Megan J. Bywater

Inhibition of RNA Polymerase I as a Therapeutic Strategy to Promote Cancer-Specific Activation of p53

Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.

Targeting RNA Polymerase I with an Oral Small Molecule CX-5461 Inhibits Ribosomal RNA Synthesis and Solid Tumor Growth

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.

AKT Promotes rRNA Synthesis and Cooperates with c-MYC to Stimulate Ribosome Biogenesis in Cancer

In addition to promoting translation, AKT also stimulates protein synthesis and cell growth by enhancing ribosome biogenesis. Building the Building Blocks Ribosomes translate mRNA into protein, and the activity of signaling pathways that promote ribosome formation (or biogenesis) is often increased in cancer cells, which have high rates of protein synthesis and cell growth. Thus, each step of ribosome biogenesis can limit cell growth, including the synthesis of ribosomal RNA (rRNA), which encodes the RNA components of the ribosome. Chan et al. found that the kinase AKT, which is frequently activated in cancer cells and was previously implicated in promoting protein translation, also promotes rRNA synthesis. Cells with increased AKT activity showed increased rRNA abundance and more ribosomes. The transcription factor c-MYC is required for ribosome biogenesis, and the gene encoding c-MYC is frequently mutated in tumors. The ability of c-MYC to promote ribosome biogenesis and cell growth in a mouse model of lymphoma was attenuated by an AKT inhibitor. These results suggest that reducing ribosome biogenesis may in part underlie the therapeutic efficacy of anticancer drugs that target AKT signaling. Precise regulation of ribosome biogenesis is fundamental to maintain normal cell growth and proliferation, and accelerated ribosome biogenesis is associated with malignant transformation. Here, we show that the kinase AKT regulates ribosome biogenesis at multiple levels to promote ribosomal RNA (rRNA) synthesis. Transcription elongation by RNA polymerase I, which synthesizes rRNA, required continuous AKT-dependent signaling, an effect independent of AKT’s role in activating the translation-promoting complex mTORC1 (mammalian target of rapamycin complex 1). Sustained inhibition of AKT and mTORC1 cooperated to reduce rRNA synthesis and ribosome biogenesis by additionally limiting RNA polymerase I loading and pre-rRNA processing. In the absence of growth factors, constitutively active AKT increased synthesis of rRNA, ribosome biogenesis, and cell growth. Furthermore, AKT cooperated with the transcription factor c-MYC to synergistically activate rRNA synthesis and ribosome biogenesis, defining a network involving AKT, mTORC1, and c-MYC as a master controller of cell growth. Maximal activation of c-MYC–dependent rRNA synthesis in lymphoma cells required AKT activity. Moreover, inhibition of AKT-dependent rRNA transcription was associated with increased lymphoma cell death by apoptosis. These data indicate that decreased ribosome biogenesis is likely to be a fundamental component of the therapeutic response to AKT inhibitors in cancer.

The mTORC1 Inhibitor Everolimus Prevents and Treats Eμ-Myc Lymphoma by Restoring Oncogene-Induced Senescence

UNLABELLED MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in Eμ-Myc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of Eμ-Myc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established Eμ-Myc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes. SIGNIFICANCE This work provides novel insights into the requirements for MYC-induced oncogenesis by showing that mTORC1 activity is necessary to bypass senescence during transformation of B lymphocytes. Furthermore, tumor eradication through senescence elicited by targeted inhibition of mTORC1 identifies a previously uncharacterized mechanism responsible for significant anticancer activity of rapamycin analogues and serves as proof-of-concept that senescence can be harnessed for therapeutic benefit

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

UNLABELLED 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.

AKT signalling is required for ribosomal RNA synthesis and progression of Eμ-Myc B-cell lymphoma in vivo.

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.

Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible “exonswitch” approach to knock in the constitutively active Pik3caH1047R mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3caH1047R mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild‐type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3caH1047R mutant mice died earlier (median survival 46.5 d post‐mutation induction) than wild‐type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.—Kinross, K. M., Montgomery, K. G., Mangiafico, S. P., Hare, L. M., Kleinschmidt, M., Bywater, M. J., Poulton, I. J., Vrahnas, C., Henneicke, H., Malaterre, J., Waring, P. M., Cullinane, C., Sims, N. A., McArthur, G. A., Andrikopoulos, S., Phillips, W. A. Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly. FASEB J. 29, 1426‐1434 (2015). www.fasebj.org

Genomic characterisation of Eμ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene

The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.

Abstract PR15: Inhibition of RNA Polymerase I as a therapeutic strategy for cancer-specific activation of p53

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 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.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.