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

Abstract

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.

Introduction

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase of the ErbB receptor family. Its activation by physiological ligands causes homo- or heterodimerization with other members of the ErbB family, resulting in activation of downstream signaling pathways including PI3K-Akt-mTOR, Ras-Raf-MEK-ERK, and STAT which regulate cellular proliferation, survival, differentiation, migration and angiogenesis. EGFR is abnormally activated in many epithelial malignancies due to gene amplification, mutations, or increased ligand production [1], [2].

Studies in non-small cell lung cancer (NSCLC), pancreatic, and colorectal cancer have shown that sensitivity to EGFR targeting therapeutics correlates with the inhibition of PI3K-Akt-mTOR signaling [3], [4]. These findings have led to the investigation of agents that target the PI3K-Akt-mTOR pathway in combination with EGFR inhibitors. Inhibitors of PI3K, Akt or mTOR have all been shown to enhance the antitumor activity of EGFR inhibitors in preclinical models of cancer [5], [6], [7]. In addition, at least one agent from each of these drug classes is currently under clinical evaluation in combination with either gefitinib or erlotinib [8], [9], [10], [11].

CK2 potentiates tumorigenesis and maintenance of the oncogenic phenotype, and has been shown to regulate EGFR activity as well as the downstream signaling pathways of EGFR through multiple mechanisms (Fig. 1) [12], [13]. CK2 phosphorylates and modulates the activity of many members of the PI3K-Akt-mTOR pathway including PTEN, p70S6K, and Akt [14], [15], [16]. CK2 also controls mTOR stability through phosphorylation of the scaffold protein TEL2 which recruits the R2TP/prefoldin-like complex and Hsp90 to mTOR [17]. Hsp90 and the Hsp90 co-chaperone Cdc37, whose clients include many members of the EGFR signaling pathway, are both direct CK2 substrates (Fig. 1) [18], [19].

Given the overlap and cooperation in pro-survival signaling between CK2 and EGFR, we hypothesized that combination of an EGFR inhibitor with an inhibitor of CK2 would result in increased killing of cancer cells compared to EGFR inhibition alone. CX-4945 is the first-in-class, clinical stage, selective, small molecule inhibitor of CK2 [20], [21]. We demonstrate that combination of CX-4945 with erlotinib results in enhanced attenuation of PI3K-Akt-mTOR signaling and induction of apoptosis at the molecular level, as well as synergistic in vitro killing of cancer cells, and an increase in in vivo antitumor efficacy in models of NSCLC and squamous cell carcinoma (SCC). Together, these findings demonstrate that inhibition of CK2 with CX-4945 can enhance the antitumor activity of EGFR inhibitors.