel drug CX-5461 in HGSOC is mediated through destabilization of DNA replication forks. The data highlights the potential of CX-5461 in overcoming a general mechanism of chemotherapeutic resistance.

Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are 400-600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.

Data from Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma

&lt;div&gt;Abstract&lt;p&gt;Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene &lt;i&gt;c-MYC&lt;/i&gt; 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-&lt;i&gt;Myc&lt;/i&gt; 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.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; 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-&lt;i&gt;Myc&lt;/i&gt; lymphoma–bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer. &lt;i&gt;Cancer Discov; 6(1); 59–70. ©2015 AACR&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 1&lt;/p&gt;&lt;/div&gt;