Publications

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.

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

Abstract Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I–driven transcription relative to Pol II–driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment. Cancer Res; 71(4); 1418–30. ©2010 AACR.

<p>This figure shows supplementary data pertinent to Figure 4 including protein and cell death analysis data.</p>

Abstract Increased transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) is a common feature of human cancer[1]. However until now no studies have directly examined the requirement for dysregulated rDNA transcription in the maintenance of the malignant phenotype. Our studies show that increased rDNA transcription is necessary for MYC oncogenic activity and can be therapeutically targeted to treat tumors. We demonstrate that restoration of hyperactivated rDNA transcription rates in Eμ-MYC lymphoma cells to the levels observed in normal B cells by knock down of Pol I transcription factors UBF and Rrn3, is rapidly selected against in vitro as determined by loss from competitive culture with parental cells. This disadvantage is due to the induction of apoptosis and can be rescued by over expression of the anti-apoptotic protein BCL2. Furthermore, treatment of Eμ-MYC lymphoma cells with a small molecule inhibitor of Pol I (CX-5461) we have recently developed[2] is able to specifically inhibit Pol I transcription (IC50=45.64nM, metabolic labeling) and rapidly induce apoptosis and subsequent cell death by 16hrs (IC50=8.4nM, PI exclusion). This apoptotic response is not an indirect consequence of ribosome insufficiency but is due to induction of the ribosome biogenesis surveillance pathway[3] characterized by rapid nucleolar disruption, as determined by immunofluorescence of Fibrillarin relocalization, and the subsequent activation of p53-dependent apoptotic signaling, as determined by increased protein levels of p53 and increased expression of p53 target genes p21, Mdm2 and Puma at the mRNA and protein level within 1hr of treatment. Using CX-5461 we show that malignant B cells have a heightened dependence on elevated rDNA transcription that can be exploited in vivo as a therapeutic target for treatment of lymphoma. Treatment of mice transplanted Eμ-MYC lymphoma with 40mg/kg CX-5461 orally every 3 days is able to delay the onset of disease (median survival of 15 days for vehicle, 31 days for drug, P<0.0001), with this delay accompanied by a period of disease remission, as determined by peripheral white blood cell counts (58×10⁁3cellsμl±4.5 for vehicle, 4.2×10⁁3cellsμl±0.29 for drug, P<0.001) and FACS analysis of the peripheral blood (62%tumor cells±2.3 for vehicle, 0.7%tumor cells±0.05 for drug, P<0.001), and maintenance of a wild type B-cell population (20%B220+cells±1.0 for vehicle, 43%B220+cells±1.5 for drug, P<0.001). Similarly in vivo, elimination of tumor cells with the inhibition of Pol I transcription results from subsequent activation of p53-dependent apoptotic signaling that specifically occurs in malignant but not normal B-lymphocytes in the spleen and bone marrow of healthy mice treated with the same dose of CX-5461. Human leukemia and lymphoma cell lines also show high sensitivity to the inhibition of Pol I transcription that is dependent on p53 mutational status. Furthermore, CX-5461 was able to delay the growth of subcutaneous MV 4;11 (human acute myeloid leukemia) xenografts in mice dosed at 125mg/kg IP weekly (tumor growth inhibition=93% compared to vehicle). Our work reveals a previously unproven paradigm that links hyperactivated rDNA transcription and nucleolar integrity to maintenance of aggressive tumors independent of ribosome levels. Critically, these results also demonstrate how activation of a ribosome biogenesis surveillance pathway by selective inhibition of rDNA transcription can be used as a novel therapeutic target for the treatment of cancer. Furthermore, patients with hematological malignancies have been identified as the appropriate first cohort for trials of CX-5461 in man commencing next year. 1. R. J. White, Nat Rev Mol Cell Biol 6, 69 (Jan, 2005). 2. D. Drygin et al., Cancer Res, (Dec 15, 2010). 3. C. Deisenroth, Y. Zhang, Oncogene 29, 4253 (Jul 29, 2010). This abstract is also presented as Poster A35. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR15.

<div>Abstract<p>Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I–driven transcription relative to Pol II–driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates <i>in vivo</i> antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment. <i>Cancer Res; 71(4); 1418–30. ©2010 AACR</i>.</p></div>

Abstract Recent findings by our group have been instrumental in the development of the novel selective inhibitors of RNA Polymerase I (Pol I) (Drygin et al., Cancer Research, 2011; Bywater et al. Cancer Cell, 2012). This work has led to the fundamental discovery that ribosomal gene transcription by Pol I is not simply a “housekeeping” process in cancer cells but is highly regulated to maintain their viability (Bywater et al. Nature Reviews Cancer, 2013). Strikingly, inhibition of Pol I transcription shows a profound selectivity for malignant over normal cells in preclinical studies. As with the majority of targeted therapies, despite initial favorable responses to approaches that target ribosome synthesis and/or function in MYC-driven lymphoma models, resistant disease emerges. It is increasingly clear that maximizing the inhibition of key signaling networks as a whole improves anti-tumor response. The well-established reliance of MYC-driven malignancies on elevated rates of ribosome biogenesis, mTORC1/eIF4E-driven protein synthesis, and cell growth makes them vulnerable to therapeutic strategies that target the ribosome. Thus we hypothesized that the simultaneous targeting of the ribosome at multiple points would antagonize the development of acquired resistance and consequently prolong survival in MYC-driven cancer models. We will present data to demonstrate that targeting both ribosome synthesis and function through the combination of novel inhibitors of RNA polymerase I transcription, and PI3K/AKT/mTOR signaling inhibitors or PIM Kinase inhibitors provides a significant increase in survival compared to treatment with single agents (Devlin et al., Cancer Discovery 2016; Rebello et al., Clinical Cancer Res. 2016). We will also discuss the molecular mechanism by which multipoint targeting of the ribosome synergizes to increase survival. Finally we will discuss our collaboration with Pimera, Inc. to develop highly selective second generation RNA Pol I inhibitors. The lead compound PMR-116 is showing exceptional activity in transgenic models of malignancy, including MLL-ENL AML and Vk*MYC driven multiple myeloma. We anticipate this compound will enter the clinic in 2017. Citation Format: Ross D. Hannan, Nadine Hein, Katherine M. Hannan, Gretchen Poortinga, Elaine Sanij, Jirawas Sornkom, Kylee MacLachlan, Andrew Cuddihy, Carleen Cullinane, Luc Furic, Denis Drygin, Mustapha Haddach, Simon Harrison, Grant McArthur, Richard B. Pearson. Drugging the ribosome at the level of synthesis and translation to treat solid and hematologic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA10.

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.

<p>This figure shows supplementary data pertinent to Figure 1 including cell death, cell signalling and mRNA transcription analysis data.</p>

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.