Discovery of S64315, a Potent and Selective Mcl-1 Inhibitor.

Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

Structure-Guided Discovery of a Selective Mcl-1 Inhibitor with Cellular Activity.

Myeloid cell leukemia 1 (Mcl-1), an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation when observed in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy, has emerged as an attractive target for cancer therapy. Here, we report the discovery of selective small molecule inhibitors of Mcl-1 that inhibit cellular activity. Fragment screening identified thienopyrimidine amino acids as promising but nonselective hits that were optimized using nuclear magnetic resonance and X-ray-derived structural information. The introduction of hindered rotation along a biaryl axis has conferred high selectivity to the compounds, and cellular activity was brought on scale by offsetting the negative charge of the anchoring carboxylate group. The obtained compounds described here exhibit nanomolar binding affinity and mechanism-based cellular efficacy, caspase induction, and growth inhibition. These early research efforts illustrate drug discovery optimization from thienopyrimidine hits to a lead compound, the chemical series leading to the identification of our more advanced compounds S63845 and S64315.

Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.

The IGF2/IR/IGF1R Pathway in Tumor Cells and Myofibroblasts Mediates Resistance to EGFR Inhibition in Cholangiocarcinoma.

Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal growth factor receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechanisms against EGFR inhibition.Experimental Design: Resistant CCA cells to EGFR inhibition were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid growth were determined in vitro, and tumor growth was evaluated in CCA xenograft models.Results: Erlotinib-resistant CCA cells displayed metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial-mesenchymal transition (EMT) and a cancer stem cell (CSC)-like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like growth factor (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expression. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated fibroblasts (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor growth and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by fibroblasts boosted IR/IGF1R signaling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated fibroblast proliferation and activation.Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribution of stromal cells. Clin Cancer Res; 24(17); 4282-96. ©2018 AACR.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth.

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Epithelial-mesenchymal transition in cholangiocarcinoma: From clinical evidence to regulatory networks.

Cholangiocarcinoma (CCA) is an aggressive tumor with a poor prognosis due to its late clinical presentation and the lack of effective non-surgical therapies. Unfortunately, most of the patients are not eligible for curative surgery owing to the presence of metastases at the time of diagnosis. Therefore, it is important to understand the steps leading to cell dissemination in patients with CCA. To metastasize from the primary site, cancer cells must acquire migratory and invasive properties by a cell plasticity-promoting phenomenon known as epithelial-mesenchymal transition (EMT). EMT is a reversible dynamic process by which epithelial cells gradually adopt structural and functional characteristics of mesenchymal cells, and has lately become a centre of attention in the field of metastatic dissemination. In the present review, we aim to provide an extensive overview of the current clinical data and the prognostic value of different EMT markers that have been analysed in CCA. We summarize all the regulatory networks implicated in EMT from the membrane receptors to the main EMT-inducing transcription factors (SNAIL, TWIST and ZEB). Furthermore, since a tumor is a complex structure not exclusively formed by tumor cells, we also address the prominent role of the main cell types of the desmoplastic stroma that characterizes CCA in the regulation of EMT. Finally, we discuss the therapeutic considerations and difficulties faced to develop an effective anti-EMT treatment due to the redundancies and bypasses among the pathways regulating EMT.

Loss of ezrin in human intrahepatic cholangiocarcinoma is associated with ectopic expression of E-cadherin.

AIMS: Ezrin connects proteins from the plasma membrane to the subcortical cytoskeleton, and contributes to epithelial integrity by interacting with the cell-cell adhesion molecule E-cadherin. In the liver, ezrin is restricted to cholangiocytes, where it regulates biliary secretory functions. During carcinogenesis, ezrin expression is impaired and associated with enhancement of cell migratory activity in cancer cells; therefore, we aimed to analyse ezrin in cholangiocarcinogenesis. METHODS AND RESULTS: Ezrin expression was evaluated by immunohistochemistry on tissue microarrays from 94 surgical specimens of intrahepatic cholangiocarcinoma (CCA), and correlated with clinicopathological factors and E-cadherin expression. Ezrin function was also analysed in human CCA cell lines. In CCA, ezrin was negative/weakly expressed in 49 cases (52%) and moderately/strongly expressed in 45 cases (48%), mostly in cell cytoplasm. The negative/weak expression of ezrin was more frequent in peripheral than in perihilar CCA (P = 0.002), and was associated with high tumour size (P = 0.001), low mucus secretion (P = 0.042), the presence of satellite nodules (P = 0.024), and ectopic cytoplasmic expression of E-cadherin (P = 0.005). In vitro, silencing of ezrin in CCA cells caused internalization of E-cadherin and favoured cell migration. CONCLUSIONS: Ezrin is down-regulated during cholangiocarcinogenesis, and its loss results in a more aggressive phenotype.

Mitogen-activated protein kinase-activated protein kinase 2 mediates resistance to hydrogen peroxide-induced oxidative stress in human hepatobiliary cancer cells.

The development and progression of liver cancer are characterized by increased levels of reactive oxygen species (ROS). ROS-induced oxidative stress impairs cell proliferation and ultimately leads to cell death. Although liver cancer cells are especially resistant to oxidative stress, mechanisms of such resistance remain understudied. We identified the MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) signaling pathway mediating defenses against oxidative stress. In addition to MK2 and Hsp27 overexpression in primary liver tumors compared to adjacent nontumorous tissues, the MK2/Hsp27 pathway is activated by hydrogen peroxide-induced oxidative stress in hepatobiliary cancer cells. MK2 inactivation or inhibition of MK2 or Hsp27 expression increases caspase-3 and PARP cleavage and DNA breaks and therefore cell death. Interestingly, MK2/Hsp27 inhibition decreases antioxidant defenses such as heme oxygenase 1 through downregulation of the transcription factor nuclear factor erythroid-derived 2-like 2. Moreover, MK2/Hsp27 inhibition decreases both phosphorylation of epidermal growth factor receptor (EGFR) and expression of its ligand, heparin-binding EGF-like growth factor. A new identified partner of MK2, the scaffold PDZ protein EBP50, could facilitate these effects through MK2/Hsp27 pathway regulation. These findings demonstrate that the MK2/Hsp27 pathway actively participates in resistance to oxidative stress and may contribute to liver cancer progression.

Mechanical induction of the tumorigenic ß-catenin pathway by tumour growth pressure.

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch(+)Apc(+/1638N) mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and ß-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of ß-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by ß-catenin nuclear translocation after 15 days. As a consequence, increased expression of ß-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic ß-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.

EGF/EGFR axis contributes to the progression of cholangiocarcinoma through the induction of an epithelial-mesenchymal transition.

BACKGROUND & AIMS: Epithelial-mesenchymal transition (EMT) is a cellular process involved in cancer progression. The first step of EMT consists in the disruption of E-cadherin-mediated adherens junctions. Cholangiocarcinoma (CCA), a cancer with a poor prognosis due to local invasion and metastasis, displays EMT features. EGFR, a receptor tyrosine kinase, plays a major role in CCA progression. The aim of the study was to determine if EMT is induced by EGFR in CCA cells. METHODS: In vivo, the expression of E-cadherin was analysed in CCA tumours of 100 patients and correlated with pathological features and EGFR expression, and in a xenograft model in mice treated with gefitinib, an inhibitor of EGFR. In vitro, the regulation of EMT by EGFR was investigated in CCA cell lines. RESULTS: In human CCA, a cytoplasmic localization of E-cadherin occurred in 50% of the tumours was associated with the peripheral type of CCA, tumour size, the presence of satellite nodules and EGFR overexpression. In xenografted tumours, E-cadherin displayed a cytoplasmic pattern whereas the treatment of mice with gefitinib restored the membranous expression of E-cadherin. In vitro, EGF induced scattering of CCA cells that resulted from the disruption of adherens junctions. Internalization and decreased expression of E-cadherin, as well as nuclear translocation of ß-catenin, were observed in EGF-treated CCA cells. In these cells, EMT-transcription factors (i.e., Slug and Zeb-1) and mesenchymal markers (i.e., N-cadherin and α-SMA) were induced, favoring cell invasiveness through cytoskeleton remodeling. All these effects were inhibited by gefitinib. CONCLUSIONS: The EGF/EGFR axis triggers EMT in CCA cells highlighting the key role of this pathway in CCA progression.

Hepatic myofibroblasts promote the progression of human cholangiocarcinoma through activation of epidermal growth factor receptor.

UNLABELLED: Intrahepatic cholangiocarcinoma (CCA) is characterized by an abundant desmoplastic environment. Poor prognosis of CCA has been associated with the presence of alpha-smooth muscle actin (α-SMA)-positive myofibroblasts (MFs) in the stroma and with the sustained activation of the epidermal growth factor receptor (EGFR) in tumor cells. Among EGFR ligands, heparin-binding epidermal growth factor (HB-EGF) has emerged as a paracrine factor that contributes to intercellular communications between MFs and tumor cells in several cancers. This study was designed to test whether hepatic MFs contributed to CCA progression through EGFR signaling. The interplay between CCA cells and hepatic MFs was examined first in vivo, using subcutaneous xenografts into immunocompromised mice. In these experiments, cotransplantation of CCA cells with human liver myofibroblasts (HLMFs) increased tumor incidence, size, and metastatic dissemination of tumors. These effects were abolished by gefitinib, an EGFR tyrosine kinase inhibitor. Immunohistochemical analyses of human CCA tissues showed that stromal MFs expressed HB-EGF, whereas EGFR was detected in cancer cells. In vitro, HLMFs produced HB-EGF and their conditioned media induced EGFR activation and promoted disruption of adherens junctions, migratory and invasive properties in CCA cells. These effects were abolished in the presence of gefitinib or HB-EGF-neutralizing antibody. We also showed that CCA cells produced transforming growth factor beta 1, which, in turn, induced HB-EGF expression in HLMFs. CONCLUSION: A reciprocal cross-talk between CCA cells and myofibroblasts through the HB-EGF/EGFR axis contributes to CCA progression.

Mitogenic insulin receptor-A is overexpressed in human hepatocellular carcinoma due to EGFR-mediated dysregulation of RNA splicing factors.

Insulin receptor (IR) exists as two isoforms resulting from the alternative splicing of IR pre-mRNA. IR-B promotes the metabolic effects of insulin, whereas IR-A rather signals proliferative effects. IR-B is predominantly expressed in the adult liver. Here, we show that the alternative splicing of IR pre-mRNA is dysregulated in a panel of 85 human hepatocellular carcinoma (HCC) while being normal in adjacent nontumor liver tissue. An IR-B to IR-A switch is frequently observed in HCC tumors regardless of tumor etiology. Using pharmacologic and siRNA approaches, we show that the autocrine or paracrine activation of the EGF receptor (EGFR)/mitogen-activated protein/extracellular signal-regulated kinase pathway increases the IR-A:IR-B ratio in HCC cell lines, but not in normal hepatocytes, by upregulating the expression of the splicing factors CUGBP1, hnRNPH, hnRNPA1, hnRNPA2B1, and SF2/ASF. In HCC tumors, there is a significant correlation between the expression of IR-A and that of splicing factors. Dysregulation of IR pre-mRNA splicing was confirmed in a chemically induced model of HCC in rat but not in regenerating livers after partial hepatectomy. This study identifies a mechanism responsible for the generation of mitogenic IR-A and provides a novel interplay between IR and EGFR pathways in HCC. Increased expression of IR-A during neoplastic transformation of hepatocytes could mediate some of the adverse effects of hyperinsulinemia on HCC.

Immunohistochemical profile of ezrin and radixin in human liver epithelia during fetal development and pediatric cholestatic diseases.

AIM: Ezrin and radixin are actin-binding proteins that contribute to the integrity of epithelia. Abnormalities of bile secretion occur primarily in cholestatic liver diseases and are associated with changes in cell cytoskeleton. Expression of these proteins during liver development and in cholestatic liver diseases remains poorly investigated. METHODS: Ezrin and radixin expression was analyzed in fetal, adult and pediatric cholestatic human liver (i.e. biliary atresia, sclerosing cholangitis) by immunohistochemistry. RESULTS: In adult and fetal livers, ezrin was expressed exclusively in the cells of the biliary lineage (i.e. biliary epithelial cells and ductal cells) whereas radixin was located not only in hepatocytes but also in cells of the biliary lineage. In the lobule of mature livers, radixin displayed a zonal distribution with predominant expression in the periportal region. In cholestatic diseases, both proteins were expressed in cells of the ductular reaction. An aberrant expression of ezrin was detected in hepatocytes of cirrhotic nodules with a CK7-positive pattern and in malignant hepatocytes in a course of cholestatic disease toward cancer. CONCLUSIONS: Among the components of the liver epithelial cells, ezrin was exclusively expressed in biliary phenotype cells, while radixin was found in biliary and hepatocytic lineages, with a periportal zonal expression. In cholestatic diseases, ezrin was expressed in hepatocytes supporting the appearance of a biliary phenotype.

Roles of the scaffolding proteins NHERF in liver biology.

Scaffold proteins are defined by the presence of specific protein-binding domains (e.g. PDZ domains) that assemble several proteins into functional complexes. Thus, scaffolds are critical for spatio-temporal organization and for proper regulation of intracellular signalling upon specific stimulus. Identified 15years ago, NHERF scaffold proteins contain several PDZ modules and were initially viewed as "passive linkers" between transmembrane proteins and the cortical cytoskeleton underlying the plasma membrane. New NHERF-binding molecules involved in cell signalling have been recently identified. Thus, NHERFs are now viewed as "active" key players in regulating cellular functions. EBP50 and PDZK1, two members of the NHERF family, are highly expressed in the liver where they link receptors, channels, transporters and cytosolic components. This review aims to give an overview of the emerging functions of NHERF proteins in liver physiology.

Ezrin-radixin-moesin-binding phosphoprotein (EBP50), an estrogen-inducible scaffold protein, contributes to biliary epithelial cell proliferation.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) anchors and regulates apical membrane proteins in epithelia. EBP50 is inducible by estrogen and may affect cell proliferation, although this latter function remains unclear. The goal of this study was to determine whether EBP50 was implicated in the ductular reaction that occurs in liver disease. EBP50 expression was examined in normal human liver, in human cholangiopathies (ie, cystic fibrosis, primary biliary cirrhosis, and primary sclerosing cholangitis), and in rats subjected to bile-duct ligation. The regulation of EBP50 by estrogens and its impact on proliferation were assessed in both bile duct-ligated rats and Mz-Cha-1 human biliary epithelial cells. Analyses of cell isolates and immunohistochemical studies showed that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and cystic fibrosis transmembrane conductance regulator, in the apical domains of cholangiocytes. In both human cholangiopathies and bile duct-ligated rats, EBP50 was redistributed to the cytoplasmic and nuclear compartments. EBP50 underwent a transient increase in rat cholangiocytes after bile-duct ligation, whereas such expression was down-regulated in ovariectomized rats. In addition, in Mz-Cha-1 cells, EBP50 underwent up-regulation and intracellular redistribution in response to 17beta-estradiol, whereas its proliferation was inhibited by siRNA-mediated EBP50 knockdown. These results indicate that both the expression and distribution of EBP50 are regulated by estrogens and contribute to the proliferative response in biliary epithelial cells.

The Kell and XK proteins of the Kell blood group are not co-expressed in the central nervous system.

The Kell blood group is constituted by two covalently linked antigens at the surface of red blood cells, Kell and Kx. Whereas Kell is a metalloprotease with demonstrated in vitro enzymatic activity, the role of Kx thereon, and/or alone, remains unknown, although its absence is linked to the McLeod syndrome, a neuroacanthocytosis. In the central nervous system, the expression of Kell and XK has been suggested, but their expression patterns remain uncharacterized, as are the post-translational pathogenic mechanisms involved in the development of the McLeod syndrome. The distributions of Kell and XK were thus studied by in situ hybridization as well as immunohistochemistry in rodent and human brain. The results reveal an independent localization of the two constituents of the Kell blood group, XK (Kx) being expressed throughout this tissue, whereas Kell expression is restricted to red blood cells in cerebral vessels. The XK protein is shown to be neuronal, located mainly in intracellular compartments, suggesting a cell specific trafficking pattern, possibly associated with specific physiological functions.

The Kell protein of the common K2 phenotype is a catalytically active metalloprotease, whereas the rare Kell K1 antigen is inactive. Identification of novel substrates for the Kell protein.

The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.