Carole Brosseau

RITA (Reactivating p53 and Inducing Tumor Apoptosis) is efficient against TP53abnormal myeloma cells independently of the p53 pathway.

BackgroundThe aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells.MethodsA large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines.ResultsBoth drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53mutated cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤19%).ConclusionThese data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.

A simple flow cytometry‐based barcode for routine authentication of multiple myeloma and mantle cell lymphoma cell lines

lines are widely used in laboratories for in vitro experi-ments, especially for investigating abnormal hallmarks in can-cer cells and identifying therapeutic targets. Human cell linesare typically derived in academic laboratories from a widerange of cancer samples. To achieve a representation of intra-cancer heterogeneity, several laboratories, including ours, haveestablished cell line collections. However, the establishmentand maintenance of such collections significantly increase therisk of cross-contaminations and misidentification of celllines, leading to the publication of false data/interpretation(1). In addition to the risk of cross-contamination, widelyused cell lines can be described in contrasting manners for aparticular feature (e.g., the JJN3 myeloma cell line appearseither TP53

Combination of lenalidomide with vitamin D3 induces apoptosis in mantle cell lymphoma via demethylation of BIK.

Mantle cell lymphoma (MCL) is a currently incurable B-cell malignancy. Lenalidomide (Len) has been demonstrated to be one of the most efficient new treatment options. Because Len and 1α,25-dihydroxyvitamin (VD3) synergize to kill breast cancer cells, we investigated whether VD3 could increase the ability of Len to induce MCL cell death. While MCL cells were weakly sensitive to Len (1 μM), the addition of VD3 at physiological dose (100 nM) strongly increased cell death, accompanied by slowdown in cell cycle progression in MCL cell lines (n=4 out of 6) and primary samples (n=5 out of 7). The Len/VD3 treatment markedly increased the expression of the BH3-only BCL2-interacting killer (Bik) without affecting the expression of other Bcl-2 molecules. Immunoprecipitation assays demonstrated that Bik was free from anti-apoptotic partners, Bcl-2 and Bcl-xL, in treated cells. Moreover, silencing of BIK prevented apoptosis induced by Len/VD3, confirming the direct involvement of Bik in cell death. Bik accumulation induced by Len/VD3 was related to an increase in BIK mRNA levels, which resulted from a demethylation of BIK CpG islands. The sensitivity of MCL cells to Len/VD3 was similar to the response to 5-azacytidine, which also induced demethylation of BIK CpG islands. These preclinical data provide the rationale to investigate the role of VD3 in vivo in the response to Len.

High Circulating CD4+CD25hiFOXP3+ T Cell Subpopulation Early After Lung Transplantation is Associated with Development of Bronchiolitis Obliterans Syndrome

Purpose Despite treatment improvement, the bronchiolitis obliterans syndrome (BOS) affects more than 50% of the lung-transplant recipient in the 5 years post-transplantation and because it cannot be predict or cure, it is the major cause of death after lung transplantation (TP). The purpose of this study was to assess whether the peripheral blood T-lymphocyte profile could predict BOS in lung transplant recipients. Methods An in-depth profiling of CD4+ and CD8+ T cells was prospectively performed on blood cells from stable and BOS patients with a longitudinal follow-up. Samples were obtained and analyzed at 1 and 6 months after transplantation, at the time of BOS diagnosis, and at an intermediate time point at 6 to 12 months before BOS diagnosis. Results Whereas no significant difference was found for T cell compartments at BOS diagnosis or several months before, we report an increase in the CD4+CD25hiFoxP3+ T cell subpopulation in BOS patients at 1 and 6 months after transplantation (3.39%±0.40 vs 1.67%±0.22 in STA, P<0.001). A CD4+CD25hiFoxP3+ T cell threshold of 2.4% discriminated BOS and stable patients at 1 month post-transplantation. This was validated on a second set of patients at 6 months post-transplantation. Patients with a proportion of CD4+CD25hiFoxP3+ T cells up to 2.4% in the 6 months following transplantation had a 2 fold higher risk of developing BOS. Conclusion This study is the first to report an increased proportion of circulating CD4+CD25hiFoxP3+ T cells early post-transplantation in lung recipients who will develop BOS within 3 years, and document support for its use as a BOS predictive biomarker.

CD9 Tetraspanin: A New Pathway for the Regulation of Inflammation?

CD9 belongs to the tetraspanin superfamily. Depending on the cell type and associated molecules, CD9 has a wide variety of biological activities such as cell adhesion, motility, metastasis, growth, signal transduction, differentiation, and sperm–egg fusion. This review focuses on CD9 expression by hematopoietic cells and its role in modulating cellular processes involved in the regulation of inflammation. CD9 is functionally very important in many diseases and is involved either in the regulation or in the mediation of the disease. The role of CD9 in various diseases, such as viral and bacterial infections, cancer and chronic lung allograft dysfunction, is discussed. This review focuses also on its interest as a biomarker in diseases. Indeed CD9 is primarily known as a specific exosome marker however, its expression is now recognized as an anti-inflammatory marker of monocytes and macrophages. It was also described as a marker of murine IL-10-competent Breg cells and IL-10-secreting CD9+ B cells were associated with better allograft outcome in lung transplant patients, and identified as a new predictive biomarker of long-term survival. In the field of cancer, CD9 was both identified as a favorable prognostic marker or as a predictor of metastatic potential depending on cancer types. Finally, this review discusses strategies to target CD9 as a therapeutic tool. Because CD9 can have opposite effects depending on the situation, the environment and the pathology, modulating CD9 expression or blocking its effects seem to be a new promising therapeutic strategy.

Abstract 793: Preclinical study of efficacy and specificity of p53-reactivating drugs in multiple myeloma, identification of biomarkers.

In this study, the efficacy of RITA (Reactivating p53 and Inducing Tumor Apoptosis) to kill multiple myeloma (MM) cells was evaluated in comparison to that of nutlin3a (MDM2 inhibitor) and in relation to the TP53 status of 32 human myeloma cell lines (HMCLs, 9 wt, 15 mutated, 6 truncated, 2 KO) and primary samples (n=19). This panel of HMCLs is representative of the molecular diversity of patients for both the chromosomal abnormalities (14q32 translocation) and the TP53 status, and thus allows us to perform an accurate evaluation of both the specificity and efficacy of molecules. TP53 status of HMCLs was established by direct sequencing of TP53 mRNA while that of primary cells was evaluated by the presence of chromosome 17p deletion (del17p), which is strong adverse prognostic value for patients with MM. Both drugs induced the apoptosis of MM cells, as shown by the loss of CD138 expression (a marker of viability for myeloma cells), Apo2.7 staining, appearance of a subG1 peak and activation of caspases 3 and 9. RITA induced cell death in about one third of HMCLs and primary samples. The apoptosis induced by RITA was not related to the t(14q32) or TP53 status of HMCLs or primary samples (p=0.52 and p=0.96, respectively). RITA failed to increase the expression level of p53 or of p53 targets i.e., Noxa, p21, Bax or DR5 in sensitive cells. In primary samples, RITA slightly increased DR5 expression in resistant samples without del17p and decreased it in sensitive samples with or without del17p. RITA failed to modulate p53 re-localization to the mitochondria or nucleus in sensitive cells. Finally, silencing of p53 in two highly sensitive TP53mutated HMCLs failed to inhibit apoptosis induced by RITA, confirming that the RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, the apoptosis induced by nutlin3a was directly linked to the TP53 status of HMCLs and primary samples (p These data show that 1) RITA effectively induced apoptosis in a subset of MM cells independently of p53 and 14q32 heterogeneity, and could be of interest for patients with del17p, who are resistant to current therapies; 2) nutlin3a was effective in myeloma cells retaining a functional p53 pathway. The efficacy and specificity of other p53-reactivating small molecules i.e., PRIMA-1met and CP-31398, are currently under evaluation. Citation Format: Sylvanie Surget, Geraldine Descamps, Carole Brosseau, Philippe Moreau, Steven Le Gouill, Martine Amiot, Catherine Pellat-Deceunynck. Preclinical study of efficacy and specificity of p53-reactivating drugs in multiple myeloma, identification of biomarkers. [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 793. doi:10.1158/1538-7445.AM2013-793