CX-4945, a Selective and Orally Bioavailable Inhibitor of Protein Kinase CK2 Inhibits PI3K/Akt, JAK-STAT and NF-κB Signaling and Induces Apoptosis In Multiple Myeloma Cells

Abstract 787

Multiple myeloma (MM) is an incurable malignancy characterized by infiltration of malignant plasma cells in the bone marrow. There are many subtypes of MM each of which relies on distinct oncogenic pathways. Oncogene addiction has been regarded as an Achilles' heel of cancer and has consequently been the focus of extensive efforts to design molecularly targeted anticancer therapies based on the premise that silencing an oncogene's expression will prove lethal in certain cancers. However, recent evidence suggests that MM may have a fatal vulnerability that is better described as non-oncogene addiction. Non-oncogenes do not directly transform cells but rather encode for normal cellular proteins that become overexpressed or otherwise deregulated in order to maintain oncogenic signaling. Protein kinase CK2 represents the prototypical non-oncogene. CK2 activity and expression levels are elevated in many cancers and it has been reported that MM cells rely on elevated CK2 for survival. CK2 is a highly conserved, constitutively active serine/threonine protein kinase that regulates multiple pathways important in MM including PI3K/AKT, JAK-STAT and NF-κB signaling cascades. Further, CK2 is elevated under hypoxic conditions regulating hypoxia-inducible transcription factor 1 alpha (HIF-1α) activity and other mediators of angiogenesis that are known to play an important role in MM pathogenesis. Given the generalized overexpression of CK2 in cancers, the central role of CK2 in multiple pro-survival pathways and the evidence that CK2 is an essential protein for MM cell survival, the development of selective CK2 inhibitors has emerged as an attractive targeted approach for the treatment of MM. Herein we describe CX-4945, a selective, orally bioavailable, small molecule inhibitor of CK2. CX-4945 is a potent, ATP-competitive inhibitor of both isoforms of the CK2 catalytic subunits CK2α and CK2α' with IC₅₀ values of 1 nM. In a panel of 13 MM cell lines, CX-4945 exhibited a range of antiproliferative activity in the low micromolar range as measured by CellTiter-Glo® Luminescent Cell Viability Assay. Molecular characterization of the MM cell line panel revealed that on average CK2α mRNA levels and CK2α protein levels were elevated 9.5-fold and 14-fold, respectively, compared to normal CD138+ plasma cells. CX-4945 effectively reduced CK2 enzymatic activity in the MM cell lines. In immunoblots, the attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific Ser129 site as well as the Ser473 and Thr308 sites. Further evidence of diminished Akt activity was demonstrated by decreased phosphorylation of the downstream Akt target protein p21 at Thr145. An additional biochemical consequence of CK2 inhibition was noted as CX-4945 selectively reduced phosphorylation of the p65 subunit of NF-κB at the CK2-specific Ser529 phosphorylation site but not at the S536 site. Moreover, CX-4945 inhibited the JAK/STAT pathway as demonstrated by the reduction in phosphorylation of regulatory sites on STAT3 (Tyr705) and JAK2 (Tyr1007/1008). PCR Arrays were used to analyze the global effect of CK2 inhibition on the NFκB and JAK/STAT mediated signal transduction pathways. Several key mediators, e.g. IL-6, CyclinD1, MYC, STAT1, STAT3, IFNγ, and IL-1R, were significantly downregulated upon treatment with CX-4945. Furthermore, CX-4945 reduced IL-6 secretion in a concentration dependent manner. In culture, MM cell lines have adapted to maintain HIF1α protein expression. CX-4945 effectively inhibited HIF1α expression and a well-known HIF1α transcriptional target, VEGF. Inhibition of CK2 by CX-4945 induced apoptosis in MM cells as shown by an increase in Caspase 3/7 activity and PARP cleavage. Interestingly, CX-4945 failed to induce apoptosis and had no effect on viability in the normal human-derived stromal cell line HS-5. These data lend further support to the CK2 non-oncogene addiction hypothesis in MM. A Phase 1 clinical trial evaluating CX-4945 as a single agent in solid tumors has already established a favorable pharmacokinetic, pharmacodynamic and safety profile. These findings, coupled with our preclinical data, suggest CX-4945 is a promising therapeutic agent for targeting aberrant CK2 activity in MM and a Phase 1 clinical trial in MM is underway.