Choline kinase alpha-Putting the ChoK-hold on tumor metabolism

Abstract

It is well established that lipid metabolism is drastically altered during tumor development and response to therapy. Choline kinase alpha (ChoKα) is a key mediator of these changes, as it represents the first committed step in the Kennedy pathway of phosphatidylcholine biosynthesis and ChoKα expression is upregulated in many human cancers. ChoKα activity is associated with drug resistant, metastatic, and malignant phenotypes, and represents a robust biomarker and therapeutic target in cancer. Effective ChoKα inhibitors have been developed and have recently entered clinical trials. ChoKα's clinical relevance was, until recently, attributed solely to its production of second messenger intermediates of phospholipid synthesis. The recent discovery of a non-catalytic scaffolding function of ChoKα may link growth receptor signaling to lipid biogenesis and requires a reinterpretation of the design and validation of ChoKα inhibitors. Advances in positron emission tomography, magnetic resonance spectroscopy, and optical imaging methods now allow for a comprehensive understanding of ChoKα expression and activity in vivo. We will review the current understanding of ChoKα metabolism, its role in tumor biology and the development and validation of targeted therapies and companion diagnostics for this important regulatory enzyme. This comes at a critical time as ChoKα-targeting programs receive more clinical interest.

Keywords: Choline kinase; Inhibitors; Lipid metabolism; Tumor metabolism.

Fig. 1

Choline kinase plays a central role in phospholipid metabolism. PtdCho is synthesized in most cells via the Kennedy pathway, where choline (top left) is phosphorylated by choline kinase. DAG is then added via a CTP-mediated two-step mechanism. PtdCho can be metabolized at a variety of cleavage sites by the phospholipase enzymes. The products of both the PtdCho catabolic and anabolic steps have important signaling functions, which can be dysregulated in diseased tissue.

Fig. 2

The metabolic roles of choline. Choline (left box) can be oxidized to betaine, acetylated to acetylcholine, or phosphorylated to PC (right box). PC can be acetylated and serve as the head-group in many glycerophospholipids (e.g. PAF, PtdCho) or sphingolipids (e.g. sphingomyelin). The fate of choline and PC varies depending of tissue type and cell status.

Fig. 3

ChoKα phosphorylates choline via an unusual ping-pong mechanism. Unprotonated Asp306 at the active site of human ChoKα can accept a phosphate group by a Mg²⁺-coordinated reaction resulting in protonation of the amino acid and subsequent ejection of ADP. The phosphate-primed enzyme can accept choline, which induces a conformational shift in the enzyme resulting in PC exit. The deprotonated ChoKα enzyme reverts to its original conformation and is again ready for ATP binding. Adapted with permission from Hudson et al., 2013 [49].

Fig. 4

ChoKα active site. (a) A surface representation of ChoKα with PC bound at the active site was colored corresponding to the electrostatic potential of the regional amino acid residues (red = −15 kT/e; blue = 15 kT/e). (b) A closer view of the choline-binding site was color-coded with hydrophobic regions in yellow and negatively charged regions in red. This view demonstrates the hydrophobic pocket and anionic rim of this atypical kinase active site. Figure adapted with permission from Malito et al. [50].

Fig. 5

Symmetric ChoKα inhibitors. The general structure of each symmetric ChoKα inhibitor class (left column) is presented with a representative compound (right column) from that class. Hemicholinium-3 is the prototypical choline mimetic, but lacks specificity for ChoKα. MN58b and TCD-717 are highly selective analogs of hemicholinium-3 with promising antiproliferative properties. JAS239 is a near infrared fluorescent choline mimetic developed as an optical imaging contrast agent for cancer applications.

Fig. 6

Asymmetric ChoKα inhibitors. A rational design approach to improve potency involved the addition of adenine moieties to MN58b (ATP-MN58b conjugate) in an effort to bind the choline and ATP binding sites simultaneously. Sahún-Roncero et al. [126] used an MN58b linker group in a mixed ATP-MN58b inhibitor (Strategy 1), while Trousil et al. [124] independently found a 12-carbon linkage (ATP-MN58b Strategy 2) was capable of inhibition in vitro. Next generation ChoKα inhibitors were developed using computational (CK37) and compound library (V-11-023,907 and V-11-0711) screening approaches.

Fig. 7

Choline metabolite accumulation as a biomarker in molecular imaging modalities. (a) Single voxel MR spectra acquired for a glioblastoma multiforme tumor or contralateral normal tissue show elevated tCho and diminished N-acetylaspartate (NAA) in the cancer tissue. (b) Gadolinium-enhanced T₁-weighted MRI image shows location of the glioblastoma multiforme tumor in the corpus callosum and contralateral normal region, with spectral regions demarcated by the right and left voxels respectively. (c) Chemical shift imaging at 3.2 ppm gives a map of choline distribution (Cho) and demonstrates elevated tCho in the tumor region. Figure adapted with permission from Horská and Barker, 2010 [150]. (d) PET scans of a patient with Grade IV glioblastoma acquired 5 and 20 min after administration of either ¹⁸F- or ¹¹C-choline (right) demonstrates comparable tumor avidity between the two tracers (Figure adapted with permission from Hara, et al., 2003 [161]). (e) An athymic nude mouse with a 9 L glioma xenograft was injected i.v. with 20 nmol JAS239, and NIR optical images were acquired after 4 h using a LiCOR Pearl (Ex 710 nm, Em 800 nm), revealing significant tumor accumulation (unpublished results).