Adam Siddiqui-Jain

Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription

The nuclease hypersensitivity element III1 upstream of the P1 promoter of c-MYC controls 85–90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

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.

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.

Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer.

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 CK2s 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.

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.

Discovery of CX-6258. A Potent, Selective, and Orally Efficacious pan-Pim Kinases Inhibitor.

Structure-activity relationship analysis in a series of 3-(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified compound 13, a pan-Pim kinases inhibitor with excellent biochemical potency and kinase selectivity. Compound 13 exhibited in vitro synergy with chemotherapeutics and robust in vivo efficacy in two Pim kinases driven tumor models.

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.

Discovery of CX-5461, the First Direct and Selective Inhibitor of RNA Polymerase I, for Cancer Therapeutics

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.