Sir Peter MacCallum Department of Oncology - Research Publications
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ItemSelective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes(CELL PRESS, 2020-08-20)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.
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ItemTargeting the RNA Polymerase I Transcription for Cancer Therapy Comes of Age(MDPI, 2020-02)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.
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ItemTranscriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila(COMPANY BIOLOGISTS LTD, 2020-06)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.
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ItemNo Preview AvailableInhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer.(AMER ASSOC CANCER RESEARCH, 2020-07)Abstract Introduction: PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient high-grade serous ovarian cancer (HGSOC). However, acquired resistance to PARPi is a major challenge in the clinic. The specific inhibitor of RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) has demonstrated single-agent antitumor activity in p53 wild-type and p53-mutant hematologic malignancies (first-in-human trial, dose escalation study of CX-5461 at Peter MacCallum Cancer Centre) (Khot et al., Cancer Discov 2019). CX-5461 has also been reported to exhibit synthetic lethality with BRCA1/2 deficiency through stabilization of G-quadruplex DNA (GQ) structures. Here, we investigate the efficacy of CX-5461 in treating HGSOC. Experimental Design: The mechanisms by which CX-5461 induces DNA damage response (DDR) and displays synthetic lethality in HR-deficient HGSOC cells are explored. We present in vivo data of mice bearing two functionally and genomically profiled HGSOC-patient-derived xenograft (PDX)s treated with CX-5461 and olaparib, alone and in combination. We also investigate CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Results: Utilizing ovarian cancer cell lines, we demonstrate that sensitivity to CX-5461 is associated with “BRCA1 mutation” and “MYC targets” gene expression signatures. In addition, sensitivity to CX-5461 is associated with high basal rates of Pol I transcription. Importantly, we demonstrate a novel mechanism for CX-5461 synthetic lethal interaction with HR deficiency mediated through the induction of replication stress at rDNA repeats. Our data reveal CX-5461-mediated DDR in HR-deficient cells does not involve stabilization of GQ structures as previously proposed. On the contrary, we show definitively that CX-5461 inhibits Pol I recruitment leading to rDNA chromatin defects including stabilization of R-loops, single-stranded DNA, and replication stress at the rDNA. Mechanistically, we demonstrate CX-5461 leads to replication-dependent DNA damage involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 has a different sensitivity spectrum to olaparib and cooperates with PARPi in exacerbating replication stress, leading to enhanced therapeutic efficacy in HR-deficient HGSOC-PDX in vivo compared to single-agent treatment of both drugs. Further, CX-5461 exhibits single-agent efficacy in olaparib-resistant HGSOC-PDX overcoming PARPi-resistance mechanisms involving fork protection. Importantly, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Conclusions: CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 also has exciting potential as a treatment option for patients with relapsed HGSOC tumors that have high MYC activity and poor clinical outcome; these patients currently have very limited effective treatment options. This abstract is also being presented as Poster A71. Citation Format: Elaine Sanij, Katherine Hannan, Jiachen Xuan, Shunfei Yan, Jessica A. Ahern, Anna S. Trigos, Natalie Brajanovski, Jinbae Son, Keefe T. Chan, Olga Kondrashova, Elizabeth Lieschke, Matthew J. Wakefield, Sarah Ellis, Carleen Cullinane, Gretchen Poortinga, Kum Kum Khanna, Linda Mileshkin, Grant A. McArthur, John Soong, Els M. Berns, Ross D. Hannan, Clare L. Scott, Karen E. Sheppard, Richard B. Pearson. Inhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR13.
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ItemReprogrammed mRNA translation drives resistance to therapeutic targeting of ribosome biogenesis(WILEY, 2020-11-02)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.
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ItemPGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth(BMC, 2020-04-03)BACKGROUND: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. RESULTS: We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. CONCLUSIONS: Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.
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ItemrDNA Chromatin Activity Status as a Biomarker of Sensitivity to the RNA Polymerase I Transcription Inhibitor CX-5461(FRONTIERS MEDIA SA, 2020-07-03)Hyperactivation of RNA polymerase I (Pol I) transcription of ribosomal RNA (rRNA) genes (rDNA) is a key determinant of growth and proliferation and a consistent feature of cancer cells. We have demonstrated that inhibition of rDNA transcription by the Pol I transcription inhibitor CX-5461 selectively kills tumor cells in vivo. Moreover, the first-in human trial of CX-5461 has demonstrated CX-5461 is well-tolerated in patients and has single-agent anti-tumor activity in hematologic malignancies. However, the mechanisms underlying tumor cell sensitivity to CX-5461 remain unclear. Understanding these mechanisms is crucial for the development of predictive biomarkers of response that can be utilized for stratifying patients who may benefit from CX-5461. The rDNA repeats exist in four different and dynamic chromatin states: inactive rDNA can be either methylated silent or unmethylated pseudo-silent; while active rDNA repeats are described as either transcriptionally competent but non-transcribed or actively transcribed, depending on the level of rDNA promoter methylation, loading of the essential rDNA chromatin remodeler UBF and histone marks status. In addition, the number of rDNA repeats per human cell can reach hundreds of copies. Here, we tested the hypothesis that the number and/or chromatin status of the rDNA repeats, is a critical determinant of tumor cell sensitivity to Pol I therapy. We systematically examined a panel of ovarian cancer (OVCA) cell lines to identify rDNA chromatin associated biomarkers that might predict sensitivity to CX-5461. We demonstrated that an increased proportion of active to inactive rDNA repeats, independent of rDNA copy number, determines OVCA cell line sensitivity to CX-5461. Further, using zinc finger nuclease genome editing we identified that reducing rDNA copy number leads to an increase in the proportion of active rDNA repeats and confers sensitivity to CX-5461 but also induces genome-wide instability and sensitivity to DNA damage. We propose that the proportion of active to inactive rDNA repeats may serve as a biomarker to identify cancer patients who will benefit from CX-5461 therapy in future clinical trials. The data also reinforces the notion that rDNA instability is a threat to genomic integrity and cellular homeostasis.
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ItemCX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer(NATURE PUBLISHING GROUP, 2020-05-26)Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
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ItemA functional genetic screen defines the AKT-induced senescence signaling network(Springer Nature, 2020-02)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.