Jennifer R. Devlin

Combined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC-driven B-cell lymphomas.

Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.

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.

Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

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.

The Dual Inhibition of RNA Pol I Transcription and PIM Kinase as a New Therapeutic Approach to Treat Advanced Prostate Cancer

Purpose: The MYC oncogene is frequently overexpressed in prostate cancer. Upregulation of ribosome biogenesis and function is characteristic of MYC-driven tumors. In addition, PIM kinases activate MYC signaling and mRNA translation in prostate cancer and cooperate with MYC to accelerate tumorigenesis. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer. Experimental Design: The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective, and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription, has been successfully exploited therapeutically but only in models of hematologic malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in prostate cancer cell lines, in Hi-MYC- and PTEN-deficient mouse models and in patient-derived xenografts (PDX) of metastatic tissue obtained from a patient with castration-resistant prostate cancer. Results: CX-5461 inhibited anchorage-independent growth and induced cell-cycle arrest in prostate cancer cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced TP53 expression and activity and reduced proliferation (MKI67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN-null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of prostate cancer. Conclusions: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of prostate cancer. Clin Cancer Res; 22(22); 5539–52. ©2016 AACR.

Inhibition of Pol I transcription treats murine and human AML by targeting the leukemia-initiating cell population

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.

Abstract 4809: Combination therapy targeting ribosome biogenesis and mRNA translation provides a novel and potent therapeutic approach to treat MYC-driven malignancy

MYC-driven malignancies are associated with elevated rates of ribosome biogenesis and mTORC1/eIF4E-driven protein synthesis suggesting they may be vulnerable to therapeutic strategies that target the ribosome. We investigated the therapeutic efficacy of targeting multiple nodes of the network controlling the ribosome in mouse models of MYC-driven lymphoma (Ei-Myc) and prostate cancer (HI-MYC). Simultaneous inhibition of ribosomal RNA synthesis and repression of protein translation was achieved by utilizing the novel RNA polymerase I inhibitor CX-5461 and PI3K/AKT/mTORC1 and PIM 1 signaling inhibitors. Combined inhibition of ribosome biogenesis and function significantly improved therapeutic outcomes in lymphoma and prostate cancer models. CX-5461 and Everolimus (mTORC1 inhibitor) co-treatment more than doubled the survival of Ei-Myc lymphoma-bearing mice. While both classes of inhibitor suppress rDNA transcription, they treat MYC-driven malignancy through distinct molecular mechanisms facilitating their combinatorial effects. In contrast to CX-5461, PI3K/AKT/mTOR pathway inhibitors did not activate a nucleolar stress response and p53-dependent apoptosis but instead induce B-lymphoma cell death via the upregulation of the BH3-only protein BMF (Devlin et al Cancer Discovery 2015 Oct 21. pii: CD-14-0673. [Epub ahead of print]). PIM kinase has been shown to regulate eIF4Edriven protein synthesis on prostate cancer cell lines. Co-treatment of HI-MYC mice with CX-5461 and the PIM kinase inhibitor CX-6258 reverted highly invasive disease to low-grade prostate intraepithelial neoplasia. These findings demonstrate that MYC driven tumors are addicted to multiple regulatory steps associated with ribosome synthesis and function and coordinated targeting of these addictions provides an effective new therapeutic approach to treat MYC driven cancers. Citation Format: Jennifer R. Devlin, Richard J. Rebello, Katherine M. Hannan, Carleen Cullinane, Denis Drygin, Gail P. Risbridger, Luc Furic, Ross D. Hannan, Richard B. Pearson. Combination therapy targeting ribosome biogenesis and mRNA translation provides a novel and potent therapeutic approach to treat MYC-driven malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4809.

Abstract 4355: Inhibition of RNA Polymerase I as a strategy to treat cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Abnormalities of the nucleolus, the site of transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I), have been recognized as diagnostic for cancer for more then a century. However, a critical, unresolved question has been whether the accelerated ribosome biogenesis responsible for the nucleolar changes is required for malignancy. Here we show that the PI3K/AKT pathway, deregulated in a high proportion of human tumours, is a critical regulator of ribosome biogenesis. Active AKT is sufficient to drive rRNA synthesis and ribosome biogenesis. Furthermore, AKT cooperates with c-MYC to drive these processes identifying the AKT//MYC network as a master controller of cell growth. Consistent with this concept, AKT activity is required for maximal activation of rRNA synthesis and tumour formation in the Eμ-Myc mouse model of Burkitts lymphoma ([1][1]). Our findings raise the possibility that cancers characterized by unrestrained cellular growth may be vulnerable to therapeutic strategies that target ribosome biogenesis. To directly test this hypothesis, we used a novel selective small molecule inhibitor of Pol I transcription (CX-5461) ([2][2]), to show that Pol I can be targeted in vivo to treat tumors in mouse models of lymphoma and leukemia through the activation of p53-dependent apoptosis, while sparing normal hematologic cells. Thus, selective inhibition of Pol I transcription can serve as a novel therapeutic strategy for the treatment of cancer ([3][3]). A Phase 1 trial of this first-in-class molecule begins in 2013 at the Peter MacCallum Cancer Centre for patients with haematologic malignancies. Strikingly, allosteric inhibitors of AKT suppress rRNA synthesis independent of p53 and cooperate with CX-5461 in killing Eμ-Myc lymphomas providing a clear rationale for combining these agents in future trials. Citation Format: Megan J. Bywater, Katherine M. Hannan, Gretchen Poortinga, Jennifer R. Devlin, Carleen Cullinane, Denis Drygin, William G. Rice, Daniel Von Hoff, Ricky W. Johnstone, Grant A. McArthur, Ross D. Hannan, Richard B. Pearson. Inhibition of RNA Polymerase I as a strategy to treat cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2013-4355 [1]: #ref-1 [2]: #ref-2 [3]: #ref-3

Abstract PR16: Combined inhibition of ribosome function and ribosomal RNA gene transcription cooperate to delay relapse and extend survival in MYC-driven tumors

Introduction: We recently demonstrated that transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) can be therapeutically targeted with a novel small molecule, CX-5461, to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population (Bywater et al Cancer Cell 2012; Bywater et al Nature Reviews Cancer 2013). The therapeutic effect was a consequence of nucleolar disruption and activation of ribosomal protein (Rp)-MDM2-P53 nucleolar stress response (3) leading to apoptosis. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. As our pre-clinical data indicate immense potential of Pol I targeting as a cancer therapy, we have launched a first-in-human clinical trial of CX-5461 in patients with hematological malignancies. Despite impressive initial responses with CX-5461 as a single agent, we decided to explore its combination potential. We hypothesised that simultaneously targeting the ribosome at multiple steps would reduce the instance of acquired resistance to CX-5461 and extend survival. Thus we tested pharmacological inhibitors of the PI3K/AKT/mTOR pathway in combination with CX-5461 as the former signaling molecules are known to be potent regulators of both ribosome translational activity (Jefferies et al EMBO J, 1997; Pourdehnad M et al PNAS 2013) and ribosome biogenesis (Chan et al Science Signaling 2011; Devlin et al FEBS J 2013). Experimental procedures and new data: Using the Eμ-Myc model of B-cell lymphoma we demonstrate that multiple pharmacological inhibitors of the PI3K/AKT/mTOR pathway also suppress transcription of the rRNA genes and induce cell death to a similar extent as CX-5461. Unexpectedly however, PI3K/AKT/mTOR pathway blockade is not associated with nucleolar disruption, nor activation of the Rp/MDM2/p53 nucleolar stress pathway. We demonstrate this is because inhibition of PI3K/AKT/mTOR suppresses both rRNA synthesis and ribosomal protein synthesis equally and therefore does not result in a pool of free Rps that are necessary to suppress MDM2 E3 ligase that regulates p53 stability. Instead apoptosis induced by PI3K/AKT/mTOR was associated with up-regulated expression of the pro-apoptotic BH3-only protein BMF. Furthermore, we demonstrate that combined treatment of Eμ-Myc tumour-bearing mice with CX-5461 and Everolimus delayed relapse compare to single agent and significantly extended survival of tumor bearing mice. Conclusions: These data demonstrate that dual targeting of the ribosome by selectively inhibiting Pol I transcription and by inhibition of key signaling molecules regulating ribosome synthesis and function combine to potently treat MYC driven tumours and provides a rationale to combine such drugs in the clinic for the treatment of MYC driven cancer. Moreover these data also demonstrate that MYCs control of Pol I transcription and nucleolar integrity is required for its oncogenic potential, independent of its well described function in function in controlling ribosome levels and protein translation. This abstract is also presented as poster C64. Citation Format: Ross Hannan, Jennifer Devlin, Katherine Hannan, Nadine Hein, Megan Bywater, Gretchen Poortinga, Don Cameron, Denis Drygin, Carleen Cullinane, Sean O9Brien, Grant McArthur, Richard Pearson. Combined inhibition of ribosome function and ribosomal RNA gene transcription cooperate to delay relapse and extend survival in MYC-driven tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR16.