Contributions volontaires présentées à la 3° consultation mondiale sur la génétique forestière Canberra

Multiple myeloma (MM) is a hematological malignancy, which remains incurable because most patients eventually relapse or become refractory to current treatments. Due to heterogeneity within the cancer cell microenvironment, cancer cell populations employ a dynamic survival strategy to chemotherapeutic treatments, which frequently results in a rapid acquisition of therapy resistance. Besides resistance-conferring genetic alterations within a tumor cell population selected during drug treatment, recent findings also reveal non-mutational mechanisms of drug resistance, involving a small population of "cancer stem cells" (CSCs) which are intrinsically more refractory to the effects of a variety of anticancer drugs. Other studies have implicated epigenetic mechanisms in reversible drug tolerance to protect the population from eradication by potentially lethal exposures, suggesting that acquired drug resistance does not necessarily require a stable heritable genetic alteration. Clonal evolution of MM cells and the bone marrow microenvironment changes contribute to drug resistance. MM-CSCs may not be a static population and survive as phenotypically and functionally different cell types via the transition between stem-like and non-stem-like states in local microenvironments, as observed in other types of cancers. Targeting MM-CSCs is clinically relevant, and different approaches have been suggested to target molecular, metabolic and epigenetic signatures, and the self-renewal signaling characteristic of MM CSC-like cells. Here, we summarize epigenetic strategies to reverse drug resistance in heterogeneous multiple myeloma

Profiling activity of cellular kinases in migrating T-cells

T-Lymphocyte kinases are important checkpoints that control T-cell motility by regulating a diverse range of signal transduction pathways. The distinct configuration of kinase events in T-cell could be used to fingerprint the status of T-cells. However, only small fraction human kinases have been characterized so far and little is known about the dynamics of the kinome in motile T-cells. Although several direct and indirect strategies exist to characterize cellular kinase activities, such as RNA interference, antibody arrays, enzyme kinetics, and mass spectrometry, this chapter focuses on an alternative multiplex phosphopeptide array-based methodology, which allows the kinome-wide identification of hyper-activated kinases involved in the regulation of T-cell migration

The steroidal lactone withaferin A impedes T-cell motility by\ua0inhibiting the kinase ZAP70 and subsequent kinome signaling

Not AvailableThe steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found thatWFA dose-dependently (within the concentration range of 0.3–1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhe sion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome anal ysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, sug gesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and im pedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologiesNot Availabl

The steroidal lactone withaferin A impedes T-cell motility by inhibiting the kinase ZAP70 and subsequent kinome signaling

Not AvailableThe steroidal lactone withaferin A (WFA) is a dietary phytochemical, derived from Withania somnifera. It exhibits a wide range of biological properties, including immunomodulatory, anti-inflammatory, antistress, and anticancer activities. Here we investigated the effect of WFA on T-cell motility, which is crucial for adaptive immune responses as well as autoimmune reactions. We found thatWFA dose-dependently (within the concentration range of 0.3–1.25 μM) inhibited the ability of human T-cells to migrate via cross-linking of the lymphocyte function-associated antigen-1 (LFA-1) integrin with its ligand, intercellular adhe sion molecule 1 (ICAM-1). Coimmunoprecipitation of WFA interacting proteins and subsequent tandem mass spectrometry identified a WFA-interactome consisting of 273 proteins in motile T-cells. In particular, our data revealed significant enrichment of the zeta-chain-associated protein kinase 70 (ZAP70) and cytoskeletal actin protein interaction networks upon stimulation. Phospho-peptide mapping and kinome anal ysis substantiated kinase signaling downstream of ZAP70 as a key WFA target, which was further confirmed by bait-pulldown and Western immunoblotting assays. The WFA-ZAP70 interaction was disrupted by a disulfide reducing agent dithiothreitol, sug gesting an involvement of cysteine covalent binding interface. In silico docking predicted WFA binding to ZAP70 at cystine 560 and 564 residues. These findings provide a mechanistic insight whereby WFA binds to and inhibits the ZAP70 kinase and im pedes T-cell motility. We therefore conclude that WFA may be exploited to pharmacologically control host immune responses and potentially prevent autoimmune-mediated pathologiesNot Availabl

Ectopic MicroRNA-150-5p Transcription Sensitizes Glucocorticoid Therapy Response in MM1S Multiple Myeloma Cells but Fails to Overcome Hormone Therapy Resistance in MM1R Cells

<div><p>Glucocorticoids (GCs) selectively trigger cell death in the multiple myeloma cell line MM1S which express NR3C1/Glucocorticoid Receptor (GR) protein, but fail to kill MM1R cells which lack GR protein. Given recent demonstrations of altered microRNA profiles in a diverse range of haematological malignancies and drug resistance, we characterized GC inducible mRNA and microRNA transcription profiles in GC sensitive MM1S as compared to GC resistant MM1R cells. Transcriptome analysis revealed that GCs regulate expression of multiple genes involved in cell cycle control, cell organization, cell death and immunological disease in MM1S cells, which remain unaffected in MM1R cells. With respect to microRNAs, mir-150-5p was identified as the most time persistent GC regulated microRNA, out of 5 QPCR validated microRNAs (mir-26b, mir-125a-5p, mir-146-5p, mir-150-5p, and mir-184), which are GC inducible in MM1S but not in MM1R cells. Functional studies further revealed that ectopic transfection of a synthetic mir-150-5p mimics GR dependent gene expression changes involved in cell death and cell proliferation pathways. Remarkably, despite the gene expression changes observed, overexpression of mir-150-5p in absence of GCs did not trigger significant cytotoxicity in MM1S or MM1R cells. This suggests the requirement of additional steps in GC induced cell death, which can not be mimicked by mir-150-5p overexpression alone. Interestingly, a combination of mir-150-5p transfection with low doses GC in MM1S cells was found to sensitize therapy response, whereas opposite effects could be observed with a mir-150-5p specific antagomir. Although mir-150-5p overexpression did not substantially change GR expression levels, it was found that mir-150-5p evokes GR specific effects through indirect mRNA regulation of GR interacting transcription factors and hormone receptors, GR chaperones, as well as various effectors of unfolded protein stress and chemokine signalling. Altogether GC-inducible mir-150-5p adds another level of regulation to GC specific therapeutic responses in multiple myeloma.</p></div