Stéphane Vispé

DNA methylation inhibitors in cancer: Recent and future approaches

This review presents the different human DNA methyltransferases (DNMTs), their biological roles, their mechanisms of action and their role in cancer. The description of assays for detecting DNMT inhibitors (DNMTi) follows. The different known DNMTi are reported along with their advantages, drawbacks and clinical trials. A discussion on the features of the future DNMT inhibitors will conclude this review.

F14512, a Potent Antitumor Agent Targeting Topoisomerase II Vectored into Cancer Cells via the Polyamine Transport System

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.

Triptolide is an inhibitor of RNA polymerase I and II–dependent transcription leading predominantly to down-regulation of short-lived mRNA

Triptolide, a natural product extracted from the Chinese plant Tripterygium wilfordii, possesses antitumor properties. Despite numerous reports showing the proapoptotic capacity and the inhibition of NF-κB–mediated transcription by triptolide, the identity of its cellular target is still unknown. To clarify its mechanism of action, we further investigated the effect of triptolide on RNA synthesis in the human non–small cell lung cancer cell line A549. Triptolide inhibited both total RNA and mRNA de novo synthesis, with the primary action being on the latter pool. We used 44K human pan-genomic DNA microarrays and identified the genes primarily affected by a short treatment with triptolide. Among the modulated genes, up to 98% are down-regulated, encompassing a large array of oncogenes including transcription factors and cell cycle regulators. We next observed that triptolide induced a rapid depletion of RPB1, the RNA polymerase II main subunit that is considered a hallmark of a transcription elongation blockage. However, we also show that triptolide does not directly interact with the RNA polymerase II complex nor does it damage DNA. We thus conclude that triptolide is an original pharmacologic inhibitor of RNA polymerase activity, affecting indirectly the transcription machinery, leading to a rapid depletion of short-lived mRNA, including transcription factors, cell cycle regulators such as CDC25A, and the oncogenes MYC and Src. Overall, the data shed light on the effect of triptolide on transcription, along with its novel potential applications in cancers, including acute myeloid leukemia, which is in part driven by the aforementioned oncogenic factors. [Mol Cancer Ther 2009;8(10):2780–90]

Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis

The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.

Mammalian Rad51 protein: a RecA homologue with pleiotropic functions.

During the last years, homologues of E coli RecA have been cloned in numerous species including man. These Rad51 proteins share sequence as well as functional homologies with the bacterial protein. Human Rad51 (HsRad51) is able to catalyze strand exchange in vitro between homologous DNAs, but with a lower efficiency compared to that of RecA. This suggests the requirement of additional factors. A very interesting feature of Rad51 is its essential role in mouse which could mean that it has gained an essential function in cell growth. The interaction of HsRad51 with several tumor suppressor genes namely p53, BRCA1 and BRCA2 implies possible role(s) of this protein in tumorigenesis. Thus, the continued study of Rad51 should bring important insights not only into homologous recombination mechanisms but also into cell proliferation regulation.

Synthesis of conjugated spermine derivatives with 7-nitrobenzoxadiazole (NBD), rhodamine and bodipy as new fluorescent probes for the polyamine transport system

The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N(1)-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.

Design, Synthesis and Biological Evaluation of 4-Amino-N-(4-aminophenyl)benzamide Analogues of Quinoline-Based SGI-1027 as Inhibitors of DNA Methylation

Quinoline derivative SGI‐1027 (N‐(4‐(2‐amino‐6‐methylpyrimidin‐4‐ylamino)phenyl)‐4‐(quinolin‐4‐ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine‐5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI‐1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compounds—namely the derivatives 12, 16, 31 and 32—exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re‐expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG‐1 cells. These compounds possessed cytotoxicity against leukemia KG‐1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI‐1027. Structure–activity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one‐ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure–activity relationships of SGI‐1027 and its derivative.

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.

Antitumor activity of pyridoisoquinoline derivatives F91873 and F91874, novel multikinase inhibitors with activity against the anaplastic lymphoma kinase.

The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin–ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.

Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome pathway inhibitors

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.