Caroline Ho

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 phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures

The phosphatidylinositol 3-kinase (PI3K) pathway is activated in many human tumors and mediates processes such as cell proliferation, survival, adhesion, and motility. The natural product, wortmannin, has been widely used to study the functional consequences of PI3K inhibition in both normal and transformed cells in culture but is not a suitable cancer chemotherapeutic agent due to stability and toxicity issues. PX-866, an improved wortmannin analogue, displays significant antitumor activity in xenograft models. Here, we directly compare PX-866 and wortmannin in human cancer cell lines cultured in monolayer or as three-dimensional spheroids. Both PI3K inhibitors failed to inhibit monolayer cell growth at concentrations up to 100 nmol/L but strongly suppressed spheroid growth at low nanomolar concentrations, with PX-866 showing greater potency than wortmannin. Relative to wortmannin, PX-866 treatment results in a more sustained loss of Akt phosphorylation, suggesting that the increased potency of PX-866 is related to a more durable inhibition of PI3K signaling. PX-866 and wortmannin both inhibit spheroid growth without causing cytotoxicity, similar to known cytostatic agents, such as rapamycin. PX-866 also inhibits cancer cell motility at subnanomolar concentrations. These findings suggest that the antitumor activities of PX-866 stem from prolonged inhibition of the PI3K pathway and inhibition of cell motility. In addition, we propose that the use of three-dimensional tumor models is more predictive of in vivo growth inhibition by PI3K inhibitors in cancer cell lines lacking phosphatase and tensin homologue activity or expression. [Mol Cancer Ther 2007;6(9):2505–14]

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.

Novel potent pyrimido(4,5-c)quinoline inhibitors of protein kinase CK2: SAR and preliminary assessment of their analgesic and anti-viral properties

We describe the discovery of novel potent substituted pyrimido[4,5-c]quinoline ATP-competitive inhibitors of protein kinase CK2. A binding model of the inhibitors with the protein was elaborated on the basis of SAR and revealed various modes of interaction with the hinge region. Representative analog 14k (CK2 IC(50)=9 nM) showed anti-viral activity at nanomolar concentrations against HIV-1. Orally available compound 7e (CK2 IC(50)=3 nM) reduced pain in the phase II of a murine formalin model. These preliminary data confirm that properly optimized CK2 inhibitors may be used for anti-viral and pain therapy.

Abstract 5494: CX-4945, an inhibitor of protein kinase CK2, potentiates the antitumor activity of platinum chemotherapy in models of ovarian cancer by preventing phosphorylation of XRCC1 and MDC1 and disrupting DNA damage repair

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

Abstract 2560: The CK2 inhibitor CX-4945 enhances the antitumor activity of EGFR-targeted agents by attenuating signaling in the PI3K/AKT/mTOR pathway

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The epidermal growth factor receptor (EGFR) regulates several oncogenic signaling pathways including PI3K/AKT/mTOR, MEK/ERK, and STAT. A number of EGFR antagonists have been approved for treatment of late-stage tumors of epithelial origin. Despite these advances, there are several limitations to these therapies, including primary and acquired resistance, prompting the need to combine EGFR antagonists with agents that target pathways downstream of EGFR. CK2 modulates multiple pro-proliferative and pro-survival signals through many of these same signaling pathways, and co-overexpression of EGFR and CK2 has been frequently observed in solid tumors. Here we present the combination of EGFR-targeted agents with CX-4945, a first-in-class selective CK2 inhibitor currently under evaluation in a Phase I clinical trial. CK2 phosphorylates several key proteins at multiple levels within the PI3K/AKT/mTOR signaling pathway including AKT, PTEN, and p70S6K1. CK2 also regulates the Hsp90/Cdc37 machinery, whose clients include EGFR, AKT, and Src. We proposed that top-down inhibition of EGFR, combined with lateral inhibition of the PI3K/ATK/mTOR pathway by CX-4945, would result in an improved cancer therapy compared to EGFR antagonism alone. This hypothesis was tested in vitro and in vivo in A431 squamous cell carcinoma (SCC) and non-small cell lung carcinoma (NSCLC) models of various genetic backgrounds. Signaling pathways and induction of apoptosis were analyzed by western blot and cell proliferation was measured in a 96-hour cell viability assay. The combination of CX-4945 and erlotinib resulted in enhanced reduction in phosphorylation of AKT (T308), AKT (S473), PRAS40 (S246), mTOR (S2481), mTOR (S2448), p70S6K1 (T389), S6 (S235/6), S6 (240/4), and 4E-BP1 (T37/46), and decreased Mcl-1 levels. These effects were accompanied by decreased cell proliferation and synergistic induction of apoptosis. CX-4945 plus erlotinib exhibited enhanced antitumor activity in A431 SCC and NCI-H2170 NSCLC xenograft models; moreover, in the erlotinib-resistant NCI-H1975 NSCLC xenograft model, CX-4945 plus cetuximab resulted in enhanced efficacy. These data suggest that CX-4945 in combination with EGFR-targeted agents may improve clinical outcomes in patients with EGFR and CK2-driven cancers by inhibiting multiple nodes of the EGFR signaling pathway. 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 2560. doi:10.1158/1538-7445.AM2011-2560

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