Angelique Bruyer

DNA methylation score is predictive of myeloma cell sensitivity to 5‐azacitidine

Chee, C.E., Steensma, D.P., Wu, W., Hanson, C.A. & Tefferi, A. (2008) Neither serum ferritin nor the number of red blood cell transfusions affect overall survival in refractory anemia with ringed sideroblasts. American journal of hematology, 83, 611–613. Gattermann, N. (2008) Overview of guidelines on iron chelation therapy in patients with myelodysplastic syndromes and transfusional iron overload. International journal of hematology, 88, 24–29. Hoffbrand, A.V., Taher, A. & Cappellini, M.D. (2012) How I treat transfusional iron overload. Blood, 120, 3657–3669. Pascal, L., Beyne-Rauzy, O., Brechignac, S., Marechaux, S., Vassilieff, D., Ernst, O., Berthon, C., Gyan, E., Gourin, M.-P., Dreyfus, F., Fenaux, P. & Rose, C. (2013) Cardiac iron overload assessed by T2* magnetic resonance imaging and cardiac function in regularly transfused myelodysplastic syndrome patients. British journal of haematology, 162, 413–415. Rose, C., Brechignac, S., Vassilief, D., Pascal, L., Stamatoullas, A., Guerci, A., Larbaa, D., Dreyfus, F., Beyne-Rauzy, O., Chaury, M.P., Roy, L., Cheze, S., Morel, P., Fenaux, P. & GFM (Groupe Francophone des My elodysplasies) (2010) Does iron chelation therapy improve survival in regularly transfused lower risk MDS patients? A multicenter study by the GFM (Groupe Francophone des My elodysplasies). Leukemia research, 34, 864–870. Virtanen, J.M., Remes, K.J., It€al€a-Remes, M.A., Saunavaara, J.P., Komu, M.E., Partanen, A.M. & Parkkola, R.K. (2012) The relationship between cardiac and liver iron evaluated by MR imaging in haematological malignancies and chronic liver disease. Blood cancer journal, 2, e49. Wermke, M., Schmidt, A., Middeke, J.M., Sockel, K., von Bonin, M., Sch€ onefeldt, C., Mair, S., Plodeck, V., Laniado, M., Weiss, G., Schetelig, J., Ehninger, G., Theurl, I., Bornh€auser, M. & Platzbecker, U. (2012) MRI-based liver iron content predicts for nonrelapse mortality in MDS and AML patients undergoing allogeneic stem cell transplantation. Clinical cancer research: an official journal of the American Association for Cancer Research, 18, 6460–6468.

RECQ1 helicase is involved in replication stress survival and drug resistance in multiple myeloma

Multiple myeloma (MM) is a plasma cell cancer with poor survival, characterized by the expansion of multiple myeloma cells (MMCs) in the bone marrow. Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the most downregulated genes. RecQ helicases are DNA unwinding enzymes involved in the maintenance of chromosome stability. Here we show that RECQ1 is significantly overexpressed in MMCs compared to normal plasma cells and that increased RECQ1 expression is associated with poor prognosis in three independent cohorts of patients. Interestingly, RECQ1 knockdown inhibits cells growth and induces apoptosis in MMCs. Moreover, RECQ1 depletion promotes the development of DNA double-strand breaks, as evidenced by the formation of 53BP1 foci and the phosphorylation of ataxia-telangiectasia mutated (ATM) and histone variant H2A.X (H2AX). In contrast, RECQ1 overexpression protects MMCs from melphalan and bortezomib cytotoxicity. RECQ1 interacts with PARP1 in MMCs exposed to treatment and RECQ1 depletion sensitizes MMCs to poly(ADP-ribose) polymerase (PARP) inhibitor. DNMT inhibitor treatment results in RECQ1 downregulation through miR-203 deregulation in MMC. Altogether, these data suggest that association of DNA damaging agents and/or PARP inhibitors with DNMT inhibitors may represent a therapeutic approach in patients with high RECQ1 expression associated with a poor prognosis.

Efficient transduction of healthy and malignant plasma cells by lentiviral vectors pseudotyped with measles virus glycoproteins

A lot of genes deregulated in malignant plasma cells (PCs) involved in multiple myeloma have been reported these last years. The expression of some of these genes is associated with poor survival. A critical step is to elucidate the biological mechanisms triggered by these gene products. Such studies are hampered by the difficulty to obtain malignant PCs and to genetically modify them. Usual lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein poorly transduced healthy and malignant PCs. Here, we report that LVs pseudotyped with the hemagglutinin and fusion glycoproteins from the measles Edmonston strain (H/F-LVs) can efficiently and stably transduce healthy and primary malignant PCs, without modifying their main phenotypic characteristics. Both LV pseudotypes efficiently transduced human myeloma cell lines. Importantly, both healthy and malignant PCs expressed CD46 and SLAMF1/CD150 membrane proteins, which are critical receptors for binding and productive genetic modification by H/F-LVs. The ability to efficiently introduce and express a given gene into PCs opens the possibility to study in detail PC biology.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells

BackgroundMultiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy.MethodsSince HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment.ResultsWe report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines.ConclusionIn conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

Global miRNA expression analysis identifies novel key regulators of plasma cell differentiation and malignant plasma cell

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that attenuate expression of their mRNA targets. Here, we developed a new method and an R package, to easily infer candidate miRNA–mRNA target interactions that could be functional during a given biological process. Using this method, we described, for the first time, a comprehensive integrated analysis of miRNAs and mRNAs during human normal plasma cell differentiation (PCD). Our results reveal 63 miRNAs with significant temporal changes in their expression during normal PCD. We derived a high-confidence network of 295 target relationships comprising 47 miRNAs and 141 targets. These relationships include new examples of miRNAs that appear to coordinately regulate multiple members of critical pathways associated with PCD. Consistent with this, we have experimentally validated a role for the miRNA-30b/c/d-mediated regulation of key PCD factors (IRF4, PRDM1, ELL2 and ARID3A). Furthermore, we found that 24 PCD stage-specific miRNAs are aberrantly overexpressed in multiple myeloma (MM) tumor plasma cells compared to their normal counterpart, suggesting that MM cells frequently acquired expression changes in miRNAs already undergoing dynamic expression modulation during normal PCD. Altogether, our analysis identifies candidate novel key miRNAs regulating networks of significance for normal PCD and malignant plasma cell biology.

Mechanisms and clinical relevance of TRAIL-triggered responses in synovial fibroblasts of rheumatoid arthritis patients

Objective We have shown that TRAIL induces apoptosis only in a portion of RA fibroblast-like synoviocytes (RAFLS) and that in the surviving cells, TRAIL induced proliferation. In the present study, we compared RAFLS-resistant and RAFLS-sensible to TRAIL-induced apoptosis including levels of the TRAIL receptors (TRAIL-R) and clinical features of respective patient. We evaluated TRAIL and its soluble decoy receptor osteoprotegerin (OPG) levels in RA patients, osteoarthritis (OA), spondylarthritis (SpA). Methods FLS were extracted from synovial tissues of RA patients (n=30) and analysed by FACS for TRAIL-receptors expression. We obtained DAS28 within the 3 months of surgery for 13 patients. TRAIL-responses of FLS were analysed by AnnexinV for apoptosis, thymidine-incorporation for proliferation. TRAIL receptor activity was assessed by RNA silencing. HIC were performed to evaluate TRAIL level in synovial tissues from RA (n=7) and OA patients (n=4). ELISA was used to determine TRAIL-levels in synovial fluid of OA; n=20), SpA; (n=20) and establish RA patients (n=30). Serum levels of TRAIL and OPG were measured in 72 patients fulfilling the ACR criteria (1987) with recent (<2 years) and active (>3 swollen joints) RA that were not treated or had a stable background treatment for at least 1 month. 48 of the RA patients were followed up at 6 months. Results Disease severity of RA patients inversely correlated with susceptibility of FLS to TRAIL-induced apoptosis (r=0.753, p=0. 011). TRAIL-sensitive cells expressed significantly lower levels of the decoy TRAIL-R4 (p=0.008) and surprisingly also of TRAIL-R1 (p=0.014), one of the described death receptor. Silencing of these two receptors increased TRAIL-induced apoptosis in RAFLS. TRAIL levels were elevated in the arthritic joints of patients with established RA compared to other patients (p<0.001). A low ratio OPG/TRAIL in sera of early RA patients at baseline was associated with a better evolution of disease activity (p=0.028), but high serum levels of TRAIL at follow-up were associated with joint damages (p=0.0063). Conclusion Surprisingly, TRAIL-R1 seems to be a survival factor protecting RAFLS against TRAIL-induced apoptosis. The negative correlation between TRAIL sensitivity in vitro and RA activity suggests that RAFLS develop resistance to escape TRAIL protective role. Indeed, in early RA patients, a low OPG/TRAIL ratio at baseline was associated with remission at 6 months but persistent TRAIL serum levels are associated with joint damage. These findings suggest a dual role for TRAIL in RA and resistance of RAFLS to TRAIL-induced apoptosis is associated with a disease promoting activity of TRAIL in RA.

Hypoxia favors the generation of human plasma cells

ABSTRACT Plasma cells (PCs) generation occurs in hypoxic conditions in vivo, whereas the relevance of O2 pressure in PC differentiation remains unknown. Using our in vitro PC differentiation model, we investigated the role of hypoxia in PC generation. Hypoxia increases the generation of plasmablasts (PBs) starting from memory B cells, by increasing cell cycle and division number. Reactome analysis demonstrated a significant enrichment of genes involved in HIF1α and HIF2α transcription factor network, metabolism and MYC related pathways in hypoxic compared with normoxic PBs. Hypoxia-induced metabolism alteration and MYC pathway are involved in malignant PC pathophysiology. Therefore, the expression of 28 out of the 74 genes overexpressed in hypoxic PBs compared with normoxic ones was found to be associated with an adverse prognosis (event free survival and overall survival) in newly diagnosed multiple myeloma patients. According to the role of hypoxia in supporting PBs generation through cell cycle induction, c-MYC activation and metabolism alteration, it could be involved in plasma cell tumorigenesis.

Regulation of pleitropic effect of TRAIL on RAFLS by TRAIL-R1 and -R2

Rationale Induction of apoptosis in fibroblast-like synoviocytes (FLS) has been proposed as a therapeutic approach. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on rheumatoid arthritis FLS (RAFLS) and suggested as a potential drug. We have previously shown that exposure to TRAIL induces apoptosis only in a portion of RAFLS. In the surviving cells, TRAIL induced …