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The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01–1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 ± 20 seconds in control cells to 38 ± 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility. [Mol Cancer Ther 2008;7(7):2080–9]

Supplementary Fig. S1 from Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites

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

Angiogenesis is a key event in tumor progression and metastasis. This complex process, which constitutes a potent target for cancer therapy, is inhibited by very low concentrations of microtubule-targeting drugs (MTD). However, the intimate mechanisms of the antiangiogenic activity of MTDs remain unclear. Recently, we have shown that low antiangiogenic and noncytotoxic concentrations of paclitaxel induced an unexpected increase in microtubule dynamics in endothelial cells. In this study, we showed that vinflunine, the newest Vinca alkaloid, increased microtubule dynamic instability in human endothelial cells after 4-hour incubation at low concentrations (29% and 54% at 0.1 and 2 nmol/L). The growth and shortening rates were increased, and the percentage of time spent in pause and the mean duration of pauses were decreased, as previously observed with paclitaxel. As opposed to paclitaxel, the transition frequencies were not significantly disturbed by vinflunine. Moreover, low concentrations of vinflunine did not affect mitotic index and anaphase/metaphase ratio. Interestingly, these low vinflunine concentrations that increased microtubule dynamics exhibited an antiangiogenic effect through the inhibition of both morphogenesis and random motility. Capillary tube formation on Matrigel was decreased up to 44%. The cell speed and the random motility coefficient were decreased (13% and 19% and 13% and 33% at 0.1 and 2 nmol/L, respectively) and the persistent time was statistically increased. Altogether, our results confirm that the increase in microtubule dynamics is involved in MTD antiangiogenic activity and highlight the crucial role of interphase microtubule dynamics in angiogenesis. (Cancer Res 2006; 66(6): 3256-63)

Supplementary Figure 1 from Antiangiogenic Concentrations of Vinflunine Increase the Interphase Microtubule Dynamics and Decrease the Motility of Endothelial Cells

Vinflunine, a new microtubule-targeting drug, has a marked antitumor activity in vitro and in vivo. Here, we studied the mechanisms mediating resistance to vinflunine. We investigated the response to vinflunine of ovarian cancer cells initially selected as paclitaxel-resistant cells (A2780-TC1 cells). By comparison with A2780-wild-type (wt) cells, we showed that A2780-TC1 cells were highly resistant to vinflunine, with resistance factors reaching 800 and 1,830 for IC50 and IC70, respectively. We showed that P-glycoprotein minimally participated in this cell resistance. The examination of tubulin composition revealed increased levels of acetylated α-tubulin, βII-tubulin, and βIII-tubulin in A2780-TC1 cells before vinflunine treatment. As a consequence, vinflunine unequally affected microtubule network organization and function in A2780-wt and A2780-TC1 cells. Whereas the drug depolymerized microtubules and induced a mitotic block in A2780-wt cells, it did not depolymerize microtubules and induced a G2 block in A2780-TC1 cells. Elsewhere, the mitochondrial protein Bcl-2 was down-regulated in A2780-TC1 cells. This down-regulation was related to resistance, as A2780-TC1 cells stably transfected with a Bcl-2 construct recovered a partial sensitivity to vinflunine. Lastly, we confirmed the role played by Bcl-2 by showing that the mitochondrial membrane potential was only disrupted by vinflunine in cells expressing Bcl-2. Altogether, our results indicate that modifications acquired during treatment (i.e., paclitaxel) have significant consequences on cell response to the following drug (i.e., vinflunine). Especially, this study shows that a specific pool of tubulin subtypes and a down-regulation of Bcl-2 are associated with resistance of ovarian cancer cells to vinflunine. [Mol Cancer Ther 2006;5(11):2824–33]

Purpose: The purpose of the study was to investigate the mechanisms associated with antitumor activity and resistance to F11782, a novel dual catalytic inhibitor of topoisomerases with DNA repair-inhibitory properties.Experimental Design: For that purpose, an F11782-resistant P388 leukemia subline (P388/F11782) has been developed in vivo and characterized.Results: Weekly subtherapeutic doses of F11782 (10 mg/kg) induced complete resistance to F11782 after 8 weekly passages. This resistant P388/F11782 subline retained some in vivo sensitivity to several DNA-topoisomerase II and/or I complex-stabilizing poisons and showed marked collateral sensitivity to cisplatin, topotecan, colchicine, and Vinca alkaloids, while proving completely cross-resistant only to merbarone and doxorubicin. Therefore, resistance to F11782 did not appear to be associated with a classic multidrug resistance profile, as further reflected by unaltered drug uptake and no overexpression of resistance-related proteins or modification of the glutathione-mediated detoxification process. In vivo resistance to F11782 was, however, associated with a marked reduction in topoisomerase IIα protein (87%) and mRNA (50%) levels, as well as a diminution of the catalytic activity of topoisomerase IIα. In contrast, only minor reductions in topoisomerases IIβ and I levels were recorded. However, of major interest, nucleotide excision repair activity was decreased 3-fold in these P388/F11782 cells and was more specifically associated with a decreased (67%) level of XPG (human xeroderma pigmentosum group G complementing protein), an endonuclease involved in this DNA repair system.Conclusions: These findings suggest that both topoisomerase IIα and XPG are major targets of F11782 in vivo and further demonstrate the original mechanism of action of this novel compound.

Epithelial ovarian cancer is the fourth cause of death among cancer-bearing women, frequently associated with carboplatin resistance, underlining the need of more efficient and targeted therapies. F14512 is a novel concept of drug vectorization that we propose here to investigate in ovarian cancer models. F14512 is an epipodophyllotoxin-core linked to a spermine chain, which enters selectively tumor cells via the polyamine transport system (PTS), currently in clinical phase II evaluation in AML. We compared the effects of F14512 versus etoposide against OVCAR-3, IGROV-1, SKOV-3, A2780S and A2780R cancer cell lines. Using the F17073 PTS fluorescent probe, we determined the PTS activity of these cells and of 18 clinical samples. As results, F14512 displayed strong anti-proliferative and pro-apoptotic activities in carboplatin-resistant models, being increased in cell lines with high levels of PTS. Consistently, at a dose of 1.25 mg/kg, F14512 significantly inhibited tumor growth in cisplatin-resistant SKOV-3 xenograft model. Interestingly, ex vivo analysis indicated that 15 patients sample out of 18 presented a higher F17073 fluorescent probe incorporation into CD326-positive tumor cells as compared to normal cells, even in the case of 2 platinum-refractory patients. Therefore, F14512 is a targeted drug with a potent anti-tumor efficacy regardless the status of cisplatin resistance. The high PTS activity detected in fresh clinical samples highlights the potential of F14512 as a new therapy for PTS-positive and platinum resistant ovarian cancer patients. Citation Format: Benoît Thibault, Gregoire Zorza, Samuel Meignan, Nicolas Guilbaud, Christian Bailly, Jean-Pierre Delord, Bettina Couderc, Anna Kruczynski, Pierre Ferre, Jean-Philippe Annereau. Translational study of F14512, a novel vectorised epipodophyllotoxin, which demonstrates a marked activity on ovarian cancer models from patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2428. doi:10.1158/1538-7445.AM2015-2428

In an effort to better characterize the activity of a multi-kinase inhibitors, antiproliferative activity was evaluated against a panel of human tumor cell lines. Among the different cell lines tested we have been able to highlight that the KM12 human colorectal carcinoma present hypersensitivity to inhibition. By intersecting the results of activity of the compound and the bibliographic data, we could confirm that the activity of the compound was mediated by the inhibition of phosphorylation of the receptor NTRK1. The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the hight-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a human colon carcinoma as a transforming oncogene activated by a somatic rearrangement that fused TPM3 (non-muscle tropomyosin) gene to kinase domain of a novel tyrosine-kinase receptor. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. Using differential quantitative reverse transcriptase PCR and optimised immunohistochemistry we could confirm the existence of low frequency (0.5%) NTRK1 re-arranged / activated forms in colorectal carcinoma. A same colorectal tumor sample could contain different fusion transcripts, highlighting the molecular heterogeneity of this type of tumor Furthermore we detect a novel NTRK1 fusion in colorectal carcinoma clinical samples corresponding to oncogenic rearrangement TRK-T2 identified in papillary thyroid carcinoma. These results confirm the potential interest of the target NTRK1 in the panel of targeted therapies against colorectal cancer. Citation Format: Laurent Creancier, Caroline Dejean, Isabelle Vandenberghe, Jean-Christophe Blanchet, Yannick Aussagues, Jean Philippe Annereau, Janick Selves, Anna Kruczynski. Identification of new NTRK1 gene fusion as oncogene target in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2015-4265

Despite recent advances in the therapy of metastatic melanoma, patients have a poor prognosis and die from their disease, with a 5-year survival rate of less than 20%. Metastatic melanoma remains mainly chemoresistant, therefore novel therapeutic strategies to overcome primary or acquired resistance are required. In order to more reliably predict clinical activity of novel compounds in melanoma patients, we have established a series of patient-derived tumor xenograft models (PDTX). Surgically resected tumors samples were obtained from patients with metastatic melanoma before or after treatment. Twenty-three samples from 9 patients and representative of various stages of disease progression were implanted onto immunocompromised NSG mice. A high take rate of 56% (13/23 samples) was observed, corresponding to the samples from 8/9 patients. Tumor take was independent of tumor size, histologic parameters and B-RAF status. The first tumor generation (graft) harbouring the patient-derived sample (G1), as well as the subsequent generations were characterized for each PDTX. The pattern of expression of the melanoma differentiation markers S100, melanosome, tyrosinase and melan-A were maintained over the serial transplantations. These models also closely recapitulated the heterogeneity of patient tumors in terms of cell morphology over the first generations (G1-G5), but this heterogeneity tended to decrease with the number of generations. One of these primary PDTXs, MEL-11 exhibited metastatic spread in axillary lymph nodes. Furthermore 2 other primary melanoma PDTXs, MEL-1 and MEL-3, established from primary refractory patient metastatic melanoma retained complete resistance to temozolomide and vemurafenib, the two reference drugs for melanoma. The MEL-3 model was selected to assess the anti-melanoma activity of F-RK-4, a novel multi-kinases inhibitor (including inhibition of mutated B-RAF). Multiple i.p. administrations of F-RK-4 resulted in a significant antitumor activity, as reflected by a tumor growth inhibition of 50%. Overall these results suggest that these patient-derived melanoma xenografts represent a useful preclinical tool to identify novel anti-melanoma therapeutics. Citation Format: Bruno Gomes, Céline ROBICHON, Arnaud Pillon, Jean-Philippe Annereau, Sandrine Pourtau, Jean-Christophe Blanchet, Aline Stennevin, Karim Bedjeguelal, Philippe Rochaix, Ignacio Garrido-Stowhas, Laurence Lamant, Nicolas Meyer, Nicolas Guilbaud, Christian Bailly, Anna Kruczynski. Patient-derived tumor models of resistant metastatic melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1219. doi:10.1158/1538-7445.AM2014-1219

Rapidly proliferating cancer cells have a higher demand on polyamines resulting in an over-activated Polyamines Transport System (PTS)1. Polyamines entry can be exploited as a selective anticancer drug delivery system. We synthesized polyamines-epipodophylotoxin conjugates with improved physico-chemical and pharmacological properties. As proof of concept, we demonstrated that the conjugation of epipodophylotoxin with spermine changes the pharmacological profile of the cytotoxic moiety by increasing the solubility, enhances the cellular distribution through the PTS, and increases the capacity to inhibit topoisomerase II due to stronger DNA interaction, as compared to the closest structurally related compound etoposide. Among several conjugates, F145122 was selected as a drug candidate and is currently undergoing a phase 1/2 clinical evaluation in acute myeloid leukaemia (AML). Based on this clinically validated objective to target cancer cells with the PTS, we set up a chemical platform of conjugation of polyamine moieties. We extended our approach to 3 natural products of interest in oncology: (i) an inhibitor of elongation phase in protein translation, a modulator of redox cell regulation and an inhibitor of polyADP-ribose polymerase. This approach led to the synthesis of novel spermine-pancratistatine, -artemisinine and -PARPi conjugates and their biochemical and biological characterization in terms of protein synthesis, PARP inhibition, ROS induction, anti-proliferative effects on cancer cells and their capacity to be imported via the PTS. Cell internalisation through the PTS was measured by differential cytotoxicity on PTS+ vs. PTS− cells, or by competition with a fluorescent probe. We also evaluated selected compounds in vivo and investigated whether their modified properties translate into a higher level of antitumor activity associated with an enlarged therapeutic index in a PTS+ tumor model. For the PARP inhibitor-spermine conjugates, an increase in cellular uptake was confirmed, without impairing the cytotoxic properties. The dedicated polyamine conjugation platform presented here can be adapted to many cytotoxic scaffolds to enhance their solubility, preferential incorporation into cancer cells through the PTS, and their pharmacological activity in vitro and in vivo. The design of polyamine-vectorized cytotoxic agents will be presented. 1 Delcros J.G. et al., Biochem. J., 1993, 269 2 PCT Int. Appl. WO2005/100363 (Pierre Fabre Medicament); Barret J.-M. et al., Cancer Res. 2008, 68, 9845 Citation Format: Frédéric Liéby-Muller, Jean-Philippe Annereau, Viviane Brel, Frédéric Marion, Yves Guminski, Florence Redoules, Nathalie de Saint Jores, Karine André, Anna Kruczynski, Christian Bailly, Nicolas Guilbaud. Tumor targeting and enhanced efficacy of novel polyamine-cytotoxic conjugates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5565. doi:10.1158/1538-7445.AM2013-5565

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC F14512 is a novel polyamine-vectorized topoisomerase II inhibitor currently in phase 1/2 clinical trial in acute myeloid leukemia (AML). Since ABCB1 (also called P-glycoprotein or MDR1) has been reported to be expressed at high levels in patients with resistant leukaemia cells, and constitutes a pejorative marker of therapy, we decided to investigate whether F14512 could be exported by ABCB1 or other ABC transporters susceptible to confer drug resistance to chemotherapy, such as MRP1. In addition, we established a F14512-resistant cell line to study the mechanism of resistance to the drug. With membrane preparation enriched in functional MDR1, we found that [F14512][1] was unable to stimulate the ATPase activity of this transporter. Using a cellular assay over expressing MDR1 transporter by recombinant transfection of MDCKII cells, [F14512][1] was detectable, but the intracellular level was unchanged regardless of the MDR1 status or in presence of MDR1 competitors. Consistently, iterative selection of a [F14512][1]-resistant A-549 cell line with 36 cycles of [F14512][1] at cytotoxic doses (0.5 EC50) over 9 months, led to only moderate shift of resistance (9 fold). In the resulting selected A-549 subclones, MDR1, MRP1 and ABCG2 were detectable at the basal level. The ability of [F14512][1] to bypass MDR1 was finally assessed in a vinorelbine-resistant P388 model overexpressing high level of functional ABCB1 as indicated with the positive rhodamine export assay and cross resistance to MDR1 substrate. Moreover, [F14512][1] displays strong antileukemic activity in MDR1-positive P388 cells in vitro and additional in vivo measurements are in progress. In conclusion, the data concur to show that the antitumor activity of the targeted cytotoxic agent [F14512][1] is not impacted by the MDR1 status of cancer cells. MDR1 clearly does not affect the potency of [F14512][1] and this characteristic augurs well for the ongoing development of the drug in AML patients. Citation Format: Jean Philippe Annereau, Viviane Brel, William Riquet, Laurent Creancier, Isabelle Vandenberghe, Emmanuel Fournier, Celine Robichon, Aline Stennevin, Vanessa Offrete, Laurence Lacastaigneratte, Bruno Gomes, Anna Kruczynski, Christian Bailly, Nicolas Guilbaud. F14512, a novel vectorized topoiserase II inhibitor, bypasses MDR1 mediated resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 988. doi:10.1158/1538-7445.AM2013-988 [1]: /lookup/external-ref?link_type=GEN&access_num=F14512&atom=%2Fcanres%2F73%2F8_Supplement%2F988.atom

F14512 is a novel vectorized topoisomerase II inhibitor currently in phase I clinical study in AML. Briefly, F14512 combines a polyamine (spermine) chain with an epipodophyllotoxin warhead. An enhanced antitumor effect associated with a large therapeutic index results from this specific targeting of cancer cells through polyamine transport system. One of the most important limitations in chemotherapeutic treatment is acquired or innate drug resistance. To investigate this phenomenon for F14152, we selected A549 NSCLC cell lines upon F14512 treatment by increasing doses selection, and characterized the profile of resistance in term of ABC transporter expression and modification of topoisomerase II expression. As results, resistant cell lines were selected after 46 passages and 32 treatment of F14512 with doses ranging 0.25 to 4 IC50 of inhibition of proliferation, over a period of 9 months. As key observation, the intense selection pressure led solely to a moderated shift of EC50 of proliferation, not mediated through MDR1 nor MRP1, but more likely attributable to reduction of topoisomerase II expression. Moreover, we demonstrated that F14512 retains significant antiproliferative activity of F14512 on cell line overexpressing MDR1: CEM selected in vinblastine, A-549 in vinflunine, and P388 in vinorelbine, with a striking enhanced impact on the later model. According to these first investigations, we conclude that MDR1 would not be involved in the resistance mechanism against F14512. We are investigating hypotheses potentially explaining the collateral sensitivity to this novel polyamine derivative on MDR1 expressing P-388 cells.

New antileukemic agents are urgently needed to achieve improvement of the survival of patients suffering from Acute Myelogenous Leukemia (AML). F14512 combines an epipodophyllotoxin core with a spermine moiety that targets cells with a high demand for polyamines such as cancer cells. F14512 exhibits a high level of in vivo anti-tumor activity in a series of experimental murine and human solid tumor models and has entered into clinical phase one. In this study, we investigated the in vivo antileukemic activity of F14512 against cell line or primary human AML models and evaluated its potential in combination with the reference antileukemic agent, AraC. HL60 cells or AML cells collected from different patients were engrafted onto NSG mice. Using flow cytometry and q-PCR analysis, we demonstrated that multiple administrations of F14512 for 3 weeks resulted in an extensive reduction of AML cell number in the bone marrow and blood of treated mice. We also showed in vitro and in vivo synergistic activity of F14512 in combination with AraC in HL-60 and primary AML models when both compounds were used at suboptimal doses. The mechanisms triggering primary leukemic cell death were investigated and our results indicated that senescence could be involved. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting the Phase I clinical trials of this novel promising drug candidate. 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 2747. doi:1538-7445.AM2012-2747

Beside their widely recognised efficacy as antimalaric drugs, artemisinin derivatives are also known for their cytotoxic properties since the early nineties. More recently, artemisinin dimers (ADs) were shown to possess an increased potential as anti-cancer agents due to their ability to inhibit cancer cells proliferation in the low nanomolar range. Taking advantage of an original chemistry, we synthesized a new series of trifluoromethylated artemisnin dimers which proved to be particularly potent depending on the size and nature of the linker. Different mode of linkage were performed by either carbon 16 or carbon 10 and were tested in vitro and in vivo. Best combination was found in a hybrid linking position delivering non-symmetrical C10-C16 ADs. One of the lead compound of this series, F98458 demonstrated a moderate (but statiscally significative) anti-tumor activity in mice against a human melanoma xenograft that is, to our knowledge, the first demonstration of in vivo antitumor properties of ADs in preclinical models. Many efforts to understand the molecular origin of the cytotoxic effect of monomeric artemisinin derivatives have been undertaken. One of the aims of this study was to investigate the mechanism of action of ADs in comparison with artemisinin: the antimalaric drug artesunate, a representative monomeric derivative of artemisinin, is known to induce DNA damage and ROS generation while our ADs did not under the same experimental conditions. Moreover ADs did not induce apoptosis (caspase 3/7, annexin V), leading us to investigate cell death related to senescence and autophagy. We have also synthesized partially reduced ADs possessing only one endoperoxide functionality. These compounds did show the same potency than non reduced ADs, underlaying that a tight molecular recognition is involved in its biological response. All these observations suggest that monomeric artemisinin derivatives such as artesunate and ADs possess distinct modes of action. The high in vitro potency associated with the in vivo response led us to synthesize affinity probes in order to identify the molecular target of ADs which could provide fundamental informations for further developpement of these compounds as anti-cancer agents. This work currently underway will also be discussed. 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 1925. doi:1538-7445.AM2012-1925

Chemotherapy remains mainly used for the treatment of Acute Myelogenous Leukemia (AML). However, in the past 3 decades limited progress has been achieved in improving the long-term disease-free survival. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin core targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. In fact the polyamine moiety facilitates F14512 selective uptake by tumor cells via the polyamine transport system, a machinery frequently overactivated in cancer cells. In this study, we report the in vivo antileukemic activity of F14512 against human AML models, established from patient samples. AML cells, collected from 3 different patients, were established onto NSG mice (LAM-2, LAM-7 and LAM-18). These 3 AML samples exhibited a normal karyotype, with FLT3-ITD, NPM1 and DNMTA3 mutations which proved stable over serial transplantations in vivo. After multiple i.v. administrations of F14512, 3 times a week for 3 weeks, an extensive reduction of AML cell number (98–99%) was observed in LAM-2 and LAM-7 - bearing mice. This antileukemic activity was recorded on the basis of flow cytometry, q-PCR and histology assessments. The effects of F14512 on LAM-18 bearing mice were marginal with an inhibition of AML cell growth of 42%. We also show in vitro and in vivo synergistic effects of F14512 in combination with AraC, one of the frontline chemotherapeutic agents for AML. These results were obtained using the HL-60 cell line. The activity of F14512 in combination with AraC will be further investigated in patient AML models. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are on going with this novel promising drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A214.

Drugs specifically vectorized to cancer cells may offer a reinforced activity, resulting in an improved therapeutic index. In this context, F14512 exploits the Polyamines Transport System (PTS) to accumulate into cells and exhibits an enhanced anti-proliferative activity on a large panel of tumor cell lines as compared to etoposide. At the molecular level, the spermine tail of F14512 contributes to enhance the water solubility of the drug, and reinforces the activity of the drug toward its primary molecular target, topoisomerase II. This observation has encouraged the set up of a phase 1 clinical study with F14512 in patients with relapsed or refractory acute myeloid leukemia (AML). In order to determine how these molecular properties translate at the cellular level, we have compared the kinetic of cell cycle modulation associated with the inhibition of cell proliferation. 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 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 -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 vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 as compared to etoposide, and this difference may account for their distinct pharmacological profiles and the superior activity of F14512 in vivo. The identification of such response markers for senescence are currently pursued on in vivo MX-1 models by following a transcriptomic analysis focused on cell death cascades including apoptosis and senescence. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor response, applicable in clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B37.

Alkaloids pancratistatin and narciclasine, isolated from Amaryllidaceae, have been shown to present antitumor activity on preclinical models, including melanoma. Despite extensive investigations, pancratistatin or narciclasine analogues did not succeed to provide sufficient therapeutic benefit, [1] mostly hampered by poor water solubility and systemic toxicity [1]. To overcome those limitations, we developed a chemistry program aimed at improving both their physicochemical and pharmacological properties. A literature survey suggests that any structural modification of this highly oxigenated isocarbostiryl specific scaffold would lead to a loss of activity except on position 1, difficult to achieve but interesting to explore. A series of 40 novel 1-aminopancratistatin derivatives was synthesised starting from large quantities of narciclasine extracted from Narcissus bulbs. These novel compounds exhibit an improved pharmaceutical profile facing standard solubility, plasmatic and microsomal stability assays. Moreover, a subset of narciclasine derivatives demonstrated a significant enhancement of the antiproliferative effect with IC50 reaching the nM range, consistent with a major induction of pro-apoptotic signals, including mitochondrial depolarisation. To explore the mode of action of these alkaloids derivatives, we tested their capacity to inhibit protein synthesis, compared their pharmacological profile with 80 anticancer compounds on 16 tumor cell lines (oncoprofile.16) and explored their impact on major cellular functions such as cellular integrity, cell cycle, topoisomerases inhibition, DNA damage and synthesis. We demonstrated thus that inhibition of DNA synthesis is a key component of their mechanism of action. Based on the pharmacological properties, a structure-activity card can be built to select different categories of pancrastistatin derivatives depending on their ability to inhibit preferentially protein synthesis or/and DNA synthesis. Interestingly, the dimethylaminomethyl benzamide derivative F98604 exhibited a significant antiproliferative activity on the murine B16 and human A375 melanoma xenograft models associated with an increased therapeutic index as compared with the natural compound narciclasine. In conclusion, modulation of position 1 on the narciclasine pharmacophore overcomes the solubility issue and is a key position to modify their pharmacological profiles, this opening new perspectives as anticancer agents active against melanoma. 1. A. Kornienko, A. Evidente Chem. Rev. 2008, 108, 1982 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 3261. doi:10.1158/1538-7445.AM2011-3261

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Targeted anticancer therapies represent an increasing subject of interest in order to improve tumor cell selectivity. Extensive study suggests that the Polyamine Transport System (PTS) is an energy-dependent machinery generally hyper-activated in cancer cells with a high demand for polyamines. This system can be viewed as a suitable molecular target to deliver selectively polyamine-based molecules into cancer cells. We exploited this strategy to target a potent cytotoxic agent to PTS-positive tumor cells with [F14512][1], a novel polyamine-epipodophyllotoxin conjugate that exhibits a high in vivo anti-tumor activity in a series of experimental murine and human tumors. [F14512][1] has recently entered into clinical development. This preclinical study was undertaken to investigate its potential in combination chemotherapy therapies. The in vitro cytotoxicity of [F14512][1] against the A549 human non-small cell lung cancer cell line was investigated following simultaneous incubation with a variety of cytotoxic/cytostatic drugs. When using median effect analysis, [F14512][1] in combination with 5-fluorouracil, camptothecin, gemcitabine or mitoxantrone showed synergistic cytotoxicity. Additive effects were demonstrated when [F14512][1] was combined with bortezomib, doxorubicin or SAHA while antagonistic anti-proliferative effects were observed only with co-incubation of [F14512][1] and etoposide, vinorelbine or paclitaxel. Combinations inducing synergistic anti-proliferative activities were also investigated against the HL-60 human acute myeloid leukemia cell line, confirming the synergy with gemcitabine. Cellular effects of [F14512][1] are characterized by a DNA damage induction, a cell cycle blockage in G2 phase and a cell death induction. Such characteristics were investigated in A549 cells treated with this combination in order to propose mechanistic explanation of this synergy. In vivo, [F14512][1] combined with irinotecan, gemcitabine, mitoxantrone or doxorubicin showed a gain of anti-tumor activity against P388 murine leukemia grafted intravenously. As an example, the administration of sub-optimal doses of [F14512][1] (0.63 mg/kg, q1d4 schedule) and mitoxantrone (1.25 mg/kg, single dose) alone resulted in an increased life span of only 29% compared to untreated mice, while combined treatments improved survival of tumor bearing mice with an increase life span of 57%. Moreover, [F14512][1] combined with doxorubicin displayed an increased antitumor activity against MX-1 human breast cancer in nude mice.F14512 appears to be a promising candidate for combination chemotherapy, especially with DNA-damaging agents and antimetabolites. 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 3518. doi:10.1158/1538-7445.AM2011-3518 [1]: /lookup/external-ref?link_type=GEN&access_num=F14512&atom=%2Fcanres%2F71%2F8_Supplement%2F3518.atom

The Amaryllidaceous alkaloids pancratistatin and narciclasine are known to exert marginal antitumor properties but associated with toxicities. Despite extensive investigations, pancratistatin or analogues did not succeed to provide sufficient therapeutic benefit although a new pro-drug strategy was applied recently to narciclasine. We decided to undertake a chemistry program aimed at modifying the skeleton of this attractive pharmacophore in order to improve its therapeutic application. At first sight, literature survey suggests that any change in the structure would lead to a loss of activity but a careful examination of published data indicated that position −1 of the molecule may be of potential interest although poorly investigated. We report here the synthesis of a series of 39 novel derivatives of 1-aminopancratistatin by chemical modification of the naturally available narciclasine extracted from Narcissus bulbs. These new compounds present improved pharmaceutical properties such as higher aqueous solubility (from 300 µg/ml for narciclasine up to 2000 µg/ml) without loss of stability tested on human and murine microsomal assays (70-90% stability after 1h incubation). A sub-set of these novel derivatives demonstrated a drastic increase of the cytotoxic activity reaching nM range tested on a panel of 8 different cell lines. The higher antiproliferative activity translates towards the induction of stronger pro-apoptotic signals evidenced with Annexin V, caspase 3/7 and JC-1 assay. Besides the previously described inhibition of protein synthesis, our studies originally demonstrated that part of the mechanism of action involves inhibition of DNA synthesis as well. The contribution of these two pharmacological properties elaborates a SAR (structure - activity relationship) and segregates our new pancrastistatin derivatives into different categories depending on their ability to inhibit protein synthesis, DNA synthesis or both. Based on their differential in vitro activity profile, the compounds were selected for evaluation of their in vivo antitumor properties on murine and xenografted models. As a major result F 98604 (dimethylaminomethyl benzamide derivative of pancratistatin) exhibited significant and reproducible activity on the murine B16 sc and human A375 melanoma models in terms of tumor growth inhibition as well as increased therapeutic index. The present SAR suggests that modification of the natural pancratistatin or narciclasine pharmacophore at the position −1 open new perspectives as potential anticancer agents with novel pharmacological profiles. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5461.

One of the major concerns for chemotherapy is the selective targeting of drugs into highly proliferative cancer cells. Natural polyamines (spermine, spermidine, putrescine) are essential for the regulation of cellular growth and differentiation. Due to their highly proliferative nature, cancer cells have a pronounced need to import polyamines from their external environment, through the polyamine transporter system (PTS). On the basis of this biological mechanism, we vectorized the new cytotoxic anticancer compound F14512, a epipodophyllotoxin — spermine conjugate, into tumor cells.1 Here we present the synthesis and the structure-activity relationships of a new series of compounds constituted by an podophyllotoxin core tethered with a polyamine moiety with a variable spacer. Two synthetic strategies with protected polyamines, and a direct 3 steps synthesis of F14512 from natural podophyllotoxin and spermine without any protection are presented.2 This series of topoisomerase II inhibitors were checked for their cytotoxicity on A549 lung cancer cell line, displaying marked potency up to nM range. Cancer cell internalization through PTS was assessed by selective cytotoxicity on different PTS expressing cell lines, and by competition experiments. Our results displayed a potent specificity for the conjugated tetramine (spermine) compounds, which were more recognized than the triamine (spermidine) ones, while mono and diamines showed no selectivity. Lead compounds were also tested in vivo and proved potent antitumor activity. This series of new water-soluble cytotoxic compounds culminates with the selection of F14512 for clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A87.

Despite advances in the therapy of acute myeloid leukemia (AML), the majority of patients die from their disease. Therefore, the lack of effective therapy mandates the development of novel compounds to improve the outcome of patients with relapsed and refractory leukemias. F14512 is a potent spermine-epipodophyllotoxin conjugate exploiting the polyamine transport system for tumor cell delivery. In this study, we report the in vivo antitumor activity of F14512 against experimental models of AML cell lines and of patient AML samples. F14512 markedly reduced the growth of HL-60 and U937 cell lines in an in vivo xenotransplantation model, resulting in a highly significant increase of survival of leukemia-bearing mice. Etoposide evaluated concurrently demonstrated only moderate in vivo activity against these models. F14512 induced in vivo apoptosis of HL-60 cells, as shown by caspase-3 activation and PARP cleavage. In an effort to mimic the human disease, we injected approximately 106 AML cells collected from a patient into NOD/SCID mice and allowed them to establish as xenografts for 8 weeks. Subsequent treatment with F14512 was carried out for 2 or 3 weeks followed by the analysis at the end of treatment and 1 week after the end of treatment. Two human AML samples were analyzed. Multiple i.v. administrations of F14512 at 0.32 mg/kg, induced an extensive reduction of the number of leukemic cells in mouse bone marrow and blood (97–99%), assessed by flow cytometry analysis, quantitative RT-PCR and histology. To identify leukemic cells expressing an active polyamine transport system, we developed a functional method based on the measurement of the cellular uptake of a nitrobenzoxadiazole fluorescent probe (F96982) combining the same spermine moiety as F14512. The level of fluorescence emitted by the probe F96982 was high in HL-60 cells as well in the 2 patient AML samples that proved to be sensitive to F14512 in vivo. Collectively, these results demonstrated that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are now initiated with this novel promising drug candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B186.

Topoisomerases inhibitors continue to play a major role in cancer chemotherapy. A large number of drug regimens, routinely used for the management of many forms of solid tumors in particular, include a camptothecin analog targeting topoisomerase I or an anthracycline derivative targeting topoisomerase II. This is the case also for the podophyllotoxin derivative etoposide marketed more than 25 years ago and which remains prescribed for the treatment of a variety of malignancies, including leukaemia. These topoisomerases poisons form an irreplaceable class of antitumor agents, almost all derived from natural products isolated from plants or microorganisms (1). For the past 20 years, efforts have been concentrated on the discovery and conception of novel, more potent, topoisomerases inhibitors but with a limited success. Camptothecins are still unique among topoisomerase I poisons used in the clinic. Anthracyclines and podophyllotoxines remain the two most robust classes of topoisomerase II modulators. Novel compounds have been introduced into the clinical pipeline, such as the naphthyridine derivatives ARC-111 and voreloxin (SNS-595), targeting respectively topoisomerases I and II, to cite only these two novel molecules. Novel compounds interfering with the functions of these DNA manipulating enzymes are continuously reported: the alkaloids thaspine and lamellarins, the yeast-derived compound simocyclinone D-8, calothrixins, etc. An alternative approach to the design of novel categories of topoisomerases-based antitumor agents is to maintain a known chemical archetype associated with a marked target selectivity profile (e.g. selectivity of camptothecin for toposiomerase I) but to coupled the pharmacophore with additional chemical groups susceptible to modulate target-independent properties such as cell selectivity, biodistribution, stability, pharmacokinetics, or to facilitate its handling for example. With this idea in mind, we have engineered a novel group of highly potent podophyllotoxin derivatives equipped with a cell delivery vector. Three points were considered in the design strategy: (i) improving the aqueous solubility of the molecule and its pharmaceutical and clinical use, (ii) reinforcing the drug-target interaction, so as to consolidate its DNA-damaging activity, and (iii) conferring a selectivity for tumor cells via a novel delivery strategy. The third point is obviously essential, with a general applicability to transform conventional cytotoxic agents into what we call “targeted cytotoxics”, i.e., cytotoxic molecules endowed with a pronounced selectivity for cancer cells. At the molecular level, the concept consisted to link the pharmacophore to a polyamine guide providing simultaneously the desired three properties. A suitable connector was constructed to associate the two molecular entities, without affecting their intrinsic recognition properties. Polyamines, such as spermine, are highly water-soluble molecules. As cations, they binds to nucleic acid polymers, in particular DNA through minor groove interactions. Polyamine conjugates are usually potent DNA binders. But most importantly, polyamines and in particular the naturally-occurring ones (spermine, spermidine, putrescine), are essential for cell proliferation and differentiation. Their metabolism is frequently exacerbated in tumors. Different types of cancer cells heavily rely on polyamines for growth and survival. Most importantly, an efficient polyamine transport system (PTS) has been functionally characterized in many tumors cells. Polyamine transporters, import and export systems, are poorly defined at the molecular level in human, but the PTS is now well documented functionally. This polyamine-based strategy has open novel horizons to the rational design of topoisomerase II inhibitors, acting as potent DNA damaging drugs. Here we will present the strategy, the design and pharmacological properties of the lead compound in the series, designated F14512 (2). This compound exploits the PTS for entering and accumulating into tumor cells, functions as a potent topoisomerase II poison, triggers cell death and displays remarkable antitumor activities in vivo, in a large panel of xenografts models (2,3). This molecule is now entering clinical development. As far as we know, this is the first molecule exploiting the PTS for a tumor-selective delivery to reach clinical development in oncology. In addition, a functional screen has been set up to identify PTS(+) cancer cells, using fluorescent polyamine derivatives and cytometry assays (4). A clinical procedure has been designed to select patients with PTS(+) tumors eligible for treatment with F14512. This method is particularly well suited to identify leukemia cells expressing an active PTS. Along these lines, recently we showed that human acute myeloid leukemia (AML) animal models and human samples are sensitive to F14512, with a level of activity well superior to that of etoposide. Through this concept, we are now entering into the field of personalized medicine, with a novel generation of cell-targeted cytotoxic agents. The long history of topoisomerases inhibitors is thus revivified and novel opportunities to increase the therapeutic index of these agents can be envisioned. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN08-03.