Targeting Oxidative Stress With Auranofin or Prima-1Met to Circumvent p53 or Bax/Bak Deficiency in Myeloma Cells

Prima-1Met (APR-246) was previously shown to be dependent on glutathione inhibition and on ROS induction in cancer cells with mutated or deleted TP53. Because this ROS induction was, at least in part, due to a direct interference with the thioredoxin reductase enzyme, we investigated whether activity of Prima-1Met could be mimicked by auranofin, an inhibitor of the thioredoxin reductase. We thus compared the activity of auranofin and Prima-1Met in 18 myeloma cell lines and in 10 samples from patients with multiple myeloma or plasma cell leukemia. We showed that, similar to Prima-1Met, the activity of auranofin was not dependent on either TP53 status or p53 expression; was inhibited by N-acetyl-L-cysteine, a ROS scavenger; displayed a dramatic synergy with L-buthionine sulfoximine, an irreversible inhibitor of glutathione synthesis; and induced cell death that was not dependent on Bax/Bak expression. These data showed that auranofin and Prima-1Met similarly overcome cell death resistance in myeloma cells due to either p53 deficiency or to mitochondrial dysfunction

BCLXL PROTAC degrader DT2216 targets secondary plasma cell leukemia addicted to BCLXL for survival

Secondary plasma cell leukemia (sPCL) is a rare form of aggressive plasma cell malignancy arising mostly at end-stage refractory multiple myeloma and consequently presenting limited therapeutic options. We analyzed 13 sPCL for their sensitivity to BH3 mimetics targeting either BCL2 (venetoclax) or BCLXL (A1155463) and showed that 3 sPCL were efficiently killed by venetoclax and 3 sPCL by A1155463. Accordingly, BH3 profiling of 2 sPCL sensitive to BCLXL inhibition confirmed their high BCLXL primed profile. While targeting BCLXL using BH3 mimetics induces platelets on-target drug toxicity, the recent development of DT2216, a clinical-stage BCLXL proteolysis targeting chimera PROTAC compound, provides an alternative strategy to target BCLXL. Indeed, DT2216 specifically degrades BCLXL via VHL E3 ligase, without inducing thrombocytopenia. We demonstrated in human myeloma cell lines and sPCL that sensitivity to DT2216 strongly correlated with the sensitivity to A1155463. Interestingly, we showed that low doses of DT2216 (nM range) were sufficient to specifically degrade BCLXL after 48 hours of treatment, consistent with VHL expression, in all cell lines but irrespectively to DT2216 sensitivity. In myeloma cells, DT2216 induced apoptotic cell death and triggered BAX and BAK activation. In conclusion, our study demonstrated that patients with sPCL addicted to BCLXL, a small but a very challenging group, could potentially receive therapeutic benefit from DT2216. Clinical trials of DT2216 in this subset of sPCL patients are warranted

BCL2-Family Dysregulation in B-Cell Malignancies: From Gene Expression Regulation to a Targeted Therapy Biomarker

BCL2-family proteins have a central role in the mitochondrial apoptosis machinery and their expression is known to be deregulated in many cancer types. Effort in the development of small molecules that selectively target anti-apoptotic members of this family i.e., Bcl-2, Bcl-xL, Mcl-1 recently opened novel therapeutic opportunities. Among these apoptosis-inducing agents, BH3-mimetics (i.e., venetoclax) led to promising preclinical and clinical activity in B cell malignancies. However, several mechanisms of intrinsic or acquired resistance have been described ex vivo therefore predictive markers of response as well as mechanism-based combinations have to be designed. In the present study, we analyzed the expression of the BCL2-family genes across 10 mature B cell malignancies through computational normalization of 21 publicly available Affimetrix datasets gathering 1,219 patient samples. To better understand the deregulation of anti- and pro-apoptotic members of the BCL2-family in hematological disorders, we first compared gene expression profiles of malignant B cells to their relative normal control (naïve B cell to plasma cells, n = 37). We further assessed BCL2-family expression according to tissue localization i.e., peripheral blood, bone marrow, and lymph node, molecular subgroups or disease status i.e., indolent to aggressive. Across all cancer types, we showed that anti-apoptotic genes are upregulated while pro-apoptotic genes are downregulated when compared to normal counterpart cells. Of interest, our analysis highlighted that, independently of the nature of malignant B cells, the pro-apoptotic BH3-only BCL2L11 and PMAIP1 are deeply repressed in tumor niches, suggesting a central role of the microenvironment in their regulation. In addition, we showed selective modulations across molecular subgroups and showed that the BCL2-family expression profile was related to tumor aggressiveness. Finally, by integrating recent data on venetoclax-monotherapy clinical activity with the expression of BCL2-family members involved in the venetoclax response, we determined that the ratio (BCL2+BCL2L11+BAX)/BCL2L1 was the strongest predictor of venetoclax response for mature B cell malignancies in vivo

Preclinical characterization of ISB 1342, a CD38 × CD3 T-cell engager for relapsed/refractory multiple myeloma

Although treatment of multiple myeloma (MM) with daratumumab significantly extends the patient's lifespan, resistance to therapy is inevitable. ISB 1342 was designed to target MM cells from patients with relapsed/refractory MM (r/r MM) displaying lower sensitivity to daratumumab. ISB 1342 is a bispecific antibody with a high-affinity Fab binding to CD38 on tumor cells on a different epitope than daratumumab and a detuned scFv domain affinity binding to CD3ε on T cells, to mitigate the risk of life-threatening cytokine release syndrome, using the Bispecific Engagement by Antibodies based on the TCR (BEAT) platform. In vitro, ISB 1342 efficiently killed cell lines with different levels of CD38, including those with a lower sensitivity to daratumumab. In a killing assay where multiple modes of action were enabled, ISB 1342 showed higher cytotoxicity toward MM cells compared with daratumumab. This activity was retained when used in sequential or concomitant combinations with daratumumab. The efficacy of ISB 1342 was maintained in daratumumab-treated bone marrow patient samples showing lower sensitivity to daratumumab. ISB 1342 induced complete tumor control in 2 therapeutic mouse models, unlike daratumumab. Finally, in cynomolgus monkeys, ISB 1342 displayed an acceptable toxicology profile. These data suggest that ISB 1342 may be an option in patients with r/r MM refractory to prior anti-CD38 bivalent monoclonal antibody therapies. It is currently being developed in a phase 1 clinical study

Régulation de la mort et survie des plasmocytes humains normaux et de leurs équivalents tumoraux, les cellules du myélome multiple

Les plasmocytes (PC) représentent le stade terminal de différenciation des lymphocytes B activés suite à leur rencontre avec un antigène. Le processus de différenciation plasmocytaire est associé à une apoptose massive des cellules. Par ce biais, in vivo, l homéostasie du compartiment plasmocytaire est maintenue. Le but de ce travail a été de progresser dans la compréhension des mécanismes qui régissent l apoptose et la survie des cellules engagées dans le processus de différenciation plasmocytaire. Nous montrons que dès les stades précoces de la différenciation plasmocytaire, les cellules (pré-plasmoblastes) sont engagées dans le processus apoptotique avec une activation des caspases effectrices (caspase-3 et -6) associée au clivage de Mcl-1, une forte diminution de Bcl2 et une augmentation de l isoforme EL de Bim. En outre, ces cellules sont sensibles aux signaux de mort induits par l activation du récepteur de mort Fas mais résistantes à l activation des récepteurs de mort à TRAIL, DR4 et DR5. Nous mettons en évidence que les cellules dérivées des monocytes (ostéoclastes (OC), cellules dendritiques (CD) et macrophages) présentent une capacité à prévenir l apoptose spontanée de ces cellules. Toutefois, les OC paraissent capables de fournir aux PC et à ses précurseurs des signaux de survie de manière plus efficace que les macrophages tandis que les CD semblent fournir de tels signaux uniquement aux plasmoblastes. Les PC persistants au contact des OC présentent des caractéristiques phénotypiques qui évoquent le prototype du PC mature suggérant que les OC pourraient participer à la niche de survie des PC dits à longue durée de vie dans la moelle osseuse.Plasma cells are terminally differentiated final effectors of the humoral response. Plasma cell differentiation is characterized by extensive apoptosis. By this way, plasma cell homeostasis is maintained in vivo. The aim of this study was to improve our knowledge about mechanisms underlying cell survival and apoptosis during plasma cell differentiation. We show that as early as proplasmablast stage in the time course of plasma cell differentiation, cells have entered in apoptotic process characterized by effector caspase activation (caspase-3 and -6), Mcl-1 cleavage, a marked decrease of Bcl-2 and increase of Bim EL isoform. Moreover, these cells are susceptible to Fas-mediated apoptosis but resistant to TRAIL-receptor-mediated apoptosis. We demonstrated that osteoclasts, macrophages and dendritic cells, all of monocyte origin, are able to support cell survival during plasma cell differentiation. However, osteoclasts were more potent than macrophages to support the survival of plasma cells whereas dendritic cells mediated survival of plasmablasts only. Surviving plasma cells on osteoclasts displayed phenotypic features of fully differentiated plasma cells suggesting that osteoclasts could take part to the putative bone marrow niche of plasma cells.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

The origin of the plasma-cell heterogeneity

International audienceno abstrac

Targeting Oxidative Stress With Auranofin or Prima-1Met to Circumvent p53 or Bax/Bak Deficiency in Myeloma Cells

International audienc

Heat shock factor 1 is a potent therapeutic target for enhancing the efficacy of treatments for multiple myeloma with adverse prognosis

International audienceAbstractDeregulated expression of heat shock proteins (HSPs) encoding genes is frequent in multiple myeloma. HSPs, which are molecular chaperones involved in protein homeostasis pathways, have emerged recently as promising therapeutic targets. Using human myeloma cell lines and primary myeloma cells belonging to various molecular groups, we tested the efficacy of HSP90, HSP70, and heat shock factor 1 (HSF1) inhibitors alone or associated with current antimyeloma drugs. We report here that KNK-437 (an inhibitor of HSF1) and bortezomib have additive effects on apoptosis induction in cells belonging to groups with bad prognosis

Myeloma Cell Self-Renewal Depends on JAG2 Expression and Is Mediated by IGF1 or SCF Loop

International audienceThe purpose of this study was to identify the pathways associated with the ability of human myeloma cells (HMCLs) to spontaneous self-renew in a serum-free semi-solid human collagen-based assay. Among 32 HMCLs analyzed, 8 were able to grow spontaneously (from 5% to 35% of seeded cells) without any addition of cytokines or growth factors and this capacity to grow correlated with the presence of RAS mutations (p=0.04). Gene expression profile analysis of HMCLs identified one gene, JAG2, overexpressed in HMCLs that are able to self-renew. Interestingly, flow cytometry analysis of JAG2 expression showed that the level of membrane JAG2 expression positively correlated (r=0.87) to the percentage of colony formation (p=0.004). Blocking Jag-Notch interactions with Notch-Fc chimeric molecules impaired self-colony formation underlying a role for Jag-Notch pathway in colony formation. Furthermore, direct JAG2 silencing in two independent HMCLs (KMM1 and JJN3) prevented colony formation. Moreover, xenografts in SCID mice showed that JAG2 silencing fully impaired tumor growth of both KMM1 and JJN3. RT-PCR evaluation of JAG2 expression showed that 20 of 30 CD138+ purified primary myeloma cells expressed JAG2 and a Jag2+ subpopulation was identified by flow cytometry within primary CD138+ MM cells of patients at diagnosis or relapse.We further identified the growth factors involved in the self-renewal. By using blocking anti-IGF1R Ab or C-KIT inhibitor (imatinib mesylate), we showed that self-renewal of HMCLs was dependent on IGF1/IGF1R (5 of 8) or on C-KIT/SCF (1) or on both loops (2 of 8). Of note, C-KIT+ HMCLs expressed high JAG2 level at the cell membrane that was decreased by imatinib mesylate. Interestingly, none HMCL self-renewal was dependent on IL6/IL6R loop despite the high efficiency of paracrine IL6 to induce colony formation in most HMCLs. To address expression of C-KIT/SCF and IGF1R/IGF1 in primary myeloma cells, we used public data from patients at diagnosis published by Arkansas University. Expression of C-KIT and IGF1R was found in 56% and 50% of patients at diagnosis, respectively: 53% of patients express one or the other receptor, 27% express both and 20% express none. Expression of receptors is not similar with regard to the molecular classification of patients as previously shown by cytometry: indeed, MS patients underexpress C-KIT (p<0.001) but overexpress IGF1R (p<0.001), in full contrast to HY patients who overexpress C-KIT (p<0.001) but underexpress IGF1R (p<0.001). Moreover, IGF1R expression is lower on C-KIT+ patients as compared with C-KIT− ones (p=0.049). Of note, CD-1 and CD-2 patients underexpress both C-KIT (p=0.039) and IGF1R (p<0.001). IGF1 and SCF are produced by the microenvironment although IGF1 (but not SCF) mRNA was found in myeloma cells too. Altogether, these data suggest that IGF1 and SCF could be the main growth factors for 80% of the patients. Blocking these two tyrosine kinase receptors (in good agreement with their expression in patients) as well as Jag2/Notch interactions could decrease myeloma progression and/or relapse and thus increase survival