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Late effect of S49076 treatment

Pharmacokinetic parameters of S49076 administered orally to female balb/c nu/nu mice

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749–62. ©2013 AACR.

Western blot detection of total and phosphorylated MET, AXL, FGFR1/2/3 and phosphorylated FRS2 in cell lines and tumor xenografts used in the study

Tumor growth delays of subcutaneous tumors

Supplementary materials and methods and supplementary Figure and Table legends

Supplementary Figure Legends

The fibroblast growth factor receptor (FGFR) pathway has been implicated both as an escape mechanism from anti-angiogenic therapy and as a driver oncogene in different tumor types. Lucitanib is a small molecule inhibitor of vascular endothelial growth factor (VEGF) receptors 1 to 3 (VEGFR1 to 3), platelet derived growth factor α/β (PDGFRα/β) and FGFR1–3 tyrosine kinases and has demonstrated activity in a phase I/II clinical study, with objective RECIST responses in breast cancer patients with FGFR1 or FGF3/4/19 gene amplification, as well as in patients anticipated to benefit from anti-angiogenic agents. We report here the in vitro and in vivo antitumor activity of lucitanib in experimental models with or without FGFR1/2 amplification or mutations. In cell assays, lucitanib potently inhibited the growth of tumor cell lines with amplified FGFR1 or mutated/amplified FGFR2 . In all xenograft models studied, lucitanib demonstrated marked tumor growth inhibition due to potent inhibition of angiogenesis. Notably, in two lung cancer models with FGFR1 amplification, the antitumor efficacy was higher, suggesting that the simultaneous inhibition of VEGF and FGF receptors in FGFR1 dependent tumors can be therapeutically advantageous. Similar antitumor activity was observed in FGFR2 wild-type and amplified or mutated xenograft models. Pharmacokinetic studies showed lucitanib plasma concentrations in the micro/sub-micromolar range demonstrated drug accumulation following repeated lucitanib administration.

Several therapeutic agents targeting HGF/MET signaling are under clinical development as single agents or in combination, notably with anti-EGFR therapies in non–small cell lung cancer (NSCLC). However, despite increasing data supporting a link between MET, irradiation, and cancer progression, no data regarding the combination of MET-targeting agents and radiotherapy are available from the clinic. S49076 is an oral ATP-competitive inhibitor of MET, AXL, and FGFR1-3 receptors that is currently in phase I/II clinical trials in combination with gefitinib in NSCLC patients whose tumors show resistance to EGFR inhibitors. Here, we studied the impact of S49076 on MET signaling, cell proliferation, and clonogenic survival in MET-dependent (GTL16 and U87-MG) and MET-independent (H441, H460, and A549) cells. Our data show that S49076 exerts its cytotoxic activity at low doses on MET-dependent cells through MET inhibition, whereas it inhibits growth of MET-independent cells at higher but clinically relevant doses by targeting Aurora B. Furthermore, we found that S49076 improves the antitumor efficacy of radiotherapy in both MET-dependent and MET-independent cell lines in vitro and in subcutaneous and orthotopic tumor models in vivo. In conclusion, our study demonstrates that S49076 has dual antitumor activity and can be used in combination with radiotherapy for the treatment of both MET-dependent and MET-independent tumors. These results support the evaluation of combined treatment of S49076 with radiation in clinical trials without patient selection based on the tumor MET dependency status. Mol Cancer Ther; 16(10); 2107–19. ©2017 AACR.

Abexinostat, an oral pan-histone deacetylase inhibitor (HDACi), was evaluated in patients with advanced solid tumours in two single agent phase I studies (PCYC-402 and CL1-78454-002). In PCYC-402 study testing four different administration schedules, the maximum tolerated dose (MTD) was established at 75 mg/m(2) BID (twice daily) and the recommended dose at 60 mg/m(2) BID regardless of the schedule tested. The dose limiting toxicity (DLT), consistently observed across all these schedules, was reversible thrombocytopenia. The CL1-78454-002 study was initially investigating an additional schedule of 14 days on/7 days off. While testing two first cohorts, thrombocytopenia was observed without reaching DLT. To address this issue, a pharmacokinetic/pharmacodynamic (PK/PD) model was used to predict the optimal schedule allowing higher doses with minimal thrombocytopenia. Several administration schedules were simulated using this model. A 4 days on/3 days off schedule was associated with the smallest platelet decrease. Accordingly, the CL1-78454-002 study was amended. After reaching MTD1 (75 mg/m(2) BID) with the initial schedule, subsequent cohorts received abexinostat on a revised schedule of 4 days on/3 days off, starting at one dose level below MTD1 (60 mg/m(2) BID). As expected, the dose-escalation continued for two more dose levels beyond MTD1. The MTD2 reached for this optimised schedule was 105 mg/m(2) BID and the recommended dose 90 mg/m(2) BID. In conclusion, early understanding of toxicities and PK determination allowed us to build a PK/PD model of thrombocytopenia, which predicted the optimal administration schedule. This optimised schedule is currently used in the trials in solid tumours with abexinostat.

A population pharmacokinetic/pharmacodynamic (PK/PD) model was developed to describe the thrombocytopenia (dose-limiting toxicity) of abexinostat, a new histone deacetylase inhibitor. An optimal administration schedule of the drug was determined using a simulation-based approach. Early PK and PK/PD data were analysed using a sequential population modeling approach (NONMEM 7), allowing for the description of a PK profile and platelet-count decrease after abexinostat administration with various administration schedules. Simulations of platelet count with several administration schedules over 3-week treatment cycles (ASC) and over a day (ASD) were computed to define the optimal schedule that limits the depth of thrombocytopenia. An intermediate PK/PD model accurately described the data. The administration of abexinostat during the first 4 days of each week in a 3-week cycle resulted in fewer adverse events (with no influence of ASD on platelet count profiles), and corresponded to the optimal treatment schedule. This administration schedule was clinically evaluated in a phase I clinical trial and allowed for the definition of a new maximum tolerated dose (MTD), leading to a nearly 30% higher dose-intensity than that of another previously tested schedule. Lastly, a final model was built using all of the available data. The final model, characterizing the dose-effect and the dose-toxicity relationships, provides a useful modeling tool for clinical drug development.

EBV-related nasopharyngeal carcinomas (NPCs) still raise serious therapeutic problems. The therapeutic potential of the histone-deacetylase (HDAC) inhibitor Abexinostat was investigated using 5 preclinical NPC models including 2 patient-derived xenografts (C15 and C17). The cytotoxicity of Abexinostat used either alone or in combination with cis-platin or irradiation was assessed in vitro by MTT and clonogenic assays using 2 EBV-negative (CNE1 and HONE1) and 3 EBV-positive NPC models (C15, C17 and C666-1). Subsequently, the 3 EBV-positive models were used under the form of xenografts to assess the impact of systemic treatments by Abexinostat or combinations of Abexinostat with cis-platin or irradiation. Several cell proteins known to be affected by HDAC inhibitors and the small viral non-coding RNA EBER1 were investigated in the treated tumors. Synergistic cytotoxic effects of Abexinostat combined with cis-platin or irradiation were demonstrated in vitro for each NPC model. When using xenografts, Abexinostat by itself (12.5 mg/kg, BID, 4 days a week for 3 weeks) had significant anti-tumor effects against C17. Cooperative effects with cis-platin (2 mg/kg, IP, at days 3, 10 and 17) and irradiation (1 Gy) were observed for the C15 and C17 xenografts. Simultaneously two types of biological alterations were induced in the tumor tissue, especially in the C17 model: a depletion of the DNA-repair protein RAD51 and a stronger in situ detection of the small viral RNA EBER1. Overall, these results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC. A depletion of RAD51 is likely to contribute to the cooperation of Abexinostat with DNA damaging agents. Reduction of RAD51 combined to enhanced detection of EBER 1 might be helpful for early assessment of tumor response.

Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749–62. ©2013 AACR.

S49076 is an oral ATP-competitive inhibitor of the receptor tyrosine kinases MET, AXL, and FGFR1-3 currently in phase I/II clinical development in patients with advanced solid tumors. Here, we investigated the effect of S49076 on both MET- and non-MET- dependent cell lines alone and in association with ionizing radiation (IR) both in vitro and in vivo. We performed proliferation and clonogenic survival assays using the low MET-expressing H460 and A549 non-small-cell lung cancer (NSCLC) cell lines, the high MET-expressing H441 NSCLC cell line and the MET-dependent GTL16 gastric cancer cell line. Activity against the MET receptor and Aurora B kinase was assessed by analyzing the phosphorylation status of MET Tyr1234/1235 and Histone H3 Ser10 residues. Pharmacologic and RNA interference approaches against Aurora B and MET were used to determine the molecular mechanism of the cell response to S49076. S49076 inhibited MET activity in the nanomolar range (1 - 100 nM). At higher, but still clinically-relevant concentrations (0.5 - 1 μM), S49076 was also found to inhibit Aurora B kinase activity in all cell lines tested. In non MET-dependent cell lines, the antiproliferative and anti-clonogenic effects of the drug correlated with its activity against Aurora B, whereas in the MET-dependent cell line, these effects could be attributed to direct inhibition of the MET receptor. When treatment with S49076 was combined with IR, an enhancement of IR effects was observed in all cell lines tested in vitro. In vivo, a significant growth delay of subcutaneous tumors was observed at 50 mg/kg bid oral S49076 in non-MET dependent models and at 3 mg/kg bid oral S49076 in the GTL16 MET-dependent model. For non MET-dependent models, immunohistochemistry revealed that treatment with S49076 reduced Histone H3 phosphorylation on Ser10. This is consistent with the finding that S49076 concentrations above 1 μM were attained in the tumors, as determined by LC-MS/MS analysis. In all tumors tested, S49076 treatment significantly delayed tumor growth when combined to a fractionated IR (4 fractions of 2.5 Gy) compared to IR alone. An orthotopic model of lung cancer using luciferase-expressing H460 NSCLC cells was also used to evaluate the combination of S49076 (50mg/kg bid) and locally fractionated thoracic IR (4*2.5 Gy). In this model, the median survival after cell implantation was 22 days for control mice, 29.5 and 29 days for S49076 treated and irradiated mice respectively and 41 days for mice receiving the combined treatment (p Our findings indicate that S49076 improves the efficacy of radiotherapy in both MET- and non-MET- dependent cell, supporting the use of combined treatment with S49076 and radiation in clinical trials without patient selection. Citation Format: Céline Clémenson, Cyrus Chargari, Michele Mondini, Charles Ferté, Winchygn Liu, Mike Burbridge, Valérie Cattan, Anne Jacquet-Bescond, Eric Deutsch. S49076, a MET, AXL, FGFR inhibitor, potentiates radiation therapy in subcutaneous and orthotopic models of lung 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 2568. doi:10.1158/1538-7445.AM2015-2568

Background: S 49076 is a novel ATP-competitive tyrosine kinase inhibitor which displays a unique profile targeting MET, AXL and FGFR-1/2/3 (IC50 for inhibition of pathway activation was Methods: Pts are enrolled on dose-escalation cohorts, using a “3+3” design. Two dosing regimens of S 49076 administration (once a day, QD, or twice a day, BID) are tested in this study, in which pts receive oral capsule(s) of S 49076 during a continuous 21-day cycle. The PK measurements are performed at D1 and at steady state. Tumor biopsies for genomic and proteomic analyses are taken predose and on treatment. Tumor response is measured once every 2 cycles. Results: To date 49 pts have been treated at doses ranging from 15 to 270 mg QD or 7.5 to 225 mg BID. Median age is 59 years, and the most common tumor types are colorectal, NSCLC and uveal melanoma. The most frequent adverse events reported as treatment-related by the investigator have been hypomagnesemia, peripheral edema and hypoalbuminemia mostly grade (G) 1-2. Three DLTs were observed, including failure to restart S 49076 within 1 week due to G2 asymptomatic ejection fraction decrease in 30 mg QD and 225 mg BID cohorts and G3 hypotension in the 180 mg BID cohort. MTD has not been reached and the dose escalation is ongoing. Among 37 evaluable pts, 20 pts had stable disease (SD) ranging from 6 to 36 weeks of whom 3 pts with uveal melanoma had prolonged SD from 12 to 24 weeks. Preliminary PK analyses showed that the maximal plasma concentration (Cmax) occurred between 2h and 6h after oral administration. The bioavailability was estimated to be around 30% and the effective half-life around 15h. There was no evidence of non-linearity since Cmax and AUC24h increased proportionally over the range of doses tested and no evidence of time dependent PK. Based on PK data, the plasma concentrations reached in human at the higher doses are consistent with inhibition of the S 49076 targets MET, AXL and FGFR. Conclusions: The general safety profile of oral treatment with S 49076 is good at the doses tested. PK data suggest dose proportionality in exposure without marked drug accumulation. S 49076 has demonstrated preliminary anti-tumor activity, especially in uveal melanoma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C65. Citation Format: Antoine Hollebecque, Jean-Charles Soria, Ratislav Bahleda, Natividad Lopez-Busto, Anne Jacquet-Bescond, Mike F. Burbridge, Cattan Valérie, Jeanne Pauly, Maria Herranz, Analia Azaro, Stéphane Depil, Jordi Rodon. First-in-human Phase I dose-escalation study of a MET/AXL/FGFR inhibitor, S 49076, in patients with advanced solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C65.

Background: S49076 was developed as an oral ATP-competitive inhibitor of MET receptor and of other kinases involved in tumor development. It is being assessed as single agent in phase I study. We examined its potential as a radiosensitizer in non-MET dependent tumor models. Experimental Procedures: Cell viability and clonogenic survival assays were performed in two non small-cell lung carcinoma (NSCLC) cells (H460 and A549) and in breast cancer cells MDA-MB-231. Then, the drug was tested in combination with ionizing irradiation (IR) in vitro. The combination was assessed in subcutaneous tumor xenografts and in NSCLC orthotopic models of luciferine-expressing H460luc and A549luc tumors. Immunohistochemical correlates were performed and the activity of S49076 was assessed in a lung metastases model of intravenous injections of A549 cells. Results: The drug as single agent inhibited proliferation (IC50 ranging from 0.50 μM to 1.1 μM) and clonogenic survival (IC50 ranging from 0.34 μM to 0.86 μM). Transcriptomic analyses showed that antitumor effect was associated with modulation of molecular pathways involved in radiation response (p53 and ATM). S49076 and IR inhibited synergistically clonogenic survival in H460 cells: sensitivity enhancement ratio at 0.6 μM was 2 for a single radiation dose of 2 Gy. Combination was additive in A549 and MDA-MB-231 cells. S49076 at 50 mg/kg twice daily enhanced radiation-induced growth delay in subcutaneous tumor xenografts, accompanied with an increase in TUNEL staining. Dose enhancement factors were 3.2 and 1.6 in H460 and A549 tumors, respectively. In lung orthotopic models, S49076 generated significant delay in time course of bioluminescent signal and improved median estimated survival, with significant benefit of combination over either IR or S49076 alone. Finally, treatment with S49076 improved significantly median survival in the lung metastases model (14 versus 22 days, p < 0.001). Conclusions: Altogether, these data suggest that S49076 has a potential as radiosensitizer in non MET-dependent cell lines, and that this effect is accompanied with a modulation of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C219. Citation Format: Cyrus Chargari, Céline Clémenson, Michele Mondini, Charles Ferte, Anne Jacquet-Bescond, Valérie Cattan, Stéphane Depil, Eric Deutsch. Novel MET/FGFR/AXL kinase inhibitor S49076 exerts radiosensitizing activity in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C219.

Epstein-Barr virus (EBV) related nasopharyngeal carcinoma (NPC) is the third leading cause of virus-related human malignancy. On average, NPCs are more radiosensitive and chemosensitive than other head and neck tumors. However, local relapse and distant organ metastases still raise serious therapeutic problems. The aim of this study was to investigate the potential of the novel pan-HDAC inhibitor Abexinostat (S78454) for the treatment of NPC. Three types of EBV-positive NPC cells have been used as targets for in vitro and in vivo treatments. C666-1 and C17 cells were first xenotransplanted from the patients to nude mice and, in a second stage, adapted to permanent in vitro culture. In contrast, C15 cells are still propagated exclusively as xenografts; however, they can be used in short term culture assays. S78454 was applied to these cells either alone or in combination with cis-platinum. All three types of NPC cells - C666-1, C15 and C17 - were sensitive to the cytotoxic effects of Abexinostat with IC50 of 220, 150 and 200 nM, respectively. Combined treatment of Abexinostat with cis-platinum resulted in a synergistic effect with Bliss additivism index of 34, 33 and 19, respectively. In the next step, we used xenografted NPC cells for in vivo assessment of Abexinostat. Tumor-bearing mice were treated for 3 weeks. The compound was administered by intra-peritoneal injections, twice a day; 4 days a week, at a dose of 12.5 mg/Kg. Cis-platinum (2mg/Kg) was delivered once a week. In these experimental conditions, cis-platinum had no effect on tumor growth when used as a single agent. In contrast, Abexinostat by itself induced a significant decrease in tumor growth. This clinical effect was accompanied by a substantial decrease in the concentrations of Rad 51 and Rad 23B proteins in xenografted NPC cells for all 3 tumor lines. An increase in the concentration of acetylated tubulin was observed for only one type of xenografted NPC (C666-1). A strong synergistic anti-tumor effect was obtained when Abexinostat was combined with cis-platinum. It was manifested clinically by a spectacular inhibition of tumor growth and at histological examination by extensive areas of fibroblast proliferation segmenting and pushing residual tumor areas. These results support implementation of phase I/II clinical trials of Abexinostat for the treatment of NPC, especially in association with cis-platinum. The fact that Abexinostat was active against NPC xenografts at a relatively low dose (25mg/kg/day) suggests that it might have a better therapeutic index in NPC patients than in patients bearing other types of carcinomas. Down-regulation of Rad51 in tumor cells seems to be more consistent that the increase in acetylated tubulin. Additional investigations will be required to determine whether this change is predictive of a long term favourable tumor response to Abexinostat. Citation Format: Benjamin Vérillaud, Mélanie Gressette, Anne-Sophie Jimenez, Hélène Lelièvre, Kwok-Wai Lo, Anne Jacquet-Bescond, Laurence Kraus-Berthier, Stéphane Depil, Pierre Busson. Treatment of EBV-positive nasopharyngeal carcinoma xenografts with the HDAC-inhibitor Abexinostat: synergy with cis-platinum. [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 1013. doi:10.1158/1538-7445.AM2013-1013

Background: Estrogen receptors (ER) drive breast cancer development through the regulation of gene transcription and the inhibition of ER signalling is the main therapeutic strategy for hormone-dependent breast cancers. Selective ER modulators (such as tamoxifen) or aromatase inhibitors are commonly administered to patients. However, de novo and acquired resistance are frequently observed and combinatorial treatments are required to bypass this phenomenon. Acetylation plays a key role in estrogen signalling by regulating ERα and ERα expression and activity (for a recent review see Linares et al, J. Biomed. Biotech. 2011). This post-translational mark is removed by histone deacetylases (HDACs) whose enzymatic activity could be blocked by specific HDAC inhibitors (HDIs). Interestingly, it has been shown that HDIs are able to regulate ER signalling and to potentiate the anti-tumor effects of anti-estrogens in ER-positive breast cancer cells. In this study, we analyzed the effects of a new hydroxamic acid HDI S78454 on several cellular parameters regulated by estrogens in ERα expressing MCF-7 breast cancer cells. Method: The following parameters were investigated both in sensitive and in antiestrogen-resistant MCF7 cells, that we developed in our laboratory: 1) ERα expression at the mRNA and protein levels by quantitative RT-PCR and western blot, respectively 2) transcriptional activity of ERα by quantifying the expression of several estrogen-regulated mRNAs 3) HDAC expression at the mRNA level and 4) 2D cell proliferation using MTS assay. Results: In MCF-7 breast cancer cells, S78454 decreased ERα expression, modulated gene expression (E2 target genes, HDACs and HDI-regulated genes) and inhibited cell proliferation. S78454 globally behaved as the reference HDIs yielding very similar amplitudes on the parameters that we have analyzed with EC50 being comprised between those of Vorinostat and Panobinostat. Interestingly, the IC50 of S78454 on MCF-7 cell proliferation was lower in cells treated with antiestrogens than in control cells or in cells treated with E2. Moreover, in antiestrogen-resistant MCF-7 cells that we developed in our laboratory, S78454 was able to significantly restore the sensitivity to the pure antiestrogen Fulvestrant. Conclusions and perspectives: Our findings suggest that in breast tumor cells, S78454 exhibits antiproliferative effects, can exert synergistic effects with antiestrogens and can restore antiestrogen-sensitivity in a model of antiestrogen-resistant MCF7 cell line. These results suggest that the combination of S78454 and antiestrogens could be valuable in the treatment of breast cancers resistant to hormonal therapies. This effect should be validated in vivo on tumor xenografts. In addition, it would be interesting to investigate whether S78454 also reinforces the anti-growth factor activity of antiestrogens. 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 4704. doi:1538-7445.AM2012-4704

Aberrant activity of receptor tyrosine kinases (RTKs) has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. Moreover, increased expression or activation of RTKs is often associated with resistance to standard chemotherapy agents or signal transduction modulators. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of the RTKs MET, AXL and FGFR. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC50 values of less than 50 nM in the case of MET and AXL, and at below 200 nM for FGFR1/2/3. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2- dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of hepatocarcinoma cells overexpressing FGFR2 and AXL. In vivo, oral administration of S 49076 inhibits MET autophosphorylation and tumor growth in subcutaneous GTL-16 human gastric carcinoma and U87-MG human glioblastoma at 6 mg/kg/day. In FGFR2-dependent SNU-16 gastric tumors S 49076 inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth at 25 mg/kg/day. In a panel of 53 patient-derived tumors and 14 cell lines of diverse origin growing in three-dimensional in vitro culture, twenty-five (37%) were found to be sensitive to S 49076 at 1 µM or less. In the majority of cases, analysis of the expression, activation and mutation status of MET, AXL, FGFR1/2/3 and other target kinases of S 49076 as well as that of major signaling proteins enabled hypotheses to be made concerning the sensitivity or resistance to S 49076. Moreover, in many of these resistant tumors, a rationale for association of S 49076 with other targeted therapies emerged. Examples of the efficacy of such combined therapies will be shown. Based on these preclinical studies showing a favorable and novel pharmacological profile of S 49076, a phase I study is currently underway in patients with advanced solid tumors. 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 848. doi:1538-7445.AM2012-848

S78454 (also called PCI-24781) is an orally bioavailable, hydroxamate-based pan-HDACi currently being tested in clinical trials in the US and EU. Like many other HDACi, this drug induces a reversible thrombocytopenia. This thrombocytopenia, which was associated with a decrease in either GATA1 transcriptional activity or the expression of proteins of the Rho GTPase family (RhoA, Cdc42 or Rac), remains poorly understood. Given that the S78454 plasma level is above 100nM in treated patients, we performed a dose-response analysis (from 10 to 100 nM) of the drug effects on CFU-MK growth and cell proliferation. CFU-MKs were generated by ex vivo culture of CD34-positive cells and their differentiation was dose-dependently decreased after either a 24-hour treatment or a continuous exposure to S78454. This effect was associated with a dose-dependent decrease in MK proliferation. When added at day 8 of the culture, at a time-point when MK have nearly finished their endomitotic process and are entering in the cytoplasmic maturation step, S78454 induced a dose-dependent decrease (ten- to- two fold at 100nM and 20nM, respectively) in proplatelet formation, even when secondarily removed from the culture medium. The decreased proliferation was associated with an increased apoptosis, as demonstrated by the presence of a sub-G1 peak after propidium iodide staining, Annexin V binding, and caspase-3 proteolysis. We did not observed any blockade in the TPO/MPL/JAK2 signaling since ERK, Stat3 and Stat5 phosphorylation remained unaffected. Interestingly, MK exposed to S78454 displayed an increase in γH2AX foci formation and a decrease in Rad51 expression, a major mediator of homologous recombination, indicating a defective repair of DNA double-strand breaks. Consequently, P53 became phosphorylated in MKs, and the expression of its target genes BAX and P21 was increased. Altogether, these results suggest that S78454-induced thrombocytopenia may be mediated by induction of apoptosis in MK due to DNA-double-strand breaks and p53 activation. 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 2022. doi:1538-7445.AM2012-2022

Epstein-Barr Virus (EBV) related nasopharyngeal carcinoma (NPC) is the third leading cause of virus-related human malignancy. On average, NPCs are more radiosensitive and chemosensitive than other head and neck tumors. However, local relapse and distant organ metastases still raise serious therapeutic problems. Histone deacetylase (HDAC) inhibitors have recently emerged as a class of therapeutic agent for the treatment of both hematologic malignancies and solid tumors. In the present study, we investigated the antitumor effects of the novel Pan HDAC inhibitor S78454 in 3 NPC cell lines, namely C666-1 (EBV-positive NPC cell line), HONE1 and CNE1 (EBV-negative). The antiproliferative effects of S78454, used either alone or in combination with cis-platinum or irradiation, were evaluated using MTT assays and clonogenic assays. Combined treatment of S78454 with cis-platinum resulted in a synergistic inhibitory effect on cell proliferation, as well as on cell clonogenic growth. HDAC inhibition also enhanced radiosensitivity of NPC cell lines. Western blot detection of PARP cleavage revealed that the antiproliferative effects of S78454 were only partially related to apoptosis. Western blot analysis was also used to explore molecular mechanisms involved in cellular response to S78454. Quantitative assessment of tubulin acetylation confirmed that S78454 effectively increased protein acetylation, even at low concentrations. As previously reported in other tumors, RAD51 protein levels were significantly decreased after 48H of treatment, suggesting that HDAC inhibitors act, at least in part, by targeting homologous recombination. However, HDAC inhibitor-induced cell death involves not only epigenetic pathways, but numerous other mechanisms, through acetylation of a variety of non-histone and non-chromatin-associated proteins. For instance, the proteasome activity may play a role in the response to HDAC inhibitors, and the amount of HR23B - a carrier protein functionally related to the proteasome - has been identified as a determinant of HDAC inhibitors sensitivity. We found that HR23B protein levels were high in all 3 NPC cell lines. Nevertheless, there was a decrease in HR23B protein levels after treatment with S78454. Altogether, these results demonstrate that the novel HDAC inhibitor S78454, currently in Phase I/II clinical trials for the treatment of solid tumors and hematologic malignancies, has promising antitumour activity in NPC, especially in association with cis-platinum and ionizing radiation, ie the two most widely used antitumor agents in NPC. Experiments are in progress to assess the anti-tumor activity of S78454 on other types of NPC cells, including xenografted NPC tumor lines. The influence of the HDAC inhibitor on the regulation of EBV latency is also under investigation. 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 4702. doi:1538-7445.AM2012-4702

Receptor tyrosine kinases (RTKs) are key regulators of a multitude of cell processes, including survival, proliferation, migration, invasion and angiogenesis. Aberrant activity of certain of these receptor tyrosine kinases has been associated with tumor progression in a wide variety of human malignancies, making them promising drug targets for cancer therapy. Although, in some instances, specific inhibition of just one of these RTKs suffices for inhibition of tumor progression, in the majority of cases, targeting more than one RTK could be required for therapeutic efficacy. These RTKs include MET, the receptor for hepatocyte growth factor (HGF), the fibroblast growth factor receptor, FGFR, and AXL. Dysregulation of MET activity, due to its overexpression or mutation, or overexpression of its ligand, has been consistently associated with aggressive phenotype, resistance to certain anti-cancer therapies and poor outcome. The FGFRs have a well-documented role in tumor angiogenesis, and, more recently, have been implicated directly in tumor cell survival, proliferation and metastasis in a wide range of tumor types. AXL is one of a family of three tyrosine kinase receptors (TAM family) involved in the pathogenesis of several human cancers and has also recently incited interest as a cancer therapeutic target. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor of MET, FGFR1/2/3 and AXL. S 49076 blocks autophosphorylation of these RTKs and their downstream signaling in cells with IC50 values of between 1 and 200 nM depending on the target and cell line. Furthermore, in kinase binding assays, S 49076 also binds to clinically-relevant MET mutated isoforms and a number of other kinases implicated in cancer pathology at concentrations of less than 100 nM. S 49076 is not, however, a potent inhibitor of VEGFR2. Although VEGFR2 is implicated in tumor angiogenesis, it also plays a major physiological role in the maintenance of vascular tone, and its inhibition by other RTK inhibitors has limited dosing of these molecules in the clinic. The unique inhibition profile of S 49076 may allow inhibition of oncogenic RTKs potentially without toxic effects encountered by VEGFR2 inhibitors. In vitro, S 49076 inhibits the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocks MET-driven migration of lung carcinoma cells and inhibits colony formation of AXL-overexpressing hepatocarcinoma cells. In vivo, oral administration of S 49076 inhibits > 80% MET autophosphorylation in subcutaneous GTL-16 human gastric carcinoma tumors at 3 mg/kg. In both this GTL-16 model and in the MET-dependent U87-MG human glioblastoma model, S 49076 inhibits > 80% tumor growth at 6 mg/kg/day. S 49076 also inhibits FGFR2 autophosphorylation, downstream signaling and tumor growth in FGFR2-dependent SNU-16 gastric tumors, and tumor growth in LS-174T colon carcinoma. Based on these preclinical studies showing a favorable and novel pharmacological profile and the potential of S 49076 as innovative anticancer agent, a phase I study is planned soon to evaluate an oral formulation of S 49076 in patients with advanced solid tumors. 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 A238.

Glioblastoma is an aggressive primary brain tumor with poor outcome, despite a treatment associating surgery and radiotherapy combined with temozolomide. Recent data have shown an in vitro synergy of the HDAC inhibitor S78454 (also called PCI-24781) with ionizing radiation, via inhibition of homologous recombinational (HR) repair of DNA double-strand breaks. We propose in this study to investigate the in vivo radiosensitizer effect of S78454 on human glioblastoma model. For this, we generated orthotopic U87 human glioblastoma xenografts in nude mice. Intra-peritoneal (i.p.). S 78454 treatment was performed daily starting 10 days after the engraftment. Irradiation was performed in one daily 2 Gy localized fraction during 4 days followed by a second similar 4 days treatment performed after 3 days off (for a total dose of 16 Gy administrated on 11 days). Survival experiments were conducted in mice bearing xenografts in the four different groups of treatment (vehicle, S 78454 alone, radiation alone, S 78454 + radiation). Mice bearing orthotopic xenografts were sacrificed at the onset of neurological signs. Survival curves were then performed. We first determined the S78454 dosing schedule to obtain a pharmacodynamic effect of the compound in the xenograft. Inhibition of RAD 51 expression, histone H3 and tubulin acetylation in the tumor were obtained 24 hours after daily i.p. treatment with 50mg/kg S78454. These results allowed us to define the schedule treatment combining S78454 with radiotherapy : mice were treated with S78454 50 mg/kg/d i.p., daily from Days 10 to14 and then from Days 17 to 21; one 2 Gy fraction radiotherapy was performed daily from Days 11 to14 and Days 18 to 21. In these conditions of treatment, S78454 was well tolerated Survival of the mice treated with S78454 alone was not significantly increased while the survival of the mice treated with irradiation alone was significantly increased compared to the control group (P 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 B85.

Introduction: Histone deacetylases inhibitors (HDACi) are promising anti-cancer agents. Recent data showing synergy of HDACi with ionizing radiation (IR) and other DNA damaging agents have suggested that HDACi may act, in part, by inhibiting DNA repair. Tumor hypoxia is a negative prognostic marker for patients undergoing IR. Hypoxic tumor cells are less radiosensitive than well-oxygenated ones and this is correlated with a lower rate of radioinduced DNA double strand breaks (DSB) under hypoxia. The purpose of this study was to determine the effect and mechanism of action of S 78454 (also called PCI-24781), a novel HDACi, in combination to IR under normoxia and hypoxia in NSCLC cells. Material and Methods: We tested the effectiveness of combining IR and S 78454 on the human NSCLC cell line H460 and A549 using clonogenic survival assays. H460 and A549 cells were treated with vehicle control or respectively 0.1 M or 0.4μM of S 78454 for 24 hours under normoxia (21% oxygen) or hypoxia (0,1% oxygen), irradiated to 0–6Gy with a 137Cs source, and incubated for 24 hours. Cells were then incubated without S 78454 for 12 days and colonies were stained and counted. Plating efficiency and surviving fractions were calculated relative to those of unirradiated cells. Flow cytometry was performed for apoptosis analysis. γH2AX foci were assessed using immunofluorescence staining assays. Histone3 acetylated, γH2AX and Rad51 protein levels were evaluated by western blotting. Results: Pretreating H460 and A549 cells with S 78454 respectively for 24 hours did synergistically enhance cell killing after IR compared with untreated irradiated control cells irrespective of hypoxia. Under normoxia, the mean surviving fraction at 2Gy (SF2) of cells untreated by S 78454 was 0.85 and 1 for H460 and A549 cells respectively, whereas the SF2 for H460 and A549 cells treated with S 78454 was 0.73 and 0.64 respectively. Under hypoxia, the mean SF2 of cells untreated by S 78454 was 0.63 and 0.72 for H460 and A549 cells respectively, whereas the SF2 for H460 and A549 cells treated with S 78454 was 0.36 and 0.56 respectively. A dose effect relationship was observed with increased radiosensitization when increasing S 78454 doses. S 78454 alone and in combination with IR (4Gy) enhanced DNA damage as evidenced by increased expression of H2AX which increased from 4 hours to at least 24 hours after treatment. The combination delayed replication recovery and caused a moderate induction of apoptosis as shown by increased sub-G1 population and Z-VAD/annexineV assay in H460 and A549 cells. Western blot analysis showed that after 24h of treatment by S 78454 levels of Rad51 decreased in both cell lines under hypoxia. IR (4Gy) slightly increased Rad51 expression under hypoxia in both cell lines from 1 hour after IR. In 24 hours pretreated hypoxic cells Rad51 expression decreased 1 hour after IR while levels of Rad51 remained stable in normoxic cells. Conclusions: Our results indicate that inhibition of HDACs by S 78454 increases sensitivity of both normoxic and hypoxic NSCLC cells to IR and suggest that decreased DSB repair might play a key role even under hypoxia. Our data suggest that impaired DSB repair does not necessarily correlate with Rad51 diminution underscoring the fact that other DNA repair proteins might account for S 78454 induced radiosentization. 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 A107.

Protein kinase inhibitors constitute a class of anticancer agents with demonstrated clinical efficacy. However, the clinical benefit of these agents is often limited to a subset of patients with specific genomic lesions in their tumor cells rendering them sensitive to inhibition of one or more target kinases. It is therefore becoming increasingly important to identify biomarkers of patient stratification to enable personalized care. For those inhibitors targeting just one kinase this stratification strategy is relatively straightforward, although the number of patients who will benefit from these highly selective inhibitors alone is in general severely limited. In the majority of cases, the simultaneous targeting of more than one kinase, possibly by the same drug, will be required for therapeutic efficacy. However, for multi-target kinase drugs, patient stratification is considerably more complex and is becoming a major challenge during clinical development. S 49076 is a novel, potent, ATP-competitive tyrosine kinase inhibitor with activity demonstrated in cell and animal assays against the receptor tyrosine kinases MET, FGFR1/2/3 and AXL, all of which are associated with progression of a wide range of human malignancies. In kinase binding assays, S 49076 also binds to clinically-relevant MET mutated isoforms and 32 other kinases, many of which have also been implicated in cancer pathology. In the present study, the antitumor activity of S 49076 was assessed in a panel of 53 patient-derived tumors of diverse origin growing in three-dimensional in vitro culture. Sixteen (30%) of these tumors were sensitive to S 49076 at 1 μM or less. With the aim to define molecular signatures of sensitivity or resistance, all 53 tumors were characterized for expression, activation and mutation of all of the known target kinases using protein, mRNA and DNA analysis techniques. On an individual basis, no clear relationship between expression level or activity of each target kinase and sensitivity or resistance to S 49076 could be established. However, analysis of microarray data using z-score transformation enabled a clear link to emerge between the sum of the z-scores of all potential target kinases (z-score) and tumor response to S 49076. Moreover, when the presence of RAS or PIK3CA activating mutations was taken into account, the predictive power of the analysis (in particular the positive predictive value for sensitivity) was even more striking. Overall, 91% (29/32) of tumors with a z-score less than 0 were resistant to S 49076, whilst 85% (11/13) of tumors with a z-score greater than 0 and with no known mutations in RAS or PIK3CA were sensitive. Six out of the 8 tumors with a z-score greater than 0 but with mutations in RAS or PIK3CA were resistant. In vivo evaluation of the response to S 49076 of several of the sensitive tumors is in progress in xenograft studies. Together, these data demonstrate the potential power of molecular profiling and, in particular, transcriptomic analyzes in the stratification of patient tumors for treatment with a multi-kinase inhibitor. It is hoped that similar analysis of patient tumors during clinical trials of S 49076 will ultimately contribute to the progress towards a more personalized approach to cancer patient care. 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 A240.

S78454 (also called PCI-24781) is a novel, orally bioavailable, hydroxamate-based pan-HDACi currently being tested in clinical trials in the US and EU. Two single agent Phase I studies have been completed in patients with advanced solid tumors. The first study (PCYC-0402) tested 4 different administration schedules with 4-week (wk) cycles: 1) TID and 2) BID 5 days/wk 3 wks/4; 3) BID on days 1–7 & 15–21; 4) BID on days 1–5 & 15–19. The planned dose levels were 30, 45, 60, 75 and 90 mg/m2. The second study (CL1-002) tested an additional schedule: 5) BID 14 days on/7 days off in 3-week cycles. The maximum tolerated dose (MTD), defined by dose-limiting toxicity (DLT) occurring in at least 2/3 or 2/6 patients so that further dose-escalation is not undertaken, was established at 75 mg/m2 BID and the recommended dose at 60 mg/m2 BID. The DLT, consistently observed across all these schedules, was reversible thrombocytopenia, common to all HDACi and directly related to their mechanism of action. To address this issue, a pharmacokinetic/pharmacodynamic (PK/PD) model was used to predict the optimal administration schedule that could allow higher doses with minimal thrombocytopenia. Platelet-time profiles from the first study (PCYC-0402) were analyzed using a mixed effect model (Sheiner et al, 1980). This approach allowed the estimation of population mean parameters and their variability by the simultaneous fit of all data records, despite their heterogeneity. This PK/PD model consisted in a previously developed PK model of S78454 used as an input into a semi-physiological platelet time-course model (Friberg et al, 2002). This model was made up of a self-renewing progenitor compartment connected to transit compartments representing maturation process, and leading to a circulating compartment. A feedback mechanism mimicked the increase in proliferation rate when platelet count falls below baseline. The model was parameterized with baseline, feedback intensity, mean maturation time of platelets, and drug concentration/effect relationship. It adequately fitted observed platelet counts after different administration schedules and showed good prediction of adverse events related to haematological toxicity. Several administration schedules were simulated using this model. The results showed that a 4 days on/ 3 days off schedule was associated with the smallest platelet decrease. Accordingly, the protocol for study CL1–002 was amended. After reaching MTD in schedule 5, subsequent cohorts received S78454 on a revised schedule of 4 days on/ 3 days off, starting at one dose level below MTD (60 mg/m2 BID). The dose escalation followed the same design as the initial schedule with the same pre-defined dose levels. As expected, the dose escalation continued for two more dose levels beyond the MTD in schedule 5. At 105 mg/m2 BID, 2/3 patients included experienced DLTs of grade 4 thrombocytopenia, and therefore the MTD in the revised schedule was established at 105 mg/m2 BID. In conclusion, early understanding of toxicities and PK determination allowed us to build a PK/PD model of thrombocytopenia, which predicted the optimal administration schedule. This optimized schedule is planned to be used in future trials with S78454/PCI-24781. This also provides a successful example of modeling early in development that can be applied to other therapeutics. 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 A192.