Emmanuelle Ménoret

Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma.

Targeting the ubiquitin-proteasome pathway has emerged as a potent anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma. Multiple myeloma cell survival is highly dependent on Mcl-1 antiapoptotic molecules. In a recent study, proteasome inhibitors induced Mcl-1 accumulation that slowed down their proapoptotic effects. Consequently, we investigated the role of Bcl-2 family members in bortezomib-induced apoptosis. We found that bortezomib induced apoptosis in five of seven human myeloma cell lines (HMCL). Bortezomib-induced apoptosis was associated with Mcl-1 cleavage regardless of Mcl-1L accumulation. Furthermore, RNA interference mediated Mcl-1 decrease and sensitized RPMI-8226 HMCL to bortezomib, highlighting the contribution of Mcl-1 in bortezomib-induced apoptosis. Interestingly, an important induction of Noxa was found in all sensitive HMCL both at protein and mRNA level. Concomitant to Mcl-1 cleavage and Noxa induction, we also found caspase-3, caspase-8, and caspase-9 activation. Under bortezomib treatment, Mcl-1L/Noxa complexes were highly increased, Mcl-1/Bak complexes were disrupted, and there was an accumulation of free Noxa. Finally, we observed a dissociation of Mcl-1/Bim complexes that may be due to a displacement of Bim induced by Noxa. Thus, in myeloma cells, the mechanistic basis for bortezomib sensitivity can be explained mainly by the model in which the sensitizer Noxa can displace Bim, a BH3-only activator, from Mcl-1, thus leading to Bax/Bak activation.

Cell Death via DR5, but not DR4, Is Regulated by p53 in Myeloma Cells

Myeloma cells are sensitive to TRAIL through the two death receptors DR4 and DR5. Because p53 directly modulates expression of death receptors, we investigated here whether p53 can modulate myeloma sensitivity to TRAIL. We found that p53 affects the sensitivity of myeloma cells to the DR5 agonistic human antibody lexatumumab but not the DR4 antibody mapatumumab. TP53 wild-type myeloma cells overexpressed DR5 in correlation with sensitivity to lexatumumab. Both nongenotoxic (nutlin-3a) and genotoxic (melphalan) p53-inducing stresses increased DR5 expression only in TP53 wild-type cells and synergistically increased lexatumumab efficiency yet did not increase DR4 expression, nor sensitivity to mapatumumab. Silencing of p53 strongly decreased DR5 expression and induced resistance to nutlin-3a and lexatumumab but did not modulate DR4 expression or sensitivity to mapatumumab. Increase of lexatumumab efficiency induced by nutlin-3a was related to a p53-dependent increase of DR5 expression. In primary myeloma cells, nutlin-3a increased DR5 expression and lexatumumab efficiency but did not increase mapatumumab efficiency. Taken together, our findings indicate that p53 controls the sensitivity of myeloma through DR5 but not DR4 and suggest that a subset of patients with multiple myeloma may benefit from DR5 therapy.

BH3-only protein Bik is involved in both apoptosis induction and sensitivity to oxidative stress in multiple myeloma

Background:Although gene expression profile of multiple myeloma (MM) patients shows a wide range of Bik/Nbk expression, varying from absent to high, its regulation and function in myeloma cells is poorly understood. Thus, we addressed these questions in MM.Methods:Human myeloma cell lines (HMCLs) and primary purified myeloma cells were studied for Bcl-2 family protein expression by western blot and further correlation analysis was performed. Correlative study between Bik and thyrotroph embryonic factor (TEF) transcription factor expression was analysed by PCR. Stress oxidative response was analysed by flow cytometry.Results:A strong expression of Bik protein was found only in one out of three of HMCL and correlated to Bcl-2 expression (P=0.0006). We demonstrated that Bik could be regulated at the protein level by Bcl-2 and at the transcriptional level by TEF. Bik overexpression sensitises myeloma cells to oxidative stress whereas Bik silencing increases resistance to H2O2 oxidative stress. Furthermore, Bik ectopic expression disrupts Bim/Bcl-2 and Bim/Bcl-xL endogenous complexes triggering Bim release that could induce Bax and Bak activation.Conclusions:Ours results suggest that Bik has a role in both, apoptosis induction and sensitivity to oxidative stress in myeloma cells. Small BH3 mimetic molecules should be considered for further apoptosis-based therapy in myeloma cells expressing endogenous Bik/Bcl-2 complexes.

Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma

Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.

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

Abstract B16: The pro-apoptotic effect of dexamethasone mediated by GILZ and Bim up-regulation is related to genetic heterogeneity of multiple myeloma.

Multiple myeloma (MM) is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. Briefly, MM that are hyperdiploid or have a t(11;14) translocation have a better prognosis than those with t(4;14) or t(14;16) translocations. Dexamethasone (Dex) is widely used in all phases of MM treatment as induction, consolidation or maintenance. However, the mechanism of Dex sensitivity still remains elusive. In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in 33 human myeloma cell lines (HMCLs) representative of the molecular subsets carrying t(11;14), t(4;14) or t(14;16) translocations, which deregulate CCND1, MMSET and c-MAF, respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing glucocorticoid receptor (GR), glucocorticoid-induced leucin zipper (GILZ) and Bcl-2 protein family expression in the different MM molecular subtypes. Transient knock-down of glucocorticoid-responsive proteins were performed to define their role in the pro-apoptotic effect of Dex. We first demonstrated that direct pro-apoptotic effect of Dex was related to the genetic heterogeneity of MM, since sensitive cell lines were restricted to t(14;16) and t(4;14) subgroups. We next demonstrated by transcriptomic Affymetrix analysis that GR expression was heterogeneous among the different molecular subtypes of HMCLs. MAF subgroup significantly expressed higher levels of GR than all other subgroups (p=0.02). This result was also confirmed on 300 newly diagnosed MM patients (p In conclusion, Dex exerted a direct anti-tumor effect on HMCLs of t(14;16) and t(4;14) molecular subgroups while t(11;14) subgroup was insensitive to it. This result suggested that conventional therapeutic approaches should be re-evaluated within molecular subgroups of MM patients to favor potential molecular subtype therapy based on rational preclinical data. Finally, while Dex interferes with multiple pathways, we begin to unravel its complex mechanism of action demonstrating that transactivation, inducing GILZ up-regulation, plays a pivotal role in Dex induced cell death through the regulation of the Bcl-2 protein network. Citation Format: Charlotte Kervoelen, Emmanuelle Menoret, Patricia Gomez-Bougie, Regis Bataille, Philippe Moreau, Catherine Pellat-Deceunynck, Martine Amiot. The pro-apoptotic effect of dexamethasone mediated by GILZ and Bim up-regulation is related to genetic heterogeneity of multiple myeloma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B16.

Abstract C28: Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim.

Multiple myeloma (MM) is heterogeneous with respect to its causative chromosomal abnormalities and the treatment response of patients. Briefly, MM patients with chromosomal hyperdiploidy or those carrying a t(11.14) translocation have a better prognosis than those with a t(4;14) or t(14;16) translocation. Dexamethasone (Dex) is widely used in all phases of the treatment of MM as induction, consolidation or maintenance. However, the mechanisms of sensitivity and resistance to Dex still remain elusive. In the present study, we analyzed the ability of Dex to induce cell death by Apo-2.7 staining in 33 human myeloma cell lines (HMCLs) representative of the molecular subsets i.e., carrying a t(11;14), t(4;14) or t(14;16) translocation, which deregulates the expression of CCND1, MMSET, and c-MAF respectively. The mechanisms controlling Dex-induced apoptosis were evaluated by analyzing the nuclear translocation of glucocorticoid receptor (GR), signalling pathways and modulation of Bcl-2 protein family in the different MM molecular subtypes. We first demonstrated by transcriptomic analysis (Affymetrix) that GR expression was heterogeneous among the different molecular subtypes of HMCLs and patients. Indeed, t(14;16) c-MAF subgroup significantly expressed higher levels of GR than all other subgroups in both HMCLs (Kruskall-Wallis p<.02) and primary MM cells from patients at diagnosis (n=309, Kruskall-Wallis p<.0001). We further demonstrated that the direct pro-apoptotic effect of Dex was related to the genetic heterogeneity of MM, since sensitive HMCLs were restricted to t(14;16) and t(4;14) subgroups. Dex-induced apoptosis was associated with caspase 6 and 3 activation. We next demonstrated that Dex sensitivity depended on its ability to induce the down-regulation of c-MAF protein, which led to a decrease in CCND2 expression, a well-known target gene of c-MAF. The down-regulation of c-MAF mainly occurred by the increase of its proteasomal degradation. Altogether, these results were in agreement with the elevated levels of c-MAF found not only in t(14;16) translocated primary cells and HMCLs but also in t(4;14) translocated cells and HMCLs. While Bim expression (all isoforms) was up-regulated in most of cell lines by Dex irrespectively of its capacity to induce apoptosis, we demonstrated by Bim silencing that its up-regulation was required for initiating Dex-induced apoptotic program. Of note, by a long-term culture in the presence of low doses of Dex, we generated a Dex-resistant t(14;16) which was characterized by a decreased expression of GR. Dex treatment of this Dex-resistant HMCL neither induced any down-regulation of c-MAF nor any up-regulation of Bim, showing that a minimal expression level of GR was the first limiting step to induce a death response. In the present study, we demonstrated that Dex exerted a direct anti-tumor effect on myeloma cells from t(14;16) and t(4;14) molecular subgroups by controlling c-MAF level while those belonging to the t(11;14) subgroup were insensitive to Dex. This result highlights the fact that conventional therapeutic approaches could be re-evaluated in concert with the definition of molecular subgroups of MM patients to favor molecular subtype-specific therapy based on rational preclinical data. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C28. Citation Format: Charlotte Kervoelen, Emmanuelle Menoret, Patricia Gomez-Bougie, Regis Bataille, Catherine Pellat-Deceunynck, Martine Amiot. Dexamethasone-induced apoptosis in multiple myeloma cells is restricted to t(14;16) and t(4;14) molecular subgroups due to a combined down-regulation of MAF and an up-regulation of Bim. [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 C28.