Elena Mariotto

Design, synthesis, crystallization and biological evaluation of new symmetrical biscationic compounds as selective inhibitors of human Choline Kinase α1 (ChoKα1).

A novel family of compounds derivative of 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bispyridinium or –bisquinolinium bromide (10a-l) containing a pair of oxygen atoms in the spacer of the linker between the biscationic moieties, were synthesized and evaluated as inhibitors of choline kinase against a panel of cancer-cell lines. The most promising compounds in this series were 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1′-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before.

Design and Synthesis of Potent in Vitro and in Vivo Anticancer Agents Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole

A novel series of tubulin polymerization inhibitors, based on the 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold and designed as cis-restricted combretastatin A-4 analogues, was synthesized with the goal of evaluating the effects of various patterns of substitution on the phenyl at the 2-position of the imidazole ring on biological activity. A chloro and ethoxy group at the meta- and para-positions, respectively, produced the most active compound in the series (4o), with IC50 values of 0.4-3.8 nM against a panel of seven cancer cell lines. Except in HL-60 cells, 4o had greater antiproliferative than CA-4, indicating that the 3′-chloro-4′-ethoxyphenyl moiety was a good surrogate for the CA-4 B-ring. Experiments carried out in a mouse syngenic model demonstrated high antitumor activity of 4o, which significantly reduced the tumor mass at a dose thirty times lower than that required for CA-4P, which was used as a reference compound. Altogether, our findings suggest that 4o is a promising anticancer drug candidate that warrants further preclinical evaluation.

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3′,4′,5′-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3′,4′,5′-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3′,4′,5′-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5–20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug.

TP-0903 inhibits neuroblastoma cell growth and enhances the sensitivity to conventional chemotherapy

Abstract Neuroblastoma (NB) is an embryonal tumor with low cure rate for patients classified as high‐risk. This class of NB tumors shows a very complex genomic background and requires aggressive treatment strategies. In this work we evaluated the efficacy of the novel multi‐kinase inhibitor TP‐0903 in impairing NB cells’ growth, proliferation and motility. In vitro studies were performed using cell lines with different molecular background, and in vivo studies were done using the zebrafish experimental model. Our results confirmed a strong cytotoxicity of TP‐0903 already at the sub‐micro molar concentrations. The observed cytotoxicity of TP‐0903 was irreversible and the resulting apoptosis was caspase dependent. In addition, TP‐0903 impaired colony formation and neurosphere creation. Depending on the molecular background of the selected NB cell lines, TP‐0903 influenced either their capacity to migrate, to complete their cell cycle or both. Likewise, TP‐0903 reduced NB cells intravasation in vitro and in vivo. Importantly, TP‐0903 showed remarkable pharmacological efficacy not only as a mono‐treatment, but also in combination with conventional chemotherapy drugs (ATRA, cisplatin, and VP16) in different types of NB cells. In conclusion, the multi‐kinase activity of TP‐0903 allowed the impairment of several biological processes required for expansion of NB cells, making them more vulnerable to the conventional chemotherapeutics. Altogether, our results support the eligibility of TP‐0903 for further (pre)clinical assessments in NB.

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

Graphical abstract Figure. No caption available. &NA; Choline kinase alpha 1 (ChoK&agr;1) 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 ChoK&agr; 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 &;, 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 ChoK&agr; may be an interesting option for treating T‐ALL and that EB‐3D could represent a valuable therapeutic tool.

Choline Kinase Alpha Inhibition by EB-3D Triggers Cellular Senescence, Reduces Tumor Growth and Metastatic Dissemination in Breast Cancer

Choline kinase (ChoK) is the first enzyme of the Kennedy pathway leading to the biosynthesis of phosphatidylcholine (PtdCho), the most abundant phospholipid in eukaryotic cell membranes. EB-3D is a novel choline kinase α1 (ChoKα1) inhibitor with potent antiproliferative activity against a panel of several cancer cell lines. ChoKα1 is particularly overexpressed and hyperactivated in aggressive breast cancer. By NMR analysis, we demonstrated that EB-3D is able to reduce the synthesis of phosphocholine, and using flow cytometry, immunoblotting, and q-RT-PCR as well as proliferation and invasion assays, we proved that EB-3D strongly impairs breast cancer cell proliferation, migration, and invasion. EB-3D induces senescence in breast cancer cell lines through the activation of the metabolic sensor AMPK and the subsequent dephosphorylation of mTORC1 downstream targets, such as p70S6K, S6 ribosomal protein, and 4E-BP1. Moreover, EB-3D strongly synergizes with drugs commonly used for breast cancer treatment. The antitumorigenic potential of EB-3D was evaluated in vivo in the syngeneic orthotopic E0771 mouse model of breast cancer, where it induces a significant reduction of the tumor mass at low doses. In addition, EB-3D showed an antimetastatic effect in experimental and spontaneous metastasis models. Altogether, our results indicate that EB-3D could be a promising new anticancer agent to improve aggressive breast cancer treatment protocols.

AKR1C enzymes sustain therapy resistance in paediatric T-ALL

BackgroundDespite chemotherapy intensification, a subgroup of high-risk paediatric T-cell acute lymphoblastic leukemia (T-ALL) patients still experience treatment failure. In this context, we hypothesised that therapy resistance in T-ALL might involve aldo-keto reductase 1C (AKR1C) enzymes as previously reported for solid tumors.MethodsExpression of NRF2-AKR1C signaling components has been analysed in paediatric T-ALL samples endowed with different treatment outcomes as well as in patient-derived xenografts of T-ALL. The effects of AKR1C enzyme modulation has been investigated in T-ALL cell lines and primary cultures by combining AKR1C inhibition, overexpression, and gene silencing approaches.ResultsWe show that T-ALL cells overexpress AKR1C1-3 enzymes in therapy-resistant patients. We report that AKR1C1-3 enzymes play a role in the response to vincristine (VCR) treatment, also ex vivo in patient-derived xenografts. Moreover, we demonstrate that the modulation of AKR1C1-3 levels is sufficient to sensitise T-ALL cells to VCR. Finally, we show that T-ALL chemotherapeutics induce overactivation of AKR1C enzymes independent of therapy resistance, thus establishing a potential resistance loop during T-ALL combination treatment.ConclusionsHere, we demonstrate that expression and activity of AKR1C enzymes correlate with response to chemotherapeutics in T-ALL, posing AKR1C1-3 as potential targets for combination treatments during T-ALL therapy.

Abstract 1297: CDK4/CDK6 inhibition in childhood B-acute lymphoblastic leukemia: a new strategy to mediate glucocorticoid sensitivity

Unrestrained cell proliferation and cell cycle deregulation are common features in almost all human cancers. Among the CDKs that tightly control cell cycle progression, cyclin D-dependent kinases, CDK4 and CDK6 are considered important oncogenic drivers in many cancers. Although many reports successfully described CDK4/CDK6 inhibitors against a broad range of carcinomas, few studies have been performed on leukemia. Deletion and methylation of CDK4/CDK6 inhibitor CDKN2A, are frequently observed in B-acute lymphoblastic leukemia (B-ALL) and gene expression analysis performed in a cohort of childhood patients showed that cyclin D1, CDK4 and CDK6 are highly expressed. Moreover, Reverse Phase Protein Arrays (RPPA) analysis showed that cyclin D1 expression is higher in High Risk-MRD patients. These results suggest specific inhibition of cyclin D/CDK4/CDK6 axis as an attractive strategy to improve the effects of standard chemotherapy on B-ALL patients. The aim of this study was to evaluate the effect of dual inhibition of CDK4/CDK6 in B-ALL. To this purpose we treated two B-ALL dexamethasone resistant cell lines, SEM and RCH-ACV, and two B-ALL dexamethasone sensitive cell lines, RS4;11 and NALM6, with ribociclib, a highly specific CDK4/6 inhibitor. As expected, treatment with ribociclib induced a strong cell cycle arrest in G1 phase in a time-dose dependent manner along with a dose-dependent decrease in phosphorylated Rb and increase of the expression of cell cycle inhibitors p21 and p27. Moreover, a strong dose-dependent reduction of clonogenic potential was observed in SEM cell line, by CFU assay. No significant reduction in cell viability was observed. However, ribociclib exposure strongly synergized (CI The synergistic effect of ribociclib-dexamethasone combination was confirmed on primary cultures derived from pediatric patients affected by B-ALL. We are actually investigating on the mechanism of this synergistic activity, and the effect of CDK4, CDK6 and cyclin D1 silencing will be presented. Preliminary experiments show a modest increase in glucocorticoid receptor expression after ribociclib treatment or CDK6 silencing in RCH-ACV cells. Our findings support the concept that pharmacologic inhibition of CDK4/CDK6 may represent a useful therapeutic strategy to control cell proliferation in B-ALL and provide new insight in understanding potential mechanisms of glucocorticoid resistance. Citation Format: Roberta Bortolozzi, Elena Mattiuzzo, Elena Mariotto, Benedetta Accordi, Luca Trentin, Giuseppe Basso, Giampietro Viola. CDK4/CDK6 inhibition in childhood B-acute lymphoblastic leukemia: a new strategy to mediate glucocorticoid sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1297.

Abstract 1233: In vitro and in vivo pharmacological study of EB-3D: a novel choline kinase inhibitor for breast cancer treatment

Choline kinase (ChoK) catalyzes the phosphorylation of choline to phosphocholine (PCho) in the first step of the Kennedy pathway for phosphatidylcholine (PtdCho) byosyntesis. PtdCho is the most abundant phospholipid in eukaryotic cell membrane and plays a crucial role for cell division and lipid second messengers production. In mammalian cells, only the ChoKα isoform is fundamental in sustaining PtdCho synthesis, indeed ChoKβ alone cannot compensate this activity. A large number of studies have shown that ChoKα is overexpressed and hyperactivated in many cancers and that correlates not only with increased cancer cell proliferation but also with malignancy, making it a potential prognostic marker. The cholinic phenotype, characterized by increased total choline-containing compound (tCho) and ChoKα upregulation renders therefore ChoKα an attractive new therapeutic target. Indeed, the feasibility of ChoKα inhibition as antitumoral therapy is being pursued through the development of chemical inhibitors of its enzymatic activity. For this reason we evaluated both in vitro and in vivo the activity of EB-3D (1,1’-(((ethane-1,2-diylbis(oxy))bis(4,1-phenylene))bis(methylene))bis(4-(dimethylamino) pyridinium) bromide), a new choline kinase inhibitor endowed with high antiproliferative activity in two human breast cancer cell lines, MCF-7 and MDA-MB-231. EB-3D treatment induced a strong decrease in cell proliferation due to G1/G0 arrest of the cell cycle in a concentration-dependent manner in both tested cell lines, whereas only a slight increase in apoptotic cells was observed. In addition EB-3D strongly synergized with drugs commonly used in protocols for breast cancer. Reverse-phase protein array (RPPA) data revealed the activation of AMPK and the dephosporylation of mTORC1 downstream targets such as 4E-BP1(S65), p70S6K(T389) and RPS6(S235/236), suggesting that ChoKα inhibition may affect protein synthesis. To further examine the antitumorigenic potential of EB-3D in vivo, a syngeneic orthotopical EO771-C57BL/6 mouse model of breast cancer was used. Preliminary results indicated that the compound significantly reduced the tumor size, with no apparent sign of toxicity. The evaluation of EB-3D as in vivo anti-metastatic adjuvant is ongoing, since cell migration and cell invasion ability of the highly metastatic MDA-MB-231 cell line was impaired after in vitro treatment. Taken together our results suggest that EB-3D have a potent cytostatic effect and it could be a promising new anticancer agent, worthy of further development. Citation Format: Elena Mariotto, Roberta Bortolozzi, Roberto Ronca, Luisa C. Lopez-Cara, Benedetta Accordi, Valentina Serafin, Giuseppe Basso, Giampietro Viola. In vitro and in vivo pharmacological study of EB-3D: a novel choline kinase inhibitor for breast cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1233.