TLR9 Ligand Induces the Generation of CD20+ Plasmablasts and Plasma Cells from CD27+ Memory B-Cells

Plasma cells (PCs) have a heterogeneous phenotype in humans. While bone marrow PCs are CD20−CD138+, tonsil PCs are CD20+CD138+/− and peripheral plasmablasts (PBs) are CD20−CD138−. In vitro, PCs are mainly generated by the activation of CD27+ memory B-cells through transient stimulation of CD40, and their phenotype appears similar to that of bone marrow PCs. While CD20 expression is lost at the plasmablastic stage, CD138 expression appears only at the PC stage. Thus, the CD20+CD138± phenotype of tonsil PCs does not represent an intermediate stage in the differentiation of memory B-cells into PCs. Because it has been previously shown that TLR9 activation was more able than CD40 stimulation to induce the differentiation of IgM+ CD27+ B-cells, we wondered whether TLR9 or CD40 stimulation would induce the same phenotype of PCs. Thus, we compared the differentiation of CD27+ B-cells isolated from either the tonsils or peripheral blood and stimulated with either CD40L-expressing fibroblasts or a TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN). We observed that CpG ODN mainly induced CD27+ B-cell differentiation into CD20+CD38+CD138− PBs and CD20+CD38+CD138± PCs, which appear similar to tonsil PCs. Removal of CpG ODN during differentiation induced a decrease in the CD20+ plasmablastic population, and, conversely, stimulation of CD40L-induced pre-plasmablasts with CpG ODN increased the population of CD20+CD38+ PBs. Analysis of Ig secretion showed that CpG ODN induced increased IgM secretion compared to CD40L. PCs from patients with multiple myeloma, the malignant counterpart of bone marrow PCs, rarely express CD20. We show that CpG ODN did not induce or increase CD20 in nine IgG or IgA myeloma cell lines. These data strongly suggest that CpG ODN mainly targets CD27+ IgM+ B-cells

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

Selective pharmacologic targeting of CTPS1 shows single-agent activity and synergizes with BCL2 inhibition in aggressive mantle cell lymphoma

Innovative therapeutic strategies have emerged over the past decade to improve outcomes for most lymphoma patients. Nevertheless, the aggressive presentation seen in high-risk mantle cell lymphoma (MCL) patients remains an unmet medical need. The highly proliferative cells that characterize these tumors depend on nucleotide synthesis to ensure high DNA replication and RNA synthesis. To take advantage of this vulnerability, STP-B, a clinically available small molecule selectively targeting CTP synthase 1 (CTPS1) has been recently developed. CTPS1 is a key enzyme of the pyrimidine synthesis pathway mediated through its unique ability to provide enough CTP in highly proliferating cells. Herein, we demonstrated that CTPS1 was expressed in all MCL cells, and that its high expression was associated with unfavorable outcomes for patients treated with chemotherapy. Using aggressive MCL models characterized by blastoid morphology, TP53 mutation or polyresistance to targeted therapies, we showed that STP-B was highly effective at nanomolar concentrations in vitro and in vivo, irrespective of these high-risk features. Inhibition of CTPS1 rapidly leads to cell cycle arrest in early S-phase accompanied by inhibition of translation, including of the anti-apoptotic protein MCL1. Consequently, CTPS1 inhibition induced synergistic cell death in combination with the selective BCL2 inhibitor venetoclax, both in vitro and in vivo. Overall, our study identified CTPS1 as a promising target for MCL patients and provided a mechanism-based combination with the BCL2 inhibitor venetoclax for the design of future chemotherapy-free treatment regimens to overcome resistance

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

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

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