Chronic lymphocytic leukemia (CLL) is caused by the accumulation of malignant B cells due to a defect in apoptosis and the presence of small population of proliferating cells principally in the lymph ...nodes. The abnormal survival of CLL B cells is explained by a plethora of supportive stimuli produced by the surrounding cells of the microenvironment, including follicular dendritic cells (FDCs), and mesenchymal stromal cells (MSCs). This crosstalk between malignant cells and normal cells can take place directly by cell-to-cell contact (assisted by adhesion molecules such as VLA-4 or CD100), indirectly by soluble factors (chemokines such as CXCL12, CXCL13, or CCL2) interacting with their receptors or by the exchange of material (protein, microRNAs or long non-coding RNAs) via extracellular vesicles. These different communication methods lead to different activation pathways (including BCR and NFκB pathways), gene expression modifications (chemokines, antiapoptotic protein increase, prognostic biomarkers), chemotaxis, homing in lymphoid tissues and survival of leukemic cells. In addition, these interactions are bidirectional, and CLL cells can manipulate the normal surrounding stromal cells in different ways to establish a supportive microenvironment. Here, we review this complex crosstalk between CLL cells and stromal cells, focusing on the different types of interactions, activated pathways, treatment strategies to disrupt this bidirectional communication, and the prognostic impact of these induced modifications.
Interactions between chronic lymphocytic leukemia (CLL) B cells and the bone marrow (BM) microenvironment play a major function in the physiopathology of CLL. Extracellular vesicles (EVs), which are ...composed of exosomes and microparticles, play an important role in cell communication. However, little is known about their role in CLL / microenvironment interactions. In the present study, EVs purified by ultracentrifugation from BM mesenchymal stromal cell (BM-MSC) cultures were added to CLL B cells. After their integration into CLL B cells, we observed a decrease of leukemic cell spontaneous apoptosis and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib and venetoclax after 24 hours. Spontaneous (
=0.0078) and stromal cell-derived factor 1α -induced migration capacities of CLL B cells were also enhanced (
=0.0020). A microarray study highlighted 805 differentially expressed genes between leukemic cells cultured with or without EVs. Of these, genes involved in the B-cell receptor pathway such as CCL3/4, EGR1/2/3, and MYC were increased. Interestingly, this signature presents important overlaps with other microenvironment stimuli such as B-cell receptor stimulation, CLL/nurse-like cells co-culture or those provided by a lymph node microenvironment. Finally, we showed that EVs from MSCs of leukemic patients also rescue leukemic cells from spontaneous or drug-induced apoptosis. However, they induce a higher migration and also a stronger gene modification compared to EVs of healthy MSCs. In conclusion, we show that EVs play a crucial role in CLL B cells/BM microenvironment communication.
Histone deacetylases (HDACs) play a crucial role in chromatin structure and, consequently, gene expression. Their deregulation has been reported in various cancers. We performed a complete and ...comprehensive study of the expression of 18 HDACs (including Sirtuin; SIRT) by real-time PCR in a cohort of 200 chronic lymphocytic leukemia (CLL) patients with a median follow-up of 77 mo, and compared it with the results obtained from normal B cells. We also compared HDAC expression at diagnosis and after relapse. We observed significant deregulation (mostly upregulation) of HDACs in CLL. In terms of clinical significance, only HDAC6 was significantly correlated with treatment-free survival (TFS), whereas HDAC3 and SIRT2, 3 and 6 were correlated with overall survival (OS). A multivariate Cox regression stepwise analysis indicated that HDAC6, 7 and 10 and SIRT3 were TFS independent predictors. Interestingly, poor prognosis was associated with an overexpression of HDAC7 and 10 but an underexpression of HDAC6 and SIRT3. Therefore, these factors were combined in a TFS score: patients with a score of 0-1-2, 3 and 4 had a median TFS of 107, 57 and 26 mo, respectively (HR = 4.03, p < 0.0001). For OS, SIRT5 and 6 allowed stratification into 3 groups, with a median OS of > 360, 237 and 94 mo (HR = 6.38, p < 0.0001). However, we could not find statistical differences in HDAC expression after relapse. These results, validated by a 5-fold cross-validation, highlight the complex impact of HDAC expression in CLL clinical course.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Due to their immunomodulatory and regenerative properties, Mesenchymal stromal cells (MSC) have generated major interests in several clinical settings including transplantation and inflammatory ...diseases. MSC functions can be influenced by their tissue origin. Their microenvironment strongly affects their biology notably through TLR sensing. In this study, we show that MSC isolated from four different sources express another type of cytosolic pathogen recognition receptors known as retinoic acid inducible gene-I (RIG-I)-like receptors (RLR). RLR activation in MSC induces the production of Type I IFN (IFN-β) and Type III IFN (IFN-λ1). The highest producers are adipose tissue(AT)-MSC. We further show that Interferon production is induced through TBK1/IKK-ε signaling and IRF7 phosphorylation. Depending on MSC source, the knockdown of TLR3 and/or RIG-I decreases the MSC response to RLR ligand poly(I:C)/Lyovec. Among the different MSC types, AT-MSCs display the highest sensitivity to viral stimuli as shown by the alteration of their viability after prolonged stimulation. Our work indicates that this could be linked to an increase of pro-apoptotic Noxa expression. Finally, the expression of IDO1 and LIF upon RLR activation indicate the increase of MSC immunomodulatory potential, especially in AT-MSCs. Altogether, these data should be considered when designing MSC-based therapy in clinical settings where inflammation or infection are present.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but ...there is no consensus on the appropriate negative control to use that can lead to false positive results.
We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).
Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).
We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mesenchymal stromal cells (MSCs) have recently been the subject of great interest in the fields of regenerative medicine and immunotherapy due to their unique biological properties. In particular, ...MSCs possess immunoregulatory properties that can modulate immune as well as inflammatory responses. Although there are many studies about MSC immunomodulation, several complex and conflicting mechanisms have been reported. Herein, we aim to review these mechanisms and identify a link between these pathways. We focus on human studies in which bone marrow-derived MSCs and T cells were investigated. We propose that MSC-induced immunomodulation exists as a network where converging regulatory pathways compete to establish a tolerogenic state. As interleukin-10 seems to play a central role in this network, we also discuss the relationship between this cytokine and other regulatory factors in the context of immunomodulation.
Abstract
Antibody–drug conjugates (ADC) represent a fast-growing drug class in oncology. However, ADCs are associated with resistance, and therapies able to overcome it are of utmost importance. ...Recently, enfortumab vedotin-ejfv (EV) was approved in nectin-4+ metastatic urothelial cancer. We previously described PVRL4/nectin-4 as a new therapeutic target in breast cancer and produced an efficient EV-like ADC comprising a human anti–nectin-4 mAb conjugated to monomethyl auristatin-E (MMAE) named N41mab-vcMMAE. To study the consequence of the long-term treatment with this ADC, we developed a preclinical breast cancer model in mice, and report a mechanism of resistance to N41mab-vcMMAE after 9-month treatment and a way to reverse it. RNA-sequencing pointed to an upregulation in resistant tumors of ABCB1 expression, encoding the multidrug resistance protein MDR-1/P-glycoprotein (P-gp), associated with focal gene amplification and high protein expression. Sensitivity to N41mab-vcMMAE of the resistant model was restored in vitro by P-gp pharmacologic inhibitors, like tariquidar. P-gp is expressed in a variety of normal tissues. By delivering the drug to the tumor more specifically than classical chemotherapy, we hypothesized that the combined use of ADC with P-gp inhibitors might reverse resistance in vivo without toxicity. Indeed, we showed that the tariquidar/N41mab-vcMMAE combination was well tolerated and induced a rapid regression of ADC-resistant tumors in mice. In contrast, the tariquidar/docetaxel combination was toxic and poorly efficient. These results show that ABC transporter inhibitors can be safely used with ADC to reverse ADC-induced resistance and open new opportunities in the fight against multidrug resistance.
Unmutated (UM) immunoglobulin heavy chain variable region (IgHV) status or IgHV3-21 gene usage is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. Interestingly, ...IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially able to trigger cell-autonomous BCR-mediated signaling. However, this light chain has never been characterized independently of the heavy chain IgHV3-21.
We performed total RNA sequencing in 32 patients and investigated IgLV3-21 prognostic impact in terms of treatment-free survival (TFS) and overall survival (OS) in 3 other independent cohorts for a total of 813 patients. IgLV3-21 presence was tested by real-time PCR and confirmed by Sanger sequencing.
Using total RNA sequencing to characterize 32 patients with high-risk CLL, we found a high frequency (28%) of IgLV3-21 rearrangements. Gene set enrichment analysis revealed that these patients express higher levels of genes responsible for ribosome biogenesis and translation initiation (
< 0.0001) as well as MYC target genes (
= 0.0003). Patients with IgLV3-21 rearrangements displayed a significantly shorter TFS and OS (
< 0.05), particularly those with IgHV mutation. In each of the three independent validation cohorts, we showed that IgLV3-21 rearrangements-similar to UM IgHV status-conferred poor prognosis compared with mutated IgHV (
< 0.0001). Importantly, we confirmed by multivariate analysis that this was independent of IgHV mutational status or subset #2 stereotyped receptor (
< 0.0001).
We have demonstrated for the first time that a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLL patients with poor prognosis.
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