Sir Peter MacCallum Department of Oncology - Research Publications
Permanent URI for this collection
Search Results
1 - 10 of 19
-
ItemAutophagy Induction Is a Tor- and Tp53-Independent Cell Survival Response in a Zebrafish Model of Disrupted Ribosome Biogenesis(PUBLIC LIBRARY SCIENCE, 2013-02)Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
-
ItemUBF levels determine the number of active ribosomal RNA genes in mammals(ROCKEFELLER UNIV PRESS, 2008-12-29)In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.
-
ItemToo much or too little Harnessing senescence to control oncogene-driven cancer(TAYLOR & FRANCIS INC, 2012-09-01)
-
ItemAKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy(NATURE PUBLISHING GROUP, 2012-04)The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.
-
ItemRelative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity.(Hindawi Limited, 2011)The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.
-
ItemAmino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription(IMPACT JOURNALS LLC, 2016-08-02)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.
-
ItemA novel small molecule that kills a subset of MLL-rearranged leukemia cells by inducing mitochondrial dysfunction(NATURE PUBLISHING GROUP, 2019-05-16)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.
-
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.
-
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.
-
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.