ChoKα Inhibitors Research Articles

Choline Kinase α Inhibitors MN58b and RSM932A Enhances the Antitumor Response to Cisplatin in Lung Tumor Cells

Lung cancer is one of the main causes of death in developed countries, and non-small cell lung cancer (NSCLC) is the most frequent type (80% of patients). In advanced NSCLC, platinum-based chemotherapy is the frontline palliative treatment, but less than 5% of patients achieve prolonged survival. Immunotherapy has recently been proposed as the standard of care (SoC) as either monotherapy or in combination with chemotherapy for advanced NSCLC. The levels of expression of PD-L1 are the only predictive biomarkers for patient assessment. Although around 30% of patients receiving immunotherapy achieve 5-year survival, a significant number does not benefit from this novel therapeutic approach. Therefore, there is a need for novel strategies to improve clinical outcomes. The expression level of choline kinase α (ChoKα) is increased in a large number of human tumors, including NSCLC tumors, and constitutes an independent prognostic factor for early-stage NSCLC patients. Thus, ChoKα has been postulated as a new target drug in cancer therapy. The combination of cisplatin with novel targeted drugs such as choline kinase inhibitors may improve both the survival rates and the quality of life of NSCLC patients and may serve as the basis for the development of new therapeutic approaches. To that aim, we developed several in vitro and in vivo approaches to assess the antitumor activity of a novel combination regimen using cisplatin and ChoKα inhibitors. Our results suggest that a proper combination of specific inhibitors of the NSCLC prognostic factor ChoKα and platinum-based conventional chemotherapy might constitute a new, efficient treatment approach for NSCLC patients. This novel approach may help reduce the toxicity profile associated with cisplatin since, despite the advances in NSCLC management in recent years, the overall 5-year survival rate is still poor.

Identification of unprecedented ATP-competitive choline kinase inhibitors

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

Choline Kinase: An Unexpected Journey for a Precision Medicine Strategy in Human Diseases.

Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure–activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and pathogenic bacteria. The evidence for ChoKα as a novel drug target for approaches in precision medicine is discussed.

Recent advances in the design of choline kinase α inhibitors and the molecular basis of their inhibition.

Upregulated choline metabolism, characterized by an increase in phosphocholine (PCho), is a hallmark of oncogenesis and tumor progression. Choline kinase (ChoK), the enzyme responsible for PCho synthesis, has consequently become a promising drug target for cancer therapy and as such a significant number of ChoK inhibitors have been developed over the last few decades. More recently, due to the role of this enzyme in other pathologies, ChoK inhibitors have also been used in new therapeutic approaches against malaria and rheumatoid arthritis. Here, we review research results in the field of ChoKα inhibitors from their synthesis to the molecular basis of their binding mode. Strategies for the development of inhibitors and their selectivity on ChoKα over ChoKβ, the plasticity of the choline-binding site, the discovery of new exploitable binding sites, and the allosteric properties of this enzyme are highlighted. The outcomes summarized in this review will be a useful guide to develop new multifunctional potent drugs for the treatment of various human diseases.

Abstract 4790: Identification and characterization of ATP-mimetic choline kinase inhibitors

Abstract Introduction: Choline kinase alpha (ChoKα), the first enzyme in the Kennedy pathway that catalyzes the phosphorylation of free choline to phosphocholine (PCho), is responsible for the de novo biosynthesis of phosphatidylcholine (PC), the major phospholipid of cellular membranes. Aberrant choline metabolic profiles and concomitant ChoKα upregulation have been described in most human malignancies (i.e. breast, lung, ovary, liver) and have been found to correlate with advanced histological tumour grade. ChoKα, depletion by siRNA or shRNA inhibits growth and migration of different tumor cell lines both in vitro and in vivo, which is not observed for the ChoKβ isoform. Choline mimetic inhibitors of ChoKα (i.e. MN58b) have been shown to have antitumor activity in preclinical models, although their efficacy is hampered by a significant toxicity, possibly due to cross-reactivity with other choline-dependent proteins (transporters, enzymes). At NMS a high throughput screening (HTS) was performed with the objective to identify ATP-mimetic ChoKα inhibitors potentially less toxic than choline-mimetic compounds. Methods: Hits from different classes were characterized for biochemical activity on ChoKα and ChoKβ and biochemical mechanism of inhibition. The binding site of selected ATP competitive inhibitors was confirmed by co-crystallization with ChoKα. On-target mechanism of action in cells was confirmed by analysing inhibition of PCho formation. Result: Structure-based chemical expansion of Hits from one of the prioritized classes resulted in compounds with biochemical potencies in the single digit nanomolar range displaying selectivity vs ChoKβ as well as a diverse panel of protein kinases. In several tumor cell lines, the compounds were able to inhibit the formation of PCho at a concentration in agreement with that required to achieve anti-proliferative activity. The most potent compounds were tested on a panel of 24 representative breast cancer cell lines which showed differential sensitivity towards ChoKα inhibition. Analysis of genomic (DNA and RNA) and proteomic (>50 markers) expression profiles of the breast cancer cell lines is ongoing to identify predictive biomarkers of response. Conclusion: Medicinal chemistry expansion of a novel class of compounds identified by HTS allowed the development of potent ChoKα ATP-mimetic inhibitors able to modulate PCho levels in cells, which can be used to identify preferential sensitivity contexts. Medicinal chemistry activities are ongoing to further improve their potency and optimize their ADME/PK properties. Citation Format: Paola Gnocchi, Francesca Quartieri, Alessandra Badari, Roberta Bosotti, Elena Casale, Emiliana Corti, Cinzia G. Cristiani, Ulisse Cucchi, Fabio Gasparri, Laura M. Gianellini, Laura M. Giorgini, Marisa Montemartini, Giuliana Mion, Marcella Nesi, Christian Orrenius, Claudia E. Re, Daniele Donati, Eduard R. Felder, Arturo Galvani, Antonella Isacchi. Identification and characterization of ATP-mimetic choline kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4790.

Choline kinase inhibitors EB-3D and EB-3P interferes with lipid homeostasis in HepG2 cells

A full understanding of the molecular mechanism of action of choline kinase α (ChoKα) inhibitors at the cell level is essential for developing therapeutic and preventive approaches for cancer. The aim of the present study was to evaluate the effects of the ChoKα inhibitors EB-3D and EB-3P on lipid metabolism in HepG2 cells. We used [methyl-14C]choline, [1,2-14C]acetic acid and [2-3H]glycerol as exogenous precursors of the corresponding phospholipids and neutral lipids. [Methyl-14C]choline was also used to determine choline uptake. Protein levels were determined by Western blot. Ultrastructural alterations were investigated by transmission electron microscopy. In this work, we demonstrate that EB-3D and EB-3P interfere with phosphatidylcholine biosynthesis via both CDP-choline pathway and choline uptake by the cell. Moreover, the synthesis of both diacylglycerols and triacylglycerols was affected by cell exposure to both inhibitors. These effects were accompanied by a substantial decrease in cholesterol biosynthesis, as well as alterations in the expression of proteins related to cholesterol homeostasis. We also found that EB-3D and EB-3P lowered ChoKα protein levels. All these effects could be explained by the modulation of the AMP-activated protein kinase signalling pathway. We show that both inhibitors cause mitochondrial alteration and an endoplasmic reticulum stress response. EB-3D and EB-3P exert effects on ChoKα expression, AMPK activation, apoptosis, endoplasmic reticulum stress and lipid metabolism. Taken together, results show that EB-3D and EB-3P have potential anti-cancer activity through the deregulation of lipid metabolism.

EB-3D a novel choline kinase inhibitor induces deregulation of the AMPK-mTOR pathway and apoptosis in leukemia T-cells

Choline kinase alpha 1 (ChoKα1) has recently become an interesting therapeutic target since its overexpression has been associated to tumorigenesis in many cancers. Nevertheless, little is known regarding hematological malignancies. In this manuscript, we investigated the effect of a novel and selective ChoKα inhibitor EB-3D in T acute lymphoblastic leukemia (T-ALL).The effect of EB-3D was evaluated in a panel of T-leukemia cell lines and ex-vivo primary cultures derived from pediatric T-ALL patients. We also evaluated in detail, using Reverse Phase Protein Array (RPPA), protein phosphorylation level changes in T-ALL cells upon treatment.The drug exhibits a potent antiproliferative activity in a panel of T-leukemia cell lines and primary cultures of pediatric patients. Moreover, the drug strongly induces apoptosis and more importantly it enhanced T-leukemia cell sensitivity to chemotherapeutic agents, such as dexamethasone and l-asparaginase. In addition, the compound induces an early activation of AMPK, the main regulator of cellular energy homeostasis, by its phosphorylation at residue T712 of catalytic subunit α, and thus repressing mTORC1 pathway, as shown by mTOR S2448 dephosphorylation. The inhibition of mTOR in turn affects the activity of several known downstream targets, such as 4E-BP1, p70S6K, S6 Ribosomal Protein and GSK3 that ultimately may lead to a reduction of protein synthesis and cell death.Taken together, our findings suggest that targeting ChoKα may be an interesting option for treating T-ALL and that EB-3D could represent a valuable therapeutic tool.

Identification of a Unique Inhibitor-Binding Site on Choline Kinase α.

Choline kinase α (ChoKα) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic. As such, it makes a promising target for inhibiting tumor growth. Though there have been several inhibitors synthesized for ChoKα, not all of them demonstrate the same efficacy in vivo, though the reasons behind this difference in potency are not clear. One particular inhibitor, designated TCD-717, has recently completed phase I clinical trials. Cell culture and in vitro studies support the powerful inhibitory effect TCD-717 has on ChoKα, but an examination of the inhibitor's interaction with the ChoKα enzyme has been missing prior to this work. Here we detail the 2.35 Å structure of ChoKα in complex with TCD-717. Examination of this structure in conjunction with kinetic assays reveals that TCD-717 does not bind directly in the choline pocket as do previously characterized ChoKα inhibitors, but rather in a proximal but novel location near the surface of the enzyme. The unique binding site identified for TCD-717 lends insight for the future design of more potent in vivo inhibitors for ChoKα.

Near infrared fluorescent imaging of choline kinase alpha expression and inhibition in breast tumors.

Choline kinase alpha (ChoKα) overexpression is associated with an aggressive tumor phenotype. ChoKα inhibitors induce apoptosis in tumors, however validation of their specificity is difficult in vivo. We report the use of optical imaging to assess ChoKα status in cells and in vivo using JAS239, a carbocyanine-based ChoKα inhibitor with inherent near infrared fluorescence. JAS239 attenuated choline phosphorylation and viability in a panel of human breast cancer cell lines. Antibody blockade prevented cellular retention of JAS239 indicating direct interaction with ChoKα independent of the choline transporters and catabolic choline pathways. In mice bearing orthotopic MCF7 breast xenografts, optical imaging with JAS239 distinguished tumors overexpressing ChoKα from their empty vector counterparts and delineated tumor margins. Pharmacological inhibition of ChoK by the established inhibitor MN58b led to a growth inhibition in 4175-Luc+ tumors that was accompanied by concomitant reduction in JAS239 uptake and decreased total choline metabolite levels as measured using magnetic resonance spectroscopy. At higher therapeutic doses, JAS239 was as effective as MN58b at arresting tumor growth and inducing apoptosis in MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic.

Dysregulated choline metabolism in T-cell lymphoma: role of choline kinase-α and therapeutic targeting.

Cancer cells have distinct metabolomic profile. Metabolic enzymes regulate key oncogenic signaling pathways and have an essential role on tumor progression. Here, serum metabolomic analysis was performed in 45 patients with T-cell lymphoma (TCL) and 50 healthy volunteers. The results showed that dysregulation of choline metabolism occurred in TCL and was related to tumor cell overexpression of choline kinase-α (Chokα). In T-lymphoma cells, pharmacological and molecular silencing of Chokα significantly decreased Ras-GTP activity, AKT and ERK phosphorylation and MYC oncoprotein expression, leading to restoration of choline metabolites and induction of tumor cell apoptosis/necropotosis. In a T-lymphoma xenograft murine model, Chokα inhibitor CK37 remarkably retarded tumor growth, suppressed Ras-AKT/ERK signaling, increased lysophosphatidylcholine levels and induced in situ cell apoptosis/necropotosis. Collectively, as a regulatory gene of aberrant choline metabolism, Chokα possessed oncogenic activity and could be a potential therapeutic target in TCL, as well as other hematological malignancies with interrupted Ras signaling pathways.

Preclinical characterization of RSM-932A, a novel anticancer drug targeting the human choline kinase alpha, an enzyme involved in increased lipid metabolism of cancer cells.

Choline kinase α (CHKA; here designated as ChoKα) is the first enzyme in the CDP-choline pathway, implicated in phospholipids metabolism. It is overexpressed in several human tumors such as breast, lung, bladder, colorectal, prostate, ovary, and liver. The overexpression of ChoKα has oncogenic potential and synergizes with other known oncogenes. It has been proposed as a novel cancer drug target with a distinct mechanism of action. We have generated a set of ChoKα inhibitors with potent in vitro antiproliferative and in vivo antitumoral activity against human xenografts in mice, showing high efficacy with low toxicity profiles. Among these inhibitors, RSM-932A has been chosen for further clinical development due to its potent antiproliferative activity in vitro against a large variety of tumor-derived cell lines, a potent in vivo anticancer activity, and lack of toxicity at the effective doses. Here, we provide the preclinical evidence to support the use of RSM-932A as a good candidate to be tested in clinical trials as the "first in humans" drug targeting ChoKα.

Choline kinase inhibition in rheumatoid arthritis

ObjectivesLittle is known about targeting the metabolome in non-cancer conditions. Choline kinase (ChoKα), an essential enzyme for phosphatidylcholine biosynthesis, is required for cell proliferation and has been implicated in cancer...

Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells

Endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that regulates protein synthesis and maturation. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress has been involved in apoptosis induced by several cytotoxic agents. Choline kinase α (ChoKα), the first enzyme in the Kennedy pathway, is responsible for the generation of phosphorylcholine (PCho) that ultimately renders phosphatidylcholine. ChoKα overexpression and high PCho levels have been detected in several cancer types. Inhibition of ChoKα has demonstrated antiproliferative and antitumor properties; however, the mechanisms underlying these activities remain poorly understood. Here, we demonstrate that ChoKα inhibitors (ChoKIs), MN58b and RSM932A, induce cell death in cancer cells (T47D, MCF7, MDA-MB231, SW620 and H460), through the prolonged activation of ER stress response. Evidence of ChoKIs-induced ER stress includes enhanced production of glucose-regulated protein, 78 kDa (GRP78), protein disulfide isomerase, IRE1α, CHOP, CCAAT/enhancer-binding protein beta (C/EBPβ) and TRB3. Although partial reduction of ChoKα levels by small interfering RNA was not sufficient to increase the production of ER stress proteins, silencing of ChoKα levels also show a decrease in CHOP overproduction induced by ChoKIs, which suggests that ER stress induction is due to a change in ChoKα protein folding after binding to ChoKIs. Silencing of CHOP expression leads to a reduction in C/EBPβ, ATF3 and GRP78 protein levels and abrogates apoptosis in tumor cells after treatment with ChoKIs, suggesting that CHOP maintains ER stress responses and triggers the pro-apoptotic signal. Consistent with the differential effect of ChoKIs in cancer and primary cells previously described, ChoKIs only promoted a transient and moderated ER stress response in the non-tumorogenic cells MCF10A. In conclusion, pharmacological inhibition of ChoKα induces cancer cell death through a mechanism that involves the activation of exaggerated and persistent ER stress supported by CHOP overproduction.

Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

BackgroundColorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.Methodology/Principal FindingsChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Conclusion/SignificanceOur data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival.

Choline kinase alpha (ChoKα) is regarded as an attractive cancer target. The enzyme catalyses the formation of phosphocholine (PCho), an important precursor in the generation of phospholipids essential for cell growth. ChoKα has oncogenic properties and is critical for the survival of cancer cells. Overexpression of the ChoKα protein can transform noncancer cells into cells with a cancerous phenotype, and depletion of the ChoKα protein can result in cancer cell death. However, the mechanisms underlying the tumourigenic properties of ChoKα are not fully understood. ChoKα was recently demonstrated to associate with other oncogenic proteins, raising the possibility that a non-catalytic protein scaffolding function drives the tumourigenic properties of ChoKα rather than a catalytic function. In order to differentiate these two roles, we compared the impact on cancer cell survival using two tools specific for ChoKα: (1) small interfering RNA (siRNA) to knockdown the ChoKα protein levels; and (2) compound V-11-0711, a novel potent and selective ChoKα inhibitor (ChoKα IC50 20 nℳ), to impede the catalytic activity. Both treatments targeted the endogenous ChoKα protein in HeLa cells, as demonstrated by a substantial reduction in the PCho levels. siRNA knockdown of the ChoKα protein in HeLa cells resulted in significant cell death through apoptosis. In contrast, compound V-11-0711 caused a reversible growth arrest. This suggests that inhibition of ChoKα catalytic activity alone is not sufficient to kill cancer cells, and leads us to conclude that there is a role for the ChoKα protein in promoting cancer cell survival that is independent of its catalytic activity.

Kinetic and mechanistic characterisation of Choline Kinase-α

Choline Kinase is a key component of the Kennedy pathway that converts choline into a number of structural and signalling lipids that are essential for cell growth and survival. One member of the family, Choline Kinase-α (ChoKα) is frequently up-regulated in human cancers, and expression of ChoKα is sufficient to transform cells. Consequently ChoKα has been studied as a potential target for therapeutic agents in cancer research. Despite great interest in the enzyme, mechanistic studies have not been reported. In this study, a combination of initial velocity and product inhibition studies, together with the kinetic and structural characterisation of a novel ChoKα inhibitor is used to support a mechanism of action for human ChoKα. Substrate and inhibition kinetics are consistent with an iso double displacement mechanism, in which the γ-phosphate from ATP is transferred to choline in two distinct steps via a phospho-enzyme intermediate. Co-crystal structures, and existing site-specific mutation studies, support an important role for Asp306, in stabilising the phospho-enzyme intermediate. The kinetics also indicate a distinct kinetic (isomerisation) step associated with product release, which may be attributed to a conformational change in the protein to disrupt an interaction between Asp306 and the phosphocholine product, facilitating product release. This study describes a mechanism for ChoKα that is unusual amongst kinases, and highlights the availability of different enzyme states that can be exploited for drug discovery.

Acid Ceramidase as a Chemotherapeutic Target to Overcome Resistance to the Antitumoral Effect of Choline Kinase α Inhibition

We have analyzed the response of primary cultures derived from tumor specimens of non small cell lung cancer (NSCLC) patients to choline kinase α (ChoKα) inhibitors. ChoKα inhibitors have been demonstrated to increase ceramides levels specifically in tumor cells, and this increase has been suggested as the mechanism that explain its proapoptotic effect in cancer cells. Here, we have investigated the molecular mechanism associated to the intrinsic resistance, and found that other enzyme involved in lipid metabolism, acid ceramidase (ASAH1), is specifically upregulated in resistant tumors. NSCLC cells with acquired resistance to ChoKα inhibitors also display increased levels of ASAH1. Accordingly, ASAH1 inhibition synergistically sensitizes lung cancer cells to the antiproliferative effect of ChoKα inhibitors. Thus, the determination of the levels of ASAH1 predicts sensitivity to targeted therapy based on ChoKα specific inhibition and represents a model for combinatorial treatments of ChoKα inhibitors and ASAH1 inhibitors. Considering that ChoKα inhibitors have been recently approved to enter Phase I clinical trials by the Food and Drug Administration (FDA), these findings are anticipating critical information to improve the clinical outcome of this family of novel anticancer drugs under development.

Abstract 3236: Small molecule inhibitors of choline kinase lead to reduced phosphocholine levels and induction of apoptosis in cancer cells

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 >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

Abstract 3514: Combination of 5-FU with ChoKα inhibitors as a new alternative therapy in colorectal cancer

Abstract Colorectal cancer (CRC) is the third major cause of cancer related deaths worldwide. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Despite the achieved improvements over the past years in cancer treatment, the design of novel targeted and combined therapies is still necessary. Choline kinase alpha (ChoKα), a phospholipid-related enzyme that plays a role in cell proliferation and transformation, has been found overexpressed in many different tumors, including colorectal tumors. ChoKα specific inhibitors, MN58b and TCD717, have potent antitumoral activity both in vitro and in vivo against several tumor-derived cell lines including CRC-derived tumour cells. TCD717 has recently entered clinical trials (http://clinicaltrials.gov/ct2/show/NCT01215864). We have evaluated the effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors. The sequential combination of ChoKα inhibitors with 5-FU resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. Key enzymes in the metabolic pathway of 5-FU such as Thymidylate synthase (TS) and Thymidine kinase (TK1) have been related to drug resistance, one of the major problems for the lack of success of 5-FU chemotherapy. TS is a key enzyme in the synthesis of DNA and the target enzyme of 5-FU. Several studies have demonstrated that the expression of TS mRNA or protein, predicts overall survival for colon cancer and correlates with resistance to 5-FU. Acquired resistance to 5-FU can also be caused by overproduction of TS that results from gene amplification. Regarding to 5-FU developed resistance, a salvaged pathway has been reported in which TK1 plays an important role. Thus, both TS and TK1 are directly involved in resistance to 5-FU therapy. To elucidate the mechanism of the observed synergistic effect, we examined the modulation of ChoKα inhibitors on the expression levels of TS and TK1. CRC cell lines treated with the sequential combination of ChoKα inhibitors and 5-FU show both mechanisms of TS inactivation, a formation of ternary complex due to 5-FU mechanism and a significant down-regulation of TS active due to ChoKα inhibitors, resulting in a total depletion of TS protein. We also observed that ChoKα inhibitors affects the levels of TK1, and provide the basis to support a new therapy focused on the combination of ChoKα inhibitors and 5-FU as a new approach for colorectal cancer patients. In addition, we suggest a promising role for ChoKα inhibitors as a new treatment for patients who had failed 5-FU chemotherapy or display high expression levels of TS. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2011-3514

Abstract 1545: Combinatorial efficacy of TRAIL and MN58b against colon cancer to overcome TRAIL resistance

Abstract BACKGROUND: Tumor resistance is one of the major problems for cancer treatment and new approaches to overcome resistance are required. TNF-Related Apoptosis Inducing Ligand (TRAIL) has recently emerged as a promising therapy due to its selectivity towards tumor cells and is currently being evaluated in phase I and phase II clinical trials. Choline kinase alpha (ChoKα) inhibition has also recently proven to be an efficient strategy to specifically induce tumor cell death without affecting normal cells. The aim of this study is to investigate the potential use of combinatorial treatment of TRAIL and MN58b, a specific ChoKα inhibitor, to potentiate tumor cell death. EXPERIMENTAL PROCEDURES: Two different colon cancer-derived cell lines were used: DLD-1 and SW620. The antiproliferative effect of MN58b, TRAIL, or both was determined using MTT method. Western blots were carried out to measure PARP degradation and caspase-3 activation, and Annexin V- PI staining was used to verify cell death induction in response to drugs by flow cytometry. The results obtained in vitro were verified in vivo using xenograft models for both cell lines. RESULTS: Using different concentrations of both drugs, it has been established that the best strategy of combination is a simultaneous treatment. Our data shows that when MN58b and TRAIL are simultaneously combined, a strong synergistic effect is observed in DLD-1 cells, with a combination index (CI) of 0.196. Moreover, MN58b treatment is able to overcome TRAIL resistance in SW620 cell line (CI=0.155). The synergistic effect was verified by both PARP and Caspase-3 analysis as apoptosis markers. Cell death was also analyzed by flow cytometry. This in vitro synergistic effect was confirmed in vivo using xenograft models of both cell lines. DLD-1 induced tumor growth was inhibited by 42% and 51% when treated with MN58b or TRAIL respectively. When both drugs were combined the effect was much stronger, reaching 78% inhibition. These findings suggest that MN58b cooperates with TRAIL in suppressing tumor growth in DLD-1 xenograft model. SW620 xenograft response to MN58b was weaker, with 26% inhibition. TRAIL alone produced no significant decrease of tumor growth in keeping with the observation of this cell line being resistant to TRAIL. When MN58b and TRAIL were combined, there was a marked 71% tumor growth inhibition, suggesting that MN58b is able to sensitize SW620 to TRAIL-induced apoptosis in vivo, and confirming all the in vitro observed effects. CONCLUSION: ChoKα inhibition can enhance TRAIL-induced apoptosis in DLD-1 cells and is able to abrogate TRAIL resistance in SW620 cells. Considering that MN58b when used alone exhibits anticancer activities in vitro and in vivo against a wide variety of tumor-derived cell lines, this study establishes its additional potential use in combination with TRAIL on tumors that develop TRAIL resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1545.