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.

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.

Hallmark deregulated signaling in cancer cells drives exces-

Malignant transformation and maintenance of the malignant phenotype depends on oncogenic and nononcogenic 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 CK2a 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-1a) 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. Cancer Res; 70(24); 10288-98. Ó2010 AACR.

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2's small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K(i) = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.

Accelerated proliferation of solid tumor and hematologic cancer cells is linked to accelerated transcription of rDNA by the RNA polymerase I (Pol I) enzyme to produce elevated levels of rRNA (rRNA). Indeed, upregulation of Pol I, frequently caused by mutational alterations among tumor suppressors and oncogenes, is required for maintenance of the cancer phenotype and forms the basis for seeking selective inhibitors of Pol I as anticancer therapeutics. 2-(4-Methyl-[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c]fluorene-6-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)-amide (CX-5461, 7c) has been identified as the first potent, selective, and orally bioavailable inhibitor of RNA Pol I transcription with in vivo activity in tumor growth efficacy models. The preclinical data support the development of CX-5461 as an anticancer drug with potential for activity in several types of cancer.

Two types of tetracycline-controlled inducible RNAi expression systems have been developed that generally utilize multiple tetracycline operators (TetOs) or repressor fusion proteins to overcome the siRNA leakiness. Here, we report a novel system that overexpresses the tetracycline repressor (TetR) via a bicistronic construct to control siRNA expression. The high level of TetR expression ensures that the inducible promoter is tightly bound, with minimal basal transcription, allowing for regulation solely dependent on TetR rather than a TetR fusion protein via a more complicated mechanism. At the same time, this system contains only a single TetO, thus minimizing the promoter impairment occurring in existing systems due to the incorporation of multiple TetOs, and maximizing the siRNA expression upon induction. In addition, this system combines all the components required for regulation of siRNA expression into a single lentiviral vector, so that stable cell lines can be generated by a single transduction and selection, with significant reduction in time and cost. Taken together, this all-in-one lentiviral vector with the feature of TetR overexpression provides a unique and more efficient tool for conditional gene knockdown that has wide applications. We have demonstrated the high degree of robustness and versatility of this system as applied to several mammalian cells and xenograft animals.

Platinum-based chemotherapeutics are commonly used to treat solid tumors but may be restricted in their application by dose limiting toxicity and inherent or acquired resistance. Because efficient DNA damage repair mechanisms contribute to resistance, targeting components of the repair machinery has emerged as a strategy to increase the effectiveness of these and other DNA-damaging anti-cancer drugs. Protein kinase CK2 is a serine/threonine kinase that has emerged as an attractive molecular target due to its overexpression in cancer and regulatory role in key cellular processes including the cell cycle, apoptosis and DNA damage repair. Multiple lines of evidence suggest an increasingly important role for CK2 in the DNA damage response, including the phosphorylation and activation of the mediator/adaptor proteins XRCC1 and MDC1. XRCC1 is an essential component for nucleotide excision repair which is the major repair pathway responsible for removing platinum adducts. MDC1 is the predominant γ-H2AX recognition factor in mammalian cells and is essential for homologous recombination repair. CX-4945 is a first-in-class, selective, oral inhibitor of CK2 currently in Phase 1 clinical trials. We sought to determine if inhibiting CK2 activity with CX-4945 would prevent phosphorylation of XRCC1 and MDC1 and potentiate the activity of platinum-based drugs by preventing DNA damage repair. Treatment of the ovarian cancer cell lines A2780 and SKOV3 with CX-4945 led to decreased phosphorylation of XRCC1 at the CK2 specific phosphorylation sites and reduced total XRCC1 protein levels. Likewise, immunoprecipitation of MDC1 from SKOV3 cells treated with CX-4945 revealed significant reductions in phosphorylation at the CK2 specific sites, while in A2780 cells MDC1 protein levels were decreased. The reduction of MDC1 protein levels was reproduced by CK2 siRNA, confirming that CK2 activity supports MDC1 expression levels. Combined treatment of A2780 cells with CX-4945 and cycloheximide revealed a faster rate of MDC1 degradation than with cycloheximide alone but did not affect MDC1 mRNA levels, indicating that CK2 regulates MDC1 protein stability. When combined with cisplatin, CX-4945 enhanced the activation of CHK2 and increased levels of γ-H2AX and cleaved PARP. In antiproliferative experiments, CX-4945 exhibited synergy with cisplatin in A2780 and SKOV3 cell lines. The combination of CX-4945 with cisplatin or carboplatin significantly enhanced antitumor activity in ovarian xenograft models and was well tolerated. Thus, the inhibition of CK2 by CX-4945 enhanced the antitumor activity of platinum agents by preventing DNA damage repair and inducing apoptosis. These data provide compelling preclinical support for pursuing CX-4945 in combination with platinum chemotherapy in the clinic. 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 5494. doi:10.1158/1538-7445.AM2011-5494