Stéphane Depil

Polo-like Kinase 1: A Potential Therapeutic Option in Combination with Conventional Chemotherapy for the Management of Patients with Triple-Negative Breast Cancer

Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. To discover potential therapeutic targets for the poor prognosis-associated triple-negative breast cancer (TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1 (PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes. High PLK1 expression was confirmed by reverse phase protein and tissue microarrays. In triple-negative cell lines, RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increase in phosphorylated H2AX, G(2)-M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencing impaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel), and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line. When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo in two xenografts models established from biopsies of patients with TNBC. Most importantly, the administration of BI-2536, in combination with doxorubicin + cyclophosphamide chemotherapy, led to a faster complete response compared with the chemotherapy treatment alone and prevented relapse, which is the major risk associated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeutic approach, in association with conventional chemotherapy, for the management of patients with TNBC.

Determination of a HLA II promiscuous peptide cocktail as potential vaccine against EBV latency II malignancies

The Epstein-Barr virus (EBV) is associated with several malignant diseases, which can be distinguished by their patterns of viral latent gene expression. The latency II program is limited to the expression of the nonimmunodominant antigens EBNA1, LMP1 and LMP2 and is seen in EBV-positive Hodgkin disease, nasopharyngeal carcinomas, and peripheral T/NK-cell lymphomas. CD4+ T cells may play a crucial role in controlling these EBV latency II malignancies. In this study, we used the prediction software TEPITOPE to predict promiscuous major histocompatibility complex class II epitopes derived from the latency II antigens EBNA1, LMP1, and LMP2. The predicted peptides were then submitted to peptide-binding assays on HLA II purified molecules, which allowed the selection of 6 peptides (EBNA1: 3; LMP1: 1; and LMP2: 2) with a highly promiscuous capability of binding. This peptide cocktail was immunogenic in a model of HLA-DR1 transgenic mice, leading to a specific cellular and humoral TH1 response. The peptides were also recognized by human CD4+ T cells from individuals expressing various HLA II genotypes. This promiscuous peptide cocktail could be immunogenic in the majority of the population and may be used as a peptide-based vaccine in EBV latency II malignancies.

Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

Background:Oestrogen receptor-negative (ER−) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer.Methods:Gene and protein expression profiles were analysed in a panel of ER− breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively.Results:The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells.Conclusions:Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER− breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

Targeting Bcl-2/Bcl-XL Induces Antitumor Activity in Uveal Melanoma Patient-Derived Xenografts

Purpose Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-XL inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines. Experimental Design Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-XL, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC). Results S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-XL, and Mcl-1 expression were not modified after S44563 administration. Conclusion The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.

HDAC inhibition does not induce estrogen receptor in human triple-negative breast cancer cell lines and patient-derived xenografts.

Several publications have suggested that histone deacetylase inhibitors (HDACis) could reverse the repression of estrogen receptor alpha (ERα) in triple-negative breast cancer (TNBC) cell lines, leading to the induction of a functional protein. Using different HDACis, vorinostat, panobinostat, and abexinostat, we therefore investigated this hypothesis in various human TNBC cell lines and patient-derived xenografts (PDXs). We used three human TNBC cell lines and three PDXs. We analyzed the in vitro toxicity of the compounds, their effects on the hormone receptors and hormone-related genes and protein expression both in vitro and in vivo models. We then explored intra-tumor histone H3 acetylation under abexinostat in xenograft models. Despite major cytotoxicity of all tested HDAC inhibitors and repression of deactylation-dependent CCND1 gene, neither ERα nor ERβ, ESR1 or ESR2 genes respectively, were re-expressed in vitro. In vivo, after administration of abexinostat for three consecutive days, we did not observe any induction of ESR1 or ESR1-related genes and ERα protein expression by RT-qPCR and immunohistochemical methods in PDXs. This observation was concomitant to the fact that in vivo administration of abexinostat increased intra-tumor histone H3 acetylation. These observations do not allow us to confirm previous studies which suggested that HDACis are able to convert ER-negative (ER−) tumors to ER-positive (ER+) tumors, and that a combination of HDAC inhibitors and hormone therapy could be proposed in the management of TNBC patients.

Time dependent modulation of tumor radiosensitivity by a pan HDAC inhibitor: abexinostat

Despite prominent role of radiotherapy in lung cancer management, there is an urgent need for strategies increasing therapeutic efficacy. Reversible epigenetic changes are promising targets for combination strategies using HDAC inhibitors (HDACi).Here we evaluated on two NSCLC cell lines, the antitumor effect of abexinostat, a novel pan HDACi combined with irradiation in vitro in normoxia and hypoxia, by clonogenic assays, demonstrating that abexinostat enhances radiosensitivity in a time dependent way with mean SER10 between 1.6 and 2.5 for A549 and H460. We found, by immunofluorescence staining, flow cytometry assays and western blotting, in abexinostat treated cells, increasing radio-induced caspase dependent apoptosis and persistent DNA double-strand breaks associated with decreased DNA damage signalling and repair. Interestingly, we demonstrated on nude mice xenografts that abexinostat potentiates tumor growth delay in combined modality treatments associating not only abexinostat and irradiation but also when adding cisplatin.Altogether, our data demonstrate in vitro and in vivo anti-tumor effect potentiation by abexinostat combined with irradiation in NSCLC. Moreover, our work suggests for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.Despite prominent role of radiotherapy in lung cancer management, there is an urgent need for strategies increasing therapeutic efficacy. Reversible epigenetic changes are promising targets for combination strategies using HDAC inhibitors (HDACi). Here we evaluated on two NSCLC cell lines, the antitumor effect of abexinostat, a novel pan HDACi combined with irradiation in vitro in normoxia and hypoxia, by clonogenic assays, demonstrating that abexinostat enhances radiosensitivity in a time dependent way with mean SER10 between 1.6 and 2.5 for A549 and H460. We found, by immunofluorescence staining, flow cytometry assays and western blotting, in abexinostat treated cells, increasing radio-induced caspase dependent apoptosis and persistent DNA double-strand breaks associated with decreased DNA damage signalling and repair. Interestingly, we demonstrated on nude mice xenografts that abexinostat potentiates tumor growth delay in combined modality treatments associating not only abexinostat and irradiation but also when adding cisplatin. Altogether, our data demonstrate in vitro and in vivo anti-tumor effect potentiation by abexinostat combined with irradiation in NSCLC. Moreover, our work suggests for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.

TIPIN depletion leads to apoptosis in breast cancer cells

Triple‐negative breast cancer (TNBC) is the breast cancer subgroup with the most aggressive clinical behavior. Alternatives to conventional chemotherapy are required to improve the survival of TNBC patients. Gene‐expression analyses for different breast cancer subtypes revealed significant overexpression of the Timeless‐interacting protein (TIPIN), which is involved in the stability of DNA replication forks, in the highly proliferative associated TNBC samples. Immunohistochemistry analysis showed higher expression of TIPIN in the most proliferative and aggressive breast cancer subtypes including TNBC, and no TIPIN expression in healthy breast tissues. The depletion of TIPIN by RNA interference impairs the proliferation of both human breast cancer and non‐tumorigenic cell lines. However, this effect may be specifically associated with apoptosis in breast cancer cells. TIPIN silencing results in higher levels of single‐stranded DNA (ssDNA), indicative of replicative stress (RS), in TNBC compared to non‐tumorigenic cells. Upon TIPIN depletion, the speed of DNA replication fork was significantly decreased in all BC cells. However, TIPIN‐depleted TNBC cells are unable to fire additional replication origins in response to RS and therefore undergo apoptosis. TIPIN knockdown in TNBC cells decreases tumorigenicity in vitro and delays tumor growth in vivo. Our findings suggest that TIPIN is important for the maintenance of DNA replication and represents a potential treatment target for the worst prognosis associated breast cancers, such as TNBC.

Abstract 2764: The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target

Treatment of patients with triple-negative breast cancers (TNBCs) remains a major challenge for oncologists and alternative treatments to conventional chemotherapies are needed to improve their survival. The Wnt/beta-catenin signaling, recently reported to be activated in TNBCs, may represent an interesting pathway to target. We report that both LRP5 and LRP6 Wnt coreceptors are more strongly expressed in TNBCs than in other breast tumor subtypes. As very few studies have explored potential differences between LRP5 and LRP6, we investigated the effects of modulating specifically LRP5 or LRP6 expression on Wnt signaling, cell viability and tumorigenesis in HCC38 and MDA-MB-468 TNBC cells. We found that these two cell lines are more similar to TNBC biopsy specimens in terms of Wnt pathway gene expression profiles than any other tested cell line. Unlike LRP5, LRP6 was involved in activating the canonical Wnt pathway in response to Wnt3a. LRP5 knockdown induced caspase-dependent apoptosis, whereas LRP6 knockdown had no such effect. Importantly, LRP5-depleted cells were more sensitive to conventional chemotherapy than cells depleted of LRP6. The knockdown of LRP5 or LRP6 decreased tumorigenesis both in vitro and in vivo. Overall, these data suggest that the LRP5 and LRP6 coreceptors have different functions in TNBCs, with LRP5 playing a preponderant role in survival control. Our data suggest that both LRP5 and LRP6 are potential treatment targets in TNBCs, but that LRP5 may be the most useful target, given the impact of its depletion on cell survival as well as on the response to anti-cancer drugs. Citation Format: Sylvie Maubant, Virginie Maire, Bruno Tesson, Fariba Nemati, David Gentien, Berengere Marty-Prouvost, Stephane Depil, Francisco Cruzalegui, Gordon Tucker, Sergio Roman-Roman, Thierry Dubois. The depletion of LRP5, unlike that of LRP6, promotes apoptosis in triple-negative breast cancer cells, making it an interesting therapeutic target. [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 2764. doi:10.1158/1538-7445.AM2014-2764

Histone deacetylase inhibitor abexinostat affects chromatin organization and gene transcription in normal B cells and in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells.

Abstract 2921: Preclinical characteristics of NP137, a first-in-class monoclonal antibody directed against netrin-1 and inducing dependence receptors-mediated cell death

Background: Some receptors are active in the absence of ligand and actively trigger cell death through apoptosis. These receptors are called “dependence receptors” (DRs) as their expression at the cell surface renders cells critically dependent for their survival on ligand availability. Deleted in Colorectal Carcinoma (DCC) and UNC5H are prototypic DRs which induce apoptosis unless their ligand netrin-1 is present. It has been shown that netrin-1 is up-regulated in a large fraction of tumors and that interference with netrin-1/receptors interaction is associated with inhibition of tumor growth and metastasis in various preclinical models. We have generated the first humanized netrin-1 antibody, NP137, and characterized its antitumor activity. Preclinical results: NP137 is a humanized monoclonal IgG1 antibody, which binds netrin-1 in the nanomolar range affinity and efficiently inhibits the binding of netrin-1 to its receptor UNC5H2 (IC50: 0.5 nM). NP137 recognizes a netrin-1 specific epitope located in the second laminin-type EGF-like repeat. A crystal structure analysis shows that this region is crucial for the binding of netrin-1 to its receptor, providing a structural basis for the biological activity of NP137. In vitro, NP137 induced apoptosis in netrin-1-dependent cell lines as assessed by cell density measurement and caspase 3 activation. Ex vivo analysis performed on fresh human breast tumor slices (3D cultures) showed a specific and significant induction of apoptosis in tumor cells in approximately half of cases. In vivo, NP137 exhibited a significant anti-tumor effect associated with good pharmacokinetic properties in models of both solid tumors and hematologic malignancies. A dose-effect relationship was observed with an optimal pharmacologically active dose established at 10 mg/kg twice a week. We also showed that NP137 potentiates cancer cell death induced by cytotoxic drugs like doxorubicin or platinum derivatives as well as demethylating agents, which induce upregulation of netrin-1 and its receptors. In this context, a strong synergy with doxorubicin was shown in a rat syngeneic model of osteosarcoma, reversing its chemoresistant phenotype. Toxicological studies are ongoing with first results suggesting a high therapeutic margin. Conclusion: We are developing the first therapeutic antibody targeting netrin-1 and inducing dependence receptors-mediated tumor cell death. Preclinical results suggest a therapeutic potential both in solid tumors and hematological malignancies. A high therapeutic margin is expected, which may accelerate combination strategies. Based on these encouraging results, a first-in-human trial is planned at the end of 2015, with the support of EORTC. Note: This abstract was not presented at the meeting. Citation Format: Benjamen Ducarouge, Jean-Guy Delcros, Riad Abes, David Goldschneider, Benjamin Gibert, John Blachier, David Neves, Patrick Mehlen, Agnes Bernet, Stephane Depil. Preclinical characteristics of NP137, a first-in-class monoclonal antibody directed against netrin-1 and inducing dependence receptors-mediated cell death. [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 2921. doi:10.1158/1538-7445.AM2015-2921