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PDF file 43K, Phenotype of BCLs after abexinostat treatment

PDF file 86K, supplementary figure legend

PDF file 109K, Conventional molecular parameters do not predict drug-response to abexinostat

PDF file 35K, Expression level of Xist in patient-derived xenograft

PDF file 164K, Cell cycle progression was differentially altered by abexinostat according to drug-response profile

PDF file 123K, HDAC activity is impaired by treatment independently of drugresponse profiles

Purpose: Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies.Experimental Design: We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers.Results: We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population.Conclusions: Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment. Clin Cancer Res; 19(23); 6520–31. ©2013 AACR.

PDF file 126K, The effect of abexinostat on CSC is achieved through the specific inhibition of HDACs

PDF file 54K, Karyotypes of BCLs

PDF file 166K, Identification of two drug-response groups using a Gaussian mixture model (GMM)

PDF file 68K, Supplementary materials and methods

PDF file 44K, Clinical and pathological features of the breast cancer cell lines (BCLs)

PDF file 85K, Histone deacetylase inhibitors target the CSC population in lowdose sensitive BCLs

PDF file 192K, HDACi treatments define two drug-response profiles in BCLs

BACKGROUND We evaluated the functional consequences of diaphragm involvement in patients with inclusion body myositis (IBM). METHODS Ultrasound diaphragm thickening fraction (TFdi), lung function and dyspnea levels were compared between IBM patients and matched controls. Patients with IBM were grouped into "low" and "high" diaphragm activity based on TFdi values (with cutoff value being the lowest observed TFdi in the control group), and clinical characteristics were compared between groups. RESULTS 20 IBM patients were included. TFdi was significantly lower in patients and correlated with time since symptom onset (rho = 0.74, P

Case Presentation A 63-year-old man was referred for slowly progressive dyspnea on exertion that had developed over 7 years. Dyspnea was initially only present during high-intensity physical activity, but was now present while walking rapidly on a flat surface. Symptoms were accentuated while supine and when bending forward. He reported respiratory difficulties when submerged in water and a recent onset of slight symmetric lower limb weakness that was only apparent during strenuous physical activity. He also had OSA, which was adequately controlled with continuous positive airway pressure therapy. Neurologic and rheumatologic histories were otherwise unremarkable. He denied any impact accidents or trauma to the cervical spine and prior neck or thoracic surgeries.

Cancer cells contain significant alterations in their epigenomic landscape, which several enzyme families reversibly contribute to. One class of epigenetic modifying enzymes is that of histone deacetylases (HDAC), which are receiving considerable scrutiny clinically as a therapeutic target in many cancers. The underlying rationale is that inhibiting HDACs will reverse dysregulated target gene expression by modulating functional histone (or other) acetylation marks. This perspective will discuss a recent paper by Markozashvili and co-workers which appeared in Gene, which indicates that the mechanisms by which HDAC inhibitors (HDACis) alter the epigenetic landscape include widespread alternative effects beyond simply controlling regional epigenetic marks. HDACs are involved in many processes/diseases, and it is not surprising that HDACis have considerable off-target effects, and thus a major effort is being directed toward identification of inhibitors which are selective for HDAC isoforms often uniquely implicated in various cancers. This Perspective will also discuss some representative work with inhibitors targeting individual HDAC classes or isoforms. At present, it is not really clear that isoform-specific HDACis will avoid non-selective effects on other unrecognized activities of HDACs.

International audience; Background We determined the safety, pharmacokinetics, pharmacodynamics, and antitumour activity of abexinostat in B-cell lymphoma or chronic lymphocytic leukaemia. Patients and methods Thirty-five patients received oral abexinostat 30, 45, or 60 mg/m(2) bid in a 3 + 3 design in three 21-day schedules: 14 days on treatment in schedule 1 (D1-14); 10 days in schedule 2 (D1-5 and D8-12); and 12 days in schedule 3 (D1-4, D8-11, and D15-18). Safety, tumour response, plasma concentration, and histone H3 acetylation were measured. Results Two dose-limiting toxicities occurred in each schedule (one grade 3 febrile neutropenia; five grade 4 thrombocytopenia) at 60 mg/m(2) bid (maximal tolerated dose). The recommended dose was 45 mg/m(2) bid; schedule 1 was considered optimal. Non-haematological drug-related toxicities included grade 1 or 2 diarrhoea (43%), nausea (23%), and vomiting (11%); haematological toxicities included thrombocytopenia (31% grade 3, and 26% grade 4), which remained manageable and reversible on withdrawal. Of 29 evaluable patients, there were 2 complete and 6 partial responses; median duration of response was 14.6 months (range 3-16.5 months) (1 cycle is equivalent to 0.75 months). There was no evidence for nonlinear pharmacokinetics. There was a correlation between dose and histone acetylation. Conclusion Abexinostat has manageable toxicity and induced some durable complete and partial responses in B-cell lymphoma or chronic lymphocytic leukaemia. Our results suggest most favourable responses in patients with follicular lymphoma, though further research would be needed to confirm this finding.

Myeloproliferative disorders (MPD) are clonal hematopoietic diseases characterized by the proliferation and expansion of one or several myeloid cell lineages in the bone marrow. During the chronic phase, the cells follow their normal differentiation pathway and become mature blood cells. During a second phase, an acute syndrome may occur. The conventional classification separates MPDs in clinical entities. These include chronic myeloid leukemia (CML), chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). MPDs with different syndromes and molecular abnormalities are grouped in non-classical MPDs. Systemic mastocytosis is not classically included in MPDs but is a related disease.

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.

Purpose: Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies. Experimental Design: We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers. Results: We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population. Conclusions: Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment. Clin Cancer Res; 19(23); 6520–31. ©2013 AACR.

Background The t(6;8) translocation found in rare and agressive myeloproliferative disorders results in a chimeric gene encoding the FOP-FGFR1 fusion protein. This protein comprises the N-terminal region of the centrosomal protein FOP and the tyrosine kinase of the FGFR1 receptor. FOP-FGFR1 is localized at the centrosome where it exerts a constitutive kinase activity. Results We show that FOP-FGFR1 interacts with the large centrosomal protein CAP350 and that CAP350 is necessary for FOP-FGFR1 localisation at centrosome. FOP-FGFR1 activates the phosphoinositide-3 kinase (PI3K) pathway. We show that p85 interacts with tyrosine 475 of FOP-FGFR1, which is located in a YXXM consensus binding sequence for an SH2 domain of p85. This interaction is in part responsible for PI3K activation. Ba/F3 cells that express FOP-FGFR1 mutated at tyrosine 475 have reduced proliferative ability. Treatment with PI3K pathway inhibitors induces death of FOP-FGFR1 expressing cells. FOP-FGFR1 also recruits phospholipase Cγ1 (PLCγ1) at the centrosome. We show that this enzyme is recruited by FOP-FGFR1 at the centrosome during interphase. Conclusion These results delineate a particular type of oncogenic mechanism by which an ectopic kinase recruits its substrates at the centrosome whence unappropriate signaling induces continuous cell growth and MPD.

Oncogenic kinases of myeloproliferative disorders induce both protein synthesis and G1 activators

Some myeloproliferative disorders (MPD) result from a reciprocal translocation that involves the FGFR1 gene and a partner gene. The event creates a chimeric gene that encodes a fusion protein with constitutive FGFR1 tyrosine kinase activity. FGFR1-MPD is a rare disease, but its study may provide interesting clues on different processes such as cell signalling, oncogenesis and stem cell renewal. Some partners of FGFR1 are centrosomal proteins. The corresponding oncogenic fusion kinases are targeted to the centrosome. Constitutive phosphorylation at this site may perturbate centrosome function and the cell cycle. Direct attack at this small organelle may be an efficient way for oncogenes to alter regulation of signalling for proliferation and survival and get rid of checkpoints in cell cycle progression. The same effect might be triggered by other fusion kinases in other MPD and non-MPD malignancies.

Myeloproliferative disorders (MPD) are malignant diseases of hematopoietic progenitor cells. Many MPDs result from a chromosomal translocation that creates a fusion gene encoding a chimeric kinase. The fibroblast growth factor receptor 1 (FGFR1)-MPD is characterized by the fusion of the FGFR1 kinase with various partners, including FOP. We show here that both normal FOP and FOP-FGFR1 fusion kinase localize to the centrosome. The fusion kinase encounters substrates at the centrosome where it induces strong phosphorylation on tyrosine residues. Treatment with FGFR1 kinase inhibitor SU5402 abolishes FOP-FGFR1-induced centrosomal phosphorylation and suppresses the proliferative and survival potentials of FOP-FGFR1 Ba/F3 cells. We further show that FOP-FGFR1 allows cells to overcome G1 arrest. Therefore, the FOP-FGFR1 fusion kinase targets the centrosome, activates signaling pathways at this organelle, and sustains continuous entry in the cell cycle. This could represent a potential new mechanism of oncogenic transformation occurring specifically at the centrosome.

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

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

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.

Objectives. Solid tumors are believed to be organized in a cellular hierarchy with a population of cancer stem cell (CSC) driving cancer progression and resistance to conventional treatment. Deciphering molecular mechanisms related to CSC biology will help develop CSC targeted therapeutic strategies. CSC self-renewal and differentiation programs are regulated by epigenetic modulations. Our study specifically targets breast CSCs with a novel epidrug used as a differentiating agent, a histone deacetylase inhibitor (HDACi) S78454, aka PCI-24781. Methods. Seventeen breast cancer cell lines from several molecular subtypes (basal, luminal and mesenchymal) were treated for 72 hours with S78454 to determine their respective IC50s. Specific inhibition of histone deacetylation was measured by immunoblotting (histone and tubulin acetylation, p21). The effect of S78454 treatment on the breast CSC population was assayed by both ALDEFLUOR assay and tumorsphere formation. Cell cycle progression was evaluated along S78454 treatment. The induction of cell differentiation was monitored by tracking modifications in molecular phenotype using immunofluorescence. Results. Two different types of sensitivity profiles to S78454 were observed (high and low), but these were not correlated to the molecular subtype of the cell lines. After six hours of S78454 treatment, specific biological effects including increased histone and tubulin acetylation and p21 protein expression were seen. After 72 hours of treatment morphological changes were induced in cells with low sensitivity, which increased in size, grew in clusters, and changed their phenotype with a lost of cytokeratins 5/6, 14, and vimentin protein expression and a gain of E-cadherin. In cell lines with low sensitivity, S78454 treatment induced a decrease in the ALDEFLUOR-positive breast CSC population as well as a decrease in tumorsphere formation whereas no specific effect was observed on CSC population from highly sensitive cell lines. Interestingly, S78454 treatment induced a G2/M cell cycle arrest in highly sensitive cell lines whereas cell lines with a low sensitivity to S78454 present a transitory G1/S cell cycle arrest after 24 hours of treatment before achieving a complete cell division. Conclusions. In low sensitive cell lines, biological and molecular changes following S78454 might reflect the epigenetic modulation of breast cancer stem cell differentiation. Cell cycle progression from G1 to S has been defined as an important cell cycle stage controlling stem cell fate allowing equilibrium between self-renewal and committed cell fate decision. The transitory G1/S cell cycle block induced by S78454 treatment may favorized cell differentiation. In highly sensitive cell lines, S78454 treatment seems to induce an irreversible G2/M arrest inducing apoptosis in all cancer cell populations. Therefore, our findings suggest that HDACi treatment could be used as a novel therapeutic strategy through induction of differentiation of breast CSC. 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 A57.

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.