Choline Kinase Alpha (CHKα) as a Therapeutic Target in Pancreatic Ductal Adenocarcinoma: Expression, Predictive Value, and Sensitivity to Inhibitors.

Choline kinase α (CHKα) plays a crucial role in the regulation of membrane phospholipid synthesis and has oncogenic properties in vitro. We have analyzed the expression of CHKα in cell lines derived from pancreatic ductal adenocarcinoma (PDAC) and have found increased CHKα expression, associated with differentiation. CHKα protein expression was directly correlated with sensitivity to MN58b, a CHKα inhibitor that reduced cell growth through the induction of apoptosis. Accordingly, CHKα knockdown led to reduced drug sensitivity. In addition, we found that gemcitabine-resistant PDAC cells displayed enhanced sensitivity to CHKα inhibition and, in vitro, MN58b had additive or synergistic effects with gemcitabine, 5-fluorouracil, and oxaliplatin, three active drugs in the treatment of PDAC. Using tissue microarrays, CHKα was found to be overexpressed in 90% of pancreatic tumors. While cytoplasmic CHKα did not relate to survival, nuclear CHKα distribution was observed in 43% of samples and was associated with longer survival, especially among patients with well/moderately differentiated tumors. To identify the mechanisms involved in resistance to CHKα inhibitors, we cultured IMIM-PC-2 cells with increasingly higher concentrations of MN58b and isolated a subline with a 30-fold higher IC50. RNA-Seq analysis identified upregulation of ABCB1 and ABCB4 multidrug resistance transporters, and functional studies confirmed that their upregulation is the main mechanism involved in resistance. Overall, our findings support the notion that CHKα inhibition merits further attention as a therapeutic option in patients with PDAC and that expression levels may predict response.

The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy.

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models.

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Symmetrical bis-quinolinium compounds: new human choline kinase inhibitors with antiproliferative activity against the HT-29 cell line.

Studies have been aimed at the establishment of structure-activity relationships that define choline kinase inhibitory and antiproliferative activities of 40 bisquinolinium compounds. These derivatives have electron-releasing groups at position 4 of the quinolinium ring. It is found that the enzymatic inhibition is closely related to the size of the linker, the 3,3'-biphenyl moiety being the most suitable. On the other hand, the antiproliferative activity against the HT-29 cancer cell line is less influenced by the linker type and by substituent R(4). The corresponding QSAR equation was obtained for the whole set of compounds for the antiproliferative activity, the electronic parameter sigma(R) of R(4), the molar refractivity of R(8), and the lipophilic parameters clog P and pi(linker). The most potent antiproliferative agent so far described is 40 for which an IC(50) = 0.45 microM was predicted by the QSAR equation, while its experimental value is IC(50) = 0.20 microM.

Choline kinase inhibitory effect and antiproliferative activity of new 1,1',1"-(benzene-1,3,5-triylmethylene)tris[4-[(disubstituted)amino]pyridinium] tribromides.

Four derivatives of 1,1'-(benzene-1,3-diylmethylene)bis[4-[(disubstituted)amino]-pyridinium] dibromides (2-5) and six derivatives of 1,1',1"-(benzene-1,3,5-triylmethylene)-tris[4-[(disubstituted)amino]pyridinium] tribromides (6-11) were synthesised and examined for their inhibition of human choline kinase (ChoK) and antiproliferative activities. The latter are more potent as ChoK inhibitors than the former, but the antiproliferative activities against the HT-29 cell line show the opposite tendency. The higher affinity of the trispyridinium compared with the bispyridinium ones may be due to direct binding of the third pyridinium group to ChoK or may arise from a reduction of the unfavourable entropy of binding via an increase of the 'local concentration' of pyridinium groups.