Abstract 3236: Small molecule inhibitors of choline kinase lead to reduced phosphocholine levels and induction of apoptosis in cancer cells
Authors: Squillace Rachel M. , Zech Stephan , Li Feng , Kohlmann Anna , Miller David , Greenfield Matthew , Ning Yaoyu , Commodore Lois , Miret Juan , Wang Frank , Zhou Tianjun , Wang Yihan , Thomas Matthew , Qi Jiwei , Xu Yongjin , Rivera Victor , Clackson Tim , Zhu Xiaotian , Shakespeare William , Delgarno David 
Published: August 2012

Abstract
Choline Kinase (ChoK) catalyzes the synthesis of phosphocholine (pCho) as the first step in the Kennedy pathway towards synthesis of the major membrane phospholipid, phosphatidylcholine. Increased phosphorylation of choline is a hallmark of the malignant phenotype and ChoK over-expression (primarily ChoKβ) has been reported in a variety of human cancers including breast, lung, colorectal and prostate. These observations have recently motivated efforts to develop anti-cancer agents targeting ChoK. Here we summarize our efforts to further validate ChoKα as an oncogenic target by characterizing its tumorigenic potential and exploring the activity of novel small molecule inhibitors. We transduced the ChoKα gene into HEK293 cells to examine the effects of ChoK expression in vitro and in vivo. Under reduced serum conditions, over-expression of ChoK promoted cell growth, increased phospho-ERK and phospho-AKT levels, and reduced p21 levels. ChoKα, but not vector-expressing cells, formed tumors in immune-compromised mice and ChoKα expression levels were positively associated with tumor growth rates. Together, these data suggest that ChoK maintains proliferative pathways in the absence of growth factors, and itself provides an oncogenic driver capable of inducing tumor growth in the absence of other transforming mutations. Recently we identified a novel chemical series that inhibits ChoKα in both enzymatic and cellular assays. The binding of these inhibitors to ChoK protein was confirmed in surface plasmon resonance experiments. A representative member of this lead series, compound A, was characterized in more detail and demonstrated potent enzyme inhibition against ChoKα with an IC50 of 70 nM. Compound A also inhibited the growth of ChoKα -expressing breast cancer lines, MDA-MB-468 and MDA-MB-415, with GI50s of 7 and 2 uM respectively. In contrast, compound A exhibited much lower activity against the non-transformed breast epithelial cell line MCF-12A, with a GI50 &gt;40 uM. Consistent with its effects on cell growth, pCho levels in MDA-MB-415 cells, as measured by NMR, were dose-dependently inhibited up to ∼80% by 24 hours with an IC50 of ∼750 nM. In MDA-MB-415 cells, but not MCF-12A cells, levels of apoptotic markers were increased at 24 hours with compound concentrations β5 uM. In summary, we demonstrated that small molecule inhibition of ChoK results in a dose-dependent decrease of pCho levels, inhibition of proliferation and induction of apoptosis in ChoKα expressing breast cancer cells. We established that exogenous expression of ChoKα in HEK293 cells drives both oncogenic transformation and constitutive activation of proliferative signaling pathways. Taken together, these data further validate ChoKα as a potential therapeutic target in cancer and support the continued investigation into the utility of ChoKα inhibitors as anti-oncogenic agents.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3236. doi:1538-7445.AM2012-3236