John K.C. Lim

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.

CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNA-targeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy.

Drug combination therapies are commonly used for the treatment of cancers to increase therapeutic efficacy, reduce toxicity, and decrease the incidence of drug resistance. Although drug combination therapies were originally devised primarily by empirical methods, the increased understanding of drug mechanisms and the pathways they modulate provides a unique opportunity to design combinations that are based on mechanistic rationale. We have identified protein kinase CK2 as a promising therapeutic target for combination therapy, because CK2 regulates not just one but many oncogenic pathways and processes that play important roles in drug resistance, including DNA repair, epidermal growth factor receptor signaling, PI3K/AKT/mTOR signaling, Hsp90 machinery activity, hypoxia, and interleukin-6 expression. In this article, we show that CX-4945, a clinical stage selective small molecule inhibitor of CK2, blocks the DNA repair response induced by gemcitabine and cisplatin and synergizes with these agents in models of ovarian cancer. Mechanistic studies show that the enhanced activity is a result of inactivation of XRCC1 and MDC1, two mediator/adaptor proteins that are essential for DNA repair and that require phosphorylation by CK2 for their function. These data position CK2 as a valid pharmacologic target for intelligent drug combinations and support the evaluation of CX-4945 in combination with gemcitabine and platinum-based chemotherapeutics in the clinical setting. Mol Cancer Ther; 11(4); 994–1005. ©2012 AACR.

Combined inhibition of EGFR and CK2 augments the attenuation of PI3K-Akt-mTOR signaling and the killing of cancer cells

Ser/Thr protein kinase CK2 regulates multiple processes that play important roles in the sensitivity of cancer to epidermal growth factor receptor targeting therapeutics, including PI3K-Akt-mTOR signaling, Hsp90 activity, and inhibition of apoptosis. We hypothesized that top-down inhibition of EGFR, combined with lateral suppression of multiple oncogenic pathways by targeting CK2, would create a pharmacologic synthetic lethal event and result in an improved cancer therapy compared to EGFR inhibition alone. This hypothesis was tested by combining CX-4945, a first-in-class clinical stage inhibitor of CK2, with the EGFR tyrosine kinase inhibitor, erlotinib, in vitro and in vivo in models of non-small cell lung carcinoma, NCI-H2170, and squamous cell carcinoma, A431. Our results demonstrate that combination of CX-4945 with erlotinib results in enhanced attenuation of the PI3K-Akt-mTOR pathway. We also observed an increase in apoptosis, synergistic killing of cancer cells in vitro, as well as improved antitumor efficacy in vivo. Taken together, these data position CK2 as a valid pharmacologic target for drug combinations and support further evaluation of CX-4945 in combination with EGFR targeting agents.

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

RNA Polymerase I Inhibition with CX‐5461 as a Novel Therapeutic Strategy to Target MYC in Multiple Myeloma

Dysregulation of MYC is frequently implicated in both early and late myeloma progression events, yet its therapeutic targeting has remained a challenge. Among key MYC downstream targets is ribosomal biogenesis, enabling increases in protein translational capacity necessary to support the growth and self‐renewal programmes of malignant cells. We therefore explored the selective targeting of ribosomal biogenesis with the small molecule RNA polymerase (pol) I inhibitor CX‐5461 in myeloma. CX‐5461 induced significant growth inhibition in wild‐type (WT) and mutant TP53 myeloma cell lines and primary samples, in association with increases in downstream markers of apoptosis. Moreover, Pol I inhibition overcame adhesion‐mediated drug resistance and resistance to conventional and novel agents. To probe the TP53‐independent mechanisms of CX‐5461, gene expression profiling was performed on isogenic TP53 WT and knockout cell lines and revealed reduction of MYC downstream targets. Mechanistic studies confirmed that CX‐5461 rapidly suppressed both MYC protein and MYC mRNA levels. The latter was associated with an increased binding of the RNA‐induced silencing complex (RISC) subunits TARBP2 and AGO2, the ribosomal protein RPL5, and MYC mRNA, resulting in increased MYC transcript degradation. Collectively, these studies provide a rationale for the clinical translation of CX‐5461 as a novel therapeutic approach to target MYC in myeloma.

Abstract C39: Phase I clinical trial of CX‐4945: A first‐in‐class orally administered small molecule inhibitor of protein kinase CK2

Introduction: CX‐4945 is a novel, orally administered small molecule designed to potently and selectively inhibit protein kinase CK2, a previously unexploited molecular target with well documented roles in many cancers. In vitro , CX‐4945 selectively kills cancer cells by modulating key survival pathways, resulting in inhibition of proliferation, promotion of apoptosis and cell cycle arrest. Pre‐clinically, CX‐4945 has demonstrated single‐agent potency in suppressing xenograft tumor growth with a wide therapeutic window. The objectives of this phase I study are to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs), to characterize the pharmacokinetics (PKs), and to study the pharmacodynamic effects of CX‐4945. Procedures: Eligible patients with advanced solid tumors, Castleman9s disease or multiple myeloma with progressive disease, or for whom there are no available standard therapies, receive CX‐4945 in successive dose cohorts at: 90, 160, 300, 460, 700 and 1000 mg per dose. Oral doses are administered twice daily for twenty‐one consecutive days of a four week cycle. Therapy is continued in consenting patients until signs of intolerance to CX‐4945 are observed, or there is evidence of advancing disease. Response by RECIST is determined after every 2 cycles. Serial blood and plasma samples are collected on the first and final dosing days of Cycle 1 (i.e., Day 1 and Day 21) for pharmacokinetic analysis and for pharmacodynamic biomarker evaluations (specifically, total and phosphorylated forms of p21 and Akt.) Results: Thirteen patients with solid tumors (3–4 patients per cohort, from four separate dose cohorts) have received oral doses of CX‐4945. These doses have been well tolerated, with no reported adverse events of grade 3 or higher. CX‐4945 has demonstrated general linearity in PK parameters between the dose cohorts, with a terminal half life of approximately 25 hours at steady state. Conclusions: To date, CX‐4945 has shown no drug related toxicity and has dose proportional PKs. No DLTs have yet been observed, and the MTD remains to be defined in this Phase I study. Further enrollment to the planned dose escalation cohorts is ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C39.

Abstract 3765: Discovery and biological characterization of CX-8184, a potent inhibitor of protein kinase CK2

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Protein kinase CK2 regulates multiple pathways that play key roles in carcinogenesis, including cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA damage repair. Overexpression of CK2 in various cancer tissues has been frequently linked to poor prognosis. All this makes targeting of CK2 highly attractive for both single agent and combination therapies. To date therapeutic targeting of CK2 proved to be challenging, with only one compound, CX-4945, entering clinical development. In phase I clinical trials CX-4945 was well tolerated, inhibited CK2 activity in PBMC, modulated the PI3K-AKT-mTOR oncogenic pathway, reduced tumor-related serum IL-6 and IL-8 biomarkers, resulted in stable disease for at least 16 weeks in 20% of treated patients, and caused single agent tumor shrinkage. Building on these successes with the first generation CK2 inhibitor, we sought to design a second generation CK2 inhibitor with distinct pharmacologic properties. The application of a focused screening cascade and iterative medicinal chemistry efforts generated CX-8184, a selective single digit nanomolar inhibitor of the CK2 enzyme that demonstrated desirable biologic properties. When evaluated against a panel of 32 cancer cell lines, CX-8184 demonstrated broad antiproliferative activity with median IC50 = 145 nM. CX-8184 suppressed NF-κB, Hif-1α and β-catenin driven transcription at concentrations of 100-300 nM, and inhibited IL-6 expression with IC50 = 30 nM. Since CK2 is known to potentiate AKT signaling, treatment of cancer cells with CX-8184 decreased phosphorylation of AKT, as well as its substrates. The Hsp90/Cdc37 machinery is regulated by CK2 and plays an important role in activation of various kinases, including members of the ErbB RTK family. CX-8184 rapidly suppressed phosphorylation of Cdc37 by CK2, leading to the deactivation of ErbB family members. In combination with erlotinib or lapatinib, CX-8184 produced synergistic killing of lung cancer and breast cancer cells, respectively. CX-8184 also inhibited the phosphorylation of XRCC1, a scaffold protein involved in DNA strand break repair that plays an important role in sensitivity of cancer cells to DNA damaging agents. Consequently, the treatment of ovarian cancer cells pre-challenged with gemcitabine and/or cisplatin with 300 nM CX-8184 led to synergistic induction of cell death. In vivo, CX-8184 demonstrated favorable PK properties with oral bioavailability and a half-life amenable to once daily dosing in mice. CX-8184 exhibited potent anti-tumor activity and was well tolerated in A431 xenograft model. The compound has also showed an acceptable pharmacological profile as evidenced by CYP, hERG and Ames testing. The pharmacokinetic and pharmacological properties and the biological activity of CX-8184, both as single agent or in combination, support its further development as a preclinical candidate.9 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3765. doi:1538-7445.AM2012-3765

Abstract 2763: Utility of clinical biomarkers for detecting protein kinase CK2 inhibition: A report from the phase I trial of CX-4945

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: CX-4945 is a first-in-class, orally administered small molecule that potently and selectively inhibits protein kinase CK2, a previously unexploited molecular target with well documented roles in many cancers. In vitro, CX-4945 selectively kills cancer cells by modulating key survival pathways, resulting in cell cycle arrest, inhibition of cell proliferation, and promotion of apoptosis. CK2 hyperactivation of the PI3K/Akt pathway promotes the phosphorylation of several proteins which we have exploited as biomarkers for CX-4945 activity. A validated laser scanning cytometry method has been developed to quantify the phosphorylation of p21 and Akt in cells, and this has been employed to characterize these substrates in circulating blood cells collected from patients in the phase I clinical trial of CX-4945. Procedures: Eligible patients with advanced solid tumors with progressive disease, or for whom there are no available standard therapies, were administered CX-4945 in successive dose cohorts using a standard 3×3 design at: 90, 160, 300, 460, and 700 mg per dose. Oral doses were administered twice daily for twenty-one consecutive days of a four week cycle. Serial plasma samples were collected for pharmacokinetic analysis on the first and final dosing days of Cycle 1 (i.e., Day 1 and Day 21). In addition, whole blood samples were collected at pre-treatment, 4 hours and 8 hours following the first dose of CX-4945 on Day 1 and Day 21, and peripheral blood mononuclear cells (PBMCs) were isolated for pharmacodynamic biomarker evaluations (specifically, total and phosphorylated forms of p21 and Akt). Plasma samples were also collected at these time points for quantification of plasma IL-6. Results: Seventeen patients with advanced solid tumors from five separate dose cohorts have received oral doses of CX-4945 to date, and all patients in the study participated in the collection of PBMCs. Biomarkers from patients in Cohorts 3 and 4 demonstrated changes in their profile consistent with the inhibition of CK2. CX-4945 displays general linearity in PK parameters between the dose cohorts, with a terminal half life of approximately 25 hours at steady state. Conclusions: To date, no DLTs have yet been observed, and the MTD remains to be defined in this Phase I study. Early signals suggest a modulation of the biomarker profile consistent with CK2 inhibition. Patient enrollment will continue, to further characterize CX-4945s safety, tolerance, pharmacokinetics and pharmacodynamic effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2763.

Phase I clinical trial of quarfloxin administered weekly×3 of a four week cycle

14667 Background: Quarfloxin (Q) is a novel small molecule specifically designed to induce apoptosis in cancer cells by disrupting an essential protein:rDNA quadruplex complex. Pharmacokinetic para...