William G. Rice

Anticancer Activity of CX-3543: A Direct Inhibitor of rRNA Biogenesis

Hallmark deregulated signaling in cancer cells drives excessive ribosome biogenesis within the nucleolus, which elicits unbridled cell growth and proliferation. The rate-limiting step of ribosome biogenesis is synthesis of rRNA (building blocks of ribosomes) by RNA Polymerase I (Pol I). Numerous kinase pathways and products of proto-oncogenes can up-regulate Pol I, whereas tumor suppressor proteins can inhibit rRNA synthesis. In tumorigenesis, activating mutations in certain cancer-associated kinases and loss-of-function mutations in tumor suppressors lead to deregulated signaling that stimulates Pol I transcription with resultant increases in ribosome biogenesis, protein synthesis, cell growth, and proliferation. Certain anticancer therapeutics, such as cisplatin and 5-fluorouracil, reportedly exert, at least partially, their activity through disruption of ribosome biogenesis, yet many prime targets for anticancer drugs within the ribosome synthetic machinery of the nucleolus remain largely unexploited. Herein, we describe CX-3543, a small molecule nucleolus-targeting agent that selectively disrupts nucleolin/rDNA G-quadruplex complexes in the nucleolus, thereby inhibiting Pol I transcription and inducing apoptosis in cancer cells. CX-3543 is the first G-quadruplex interactive agent to enter human clinical trials, and it is currently under evaluation against carcinoid/neuroendocrine tumors in a phase II clinical trial.

CX-4945, an Orally Bioavailable Selective Inhibitor of Protein Kinase CK2, Inhibits Prosurvival and Angiogenic Signaling and Exhibits Antitumor Efficacy

Malignant transformation and maintenance of the malignant phenotype depends on oncogenic and non-oncogenic proteins that are essential to mediate oncogene signaling and to support the altered physiologic demands induced by transformation. Protein kinase CK2 supports key prosurvival signaling pathways and represents a prototypical non-oncogene. In this study, we describe CX-4945, a potent and selective orally bioavailable small molecule inhibitor of CK2. The antiproliferative activity of CX-4945 against cancer cells correlated with expression levels of the CK2α catalytic subunit. Attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific S129 site and the canonical S473 and T308 regulatory sites. CX-4945 caused cell-cycle arrest and selectively induced apoptosis in cancer cells relative to normal cells. In models of angiogenesis, CX-4945 inhibited human umbilical vein endothelial cell migration, tube formation, and blocked CK2-dependent hypoxia-induced factor 1 alpha (HIF-1α) transcription in cancer cells. When administered orally in murine xenograft models, CX-4945 was well tolerated and demonstrated robust antitumor activity with concomitant reductions of the mechanism-based biomarker phospho-p21 (T145). The observed antiproliferative and anti-angiogenic responses to CX-4945 in tumor cells and endothelial cells collectively illustrate that this compound exerts its antitumor effects through inhibition of CK2-dependent signaling in multiple pathways. Finally, CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to advance into human clinical trials, thereby paving the way for an entirely new class of targeted treatment for cancer.

The RNA Polymerase I Transcription Machinery: An Emerging Target for the Treatment of Cancer

The RNA polymerase I (Pol I) transcription machinery in the nucleolus is the key convergence point that collects and integrates a vast array of information from cellular signaling cascades to regulate ribosome production that in turn guides cell growth and proliferation. Cancer cells commonly harbor mutations that inactivate tumor suppressors, hyperactivate oncogenes, and upregulate protein kinases, all of which promote Pol I transcription and drive cell proliferation. The intimate balance between Pol I transcription and growth-factor signaling is perturbed in cancer cells, indicating that upregulation of rRNA synthesis is mandatory for all tumors. Though the emerging picture of transcriptional regulation reveals an unexpected level of complexity, we are beginning to understand the multiple links between rRNA biogenesis and cancer. In this review, we discuss experimental data and potential strategies to downregulate rRNA synthesis and induce an antiproliferative response in cancer cells.

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.

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.

Abstract 4511: CX-5461, a non-genotoxic activator of p53 through selective inhibition of RNA polymerase I for the treatment of hematological cancers

Cancer is characterized by hyperactivation of ribosome biogenesis which depends on increased RNA Polymerase I (Pol I) transcription. Inhibition of Pol I transcription causes nucleolar stress that leads to the release of ribosomal proteins from the nucleolus into the nucleoplasm where they can sequester the p53 inhibitory protein MDM2, causing activation of p53 and induction of apoptosis. The inhibition of Pol I transcription as a therapeutic approach is significant because it impacts two critically balanced processes, proliferation and apoptosis, that regulate cancer cell survival. CX-5461 is a potent and selective inhibitor of Pol I transcription. CX-5461 acts at the initiation stage of Pol I transcription through the disruption of the SL1/rDNA complex. CX-5461 is non-genotoxic and does not inhibit DNA replication, protein translation or RNA Polymerase II transcription. We have previously demonstrated that CX-5461 triggers autophagic cell death in solid tumor cell lines and exhibits antitumor activity in xenograft models, highlighting the importance of Pol I transcription in cancer (Drygin et al. Cancer Res. in press). In preparation for clinical testing we sought to identify the most sensitive indications by evaluating CX-5461 against a panel of genetically diverse cancer cell lines. CX-5461 exhibited a broad range of antiproliferative activity with wild-type (wt) p53 cells derived from hematological malignancies being the most sensitive (median IC50 = 5 nM). Other cell types, i.e. p53 mutated hematological, p53wt and p53 mutated solid tumors were less sensitive to CX-5461 (median IC50s = 94, 164 and 265 nM respectively). In contrast, the median IC50 against normal cells was 5 uM indicating that CX-5461 selectively kills cancer cells. Further molecular characterization revealed that CX-5461 treatment of p53wt hematologic cancer cells inhibited rRNA synthesis, stabilized p53, activated p21, caused cell cycle arrest and induced apoptosis. Chemical inhibition of p53 prevented the induction of apoptosis indicating that CX-5461 acts through activation of p53. Interestingly, while p53 mutations impacted the activity of CX-5461, other genetic alterations known to silence p53 response, i.e. deletion of ARF, did not affect sensitivity to CX-5461 (Bywater et al. 2010 AACR Ann Met Proceedings). Activation of p53 has long been considered an attractive approach for treating cancers because of the surveillance function of p53 to remove abnormal cells via induction of apoptotic cell death. The fact that mutations or deletions of the p53 gene are relatively rare in hematological malignancies, coupled with our data that p53wt hematologic cancer cells are particularly sensitive to CX-5461 provides compelling rationale for evaluating CX-5461 in this patient population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2011-4511

Abstract C198: CX‐4945, a novel small molecule inhibitor of CK2 protein kinase, reduces hyperactivated Akt signaling and synergizes with Akt inhibitors in breast cancer cells

Akt, a critical protein kinase in the PI3K signaling pathway regulates multiple biological processes that are important in tumorigenesis. This “Master Regulator Kinase” is often hyperactivated in cancer through various mechanisms, including mutations or deletions in Akt, PI3K or PTEN tumor suppressor. The last decade witnessed the emergence of another “Master Regulator Kinase” ‐ CK2. Like Akt, CK2 controls the growth, proliferation and survival of cancer cells. Ironically, CK2 regulates Akt through phosphorylation and down regulation of PTEN and via direct and specific phosphorylation of Akt at Ser129. This phosphorylation event by CK2 further stimulates the activity of Akt, thereby enhancing the “driver effect” of Akt in promoting oncogenic signaling. Unlike Akt, CK2 does not require phosphorylation for activation but rather its activity appears to be regulated through expression levels. Due to the unique shape of the ATP‐binding site and an incomplete understanding of the regulation of CK2 expression, pharmacological targeting of CK2 has proven to be very challenging. To date only one small molecule inhibitor of CK2, CX‐4945, has advanced into clinical development. Herein, we describe the pharmacological characterization of CX‐4945, its impact on Akt signaling and implications for combination therapies. The high incidence of abnormalities found in the PI3K pathway in breast cancers and the distinct roles that CK2 and Akt play in this disease have made it an attractive tumor type to study the effects of CX‐4945. A cell viability screen of 16 diverse but genetically well‐characterized breast cancer lines revealed that cells carrying mutations causing Akt‐activation were significantly more sensitive to CX‐4945 than those that did not. Western blot analyses of these cell lines demonstrated good correlation between the phosphorylation of Akt at Ser129 and expression of catalytic subunits of CK2. Treatment with CX‐4945 resulted in dramatic reductions of phosphorylation of Akt at Ser129 and reductions in phosphorylation of the downstream targets of Akt, e.g. p21. Upon combination of CX‐4945 with inhibitors that targeted the PI3K/Akt pathway, we observed synergistic antiproliferative activity in breast cancer cells. Thus, evaluation of the effects of CX‐4945 on the Akt pathway in breast cancer cell lines may allow for the identification of patient populations more sensitive to CX‐4945 and guide the selection of more effective combination therapies for cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C198.