Mantle cell lymphoma (MCL) is an incurable B-cell malignancy characterized by a high clinical variability. Therefore, there is a critical need to define parameters that identify high-risk patients ...for aggressive disease and therapy resistance. B-cell receptor (BCR) signaling is crucial for MCL initiation and progression and is a target for therapeutic intervention. We interrogated BCR signaling proteins (SYK, LCK, BTK, PLCγ2, p38, AKT, NF-κB p65, and STAT5) in 30 primary MCL samples using phospho-specific flow cytometry. Anti-IgM modulation induced heterogeneous BCR signaling responses among samples allowing the identification of two clusters with differential responses. The cluster with higher response was associated with shorter progression free survival (PFS) and overall survival (OS). Moreover, higher constitutive AKT activity was predictive of inferior response to the Bruton's tyrosine kinase inhibitor (BTKi) ibrutinib. Time-to-event analyses showed that MCL international prognostic index (MIPI) high-risk category and higher STAT5 response were predictors of shorter PFS and OS whilst MIPI high-risk category and high SYK response predicted shorter OS. In conclusion, we identified BCR signaling properties associated with poor clinical outcome and resistance to ibrutinib, thus highlighting the prognostic and predictive significance of BCR activity and advancing our understanding of signaling heterogeneity underlying clinical behavior of MCL.
Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the ...functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. ...B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.
Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an extremely variable clinical course. We have recently shown that high catalase (
CAT
) expression identifies patients ...with an aggressive clinical course. Elucidating mechanisms regulating
CAT
expression in CLL is preeminent to understand disease mechanisms and develop strategies for improving its clinical management. In this study, we investigated the role of the
CAT
promoter rs1001179 single nucleotide polymorphism (SNP) and of the CpG Island II methylation encompassing this SNP in the regulation of
CAT
expression in CLL. Leukemic cells harboring the rs1001179 SNP T allele exhibited a significantly higher
CAT
expression compared with cells bearing the CC genotype.
CAT
promoter harboring the T -but not C- allele was accessible to ETS-1 and GR-β transcription factors. Moreover, CLL cells exhibited lower methylation levels than normal B cells, in line with the higher CAT mRNA and protein expressed by CLL in comparison with normal B cells. Methylation levels at specific CpG sites negatively correlated with
CAT
levels in CLL cells. Inhibition of methyltransferase activity induced a significant increase in CAT levels, thus functionally validating the role of CpG methylation in regulating
CAT
expression in CLL. Finally, the CT/TT genotypes were associated with lower methylation and higher
CAT
levels, suggesting that the rs1001179 T allele and CpG methylation may interact in regulating
CAT
expression in CLL. This study identifies genetic and epigenetic mechanisms underlying differential expression of
CAT
, which could be of crucial relevance for the development of therapies targeting redox regulatory pathways in CLL.
Graphical abstract
B-cell receptor (BCR) signaling is a key determinant of variable clinical behavior and a target for therapeutic interventions in chronic lymphocytic leukemia (CLL). Endogenously produced H2O2 is ...thought to fine-tune the BCR signaling by reversibly inhibiting phosphatases. However, little is known about how CLL cells sense and respond to such redox cues and what effect they have on CLL. We characterized the response of BCR signaling proteins to exogenous H2O2 in cells from patients with CLL, using phosphospecific flow cytometry. Exogenous H2O2 in the absence of BCR engagement induced a signaling response of BCR proteins that was higher in CLL with favorable prognostic parameters and an indolent clinical course. We identified low catalase expression as a possible mechanism accounting for redox signaling hypersensitivity. Decreased catalase could cause an escalated accumulation of exogenous H2O2 in leukemic cells with a consequent greater inhibition of phosphatases and an increase of redox signaling sensitivity. Moreover, lower levels of catalase were significantly associated with a slower progression of the disease. In leukemic cells characterized by redox hypersensitivity, we also documented an elevated accumulation of ROS and an increased mitochondrial amount. Taken together, our data identified redox sensitivity and metabolic profiles that are linked to differential clinical behavior in CLL. This study advances our understanding of the redox and signaling heterogeneity of CLL and provides the rationale for the development of therapies targeting redox pathways in CLL.
•Low catalase expression confers hypersensitivity to external redox cues.•Differential redox profiles are associated with divergent clinical behaviors in CLL.
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Summary
Recently, clinical trial results have established inhibitors of B‐cell receptor (BCR)‐associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first‐line ...treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies — rituximab, ofatumumab, and obinutuzumab — inhibited BCR signalling pathways targeting preferentially pBTKY551 — but not BTKY223 — and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTKY223 to a higher extent than pBTKY551. The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL.
Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) is a member of the TNF superfamily, expressed on dendritic cells, macrophages, lymphocytes, and endothelial cells. It is the only described ligand ...for death receptor 3 (DR3), a death-domain containing receptor of the TNF-receptor superfamily, mainly expressed on lymphocytes, natural killer (NK) cells, and NK-T cells. TL1A–DR3 interaction results in co-stimulatory signaling for activated T cells, leading to amplification of inflammation and immune responses, correlated with greater pathogenicity in diverse autoimmune diseases. In contrast, in activated B cells the TL1A/DR3 system exerts inhibitory effect on cell proliferation, suggesting that TL1A may also have modulatory and homeostatic functions on B-cell expansion. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults in Western countries. It is well documented that several elements within the tumor microenvironment, including antigens, cytokines, adhesion molecules, and surface receptors, play a fundamental role in supporting the growth of CLL. In contrast, little is known on regulatory mechanisms of CLL growth. In this study, we have investigated the possible regulatory role of the TL1A/DR3 system in B cells from CLL patients.
CLL patients from the Hematology Unit at the University Hospital of Verona (Italy) were included in this study (n=37). Disease characteristics and demographic variables were collected on all patients. Purified B cells were obtained by negative selection. DR3 expression was measured on peripheral blood B cells by flow cytometry and western blot at baseline and following B cell receptor (BCR) stimulation with anti-human IgM. DR3 expression was confirmed on lymph-node CLL specimens by immunofluorescence. Metabolic activity of CLL B cells was analyzed by MTT assay. Apoptosis was analyzed by Annexin V assay. TL1A serum levels in CLL patients were measured by ELISA. Baseline analysis in vitro showed that DR3 was expressed at low levels in CLL B cells. Cell activation through stimulation of the BCR induced a significant increase of DR3 expression in a fraction of CLL B cells (p<0.001). Higher levels of DR3 expression were associated with early-stage (Rai 0) disease (p=0.019). The relevance of these findings was confirmed by immuofluorescence analysis of B-CLL lymph-node specimens showing that DR3 was expressed at high levels in some CLL B cells in vivo. Treatment of purified CLL B cells with exogenous recombinant TL1A in vitro, in the presence of BCR stimulation, induced a decreased metabolic activity in 3/8 B-CLL cell samples (37.5%). No change in CLL metabolic activity was observed following treatment with TL1A alone, in the absence of BCR stimulation. Treatment of B-CLL samples with TL1A, either in the presence or absence of BCR stimulation, induced no changes in cell viability, thus ruling out that decreased metabolic activity was due to reduced survival. A soluble form of TL1A was detected in the sera of CLL patients and higher serum levels of TL1A were significantly associated with early-stage (Rai 0) disease (p=0.023) and absence of CD38 expressions (p=0.035).
In summary, this study shows that B-CLL metabolic activity can be reduced through the activity of the TL1A/DR3 system, in the presence of the BCR stimulation. Furthermore, TL1A and DR3 levels are higher in patients with early-stage disease. Taken together, these findings suggest that the TL1A/DR3 system in vivo, in the presence of antigen stimulation, may modulate leukemic cell metabolism in early-stage CLL, thus influencing the clinical course of disease.
No relevant conflicts of interest to declare.
TNF-like ligand 1A (TL1A) and its unique receptor death receptor 3 (DR3) acts as broad T-cell costimulator involved in regulatory mechanisms of adaptive immune response under physiological and ...pathological settings. Moreover, we have recently shown that TL1A negatively regulates B-cell proliferation. Despite increasing interest on the TL1A/DR3-axis functions, very little is known on its expression and role in leukemia. In this study, we investigated the expression and function of TL1A/DR3 axis in chronic lymphocytic leukemia (CLL). DR3 was differentially expressed in activated CLL cells and predominantly detected in patients with early clinical stage disease. Soluble TL1A has been revealed in the sera of CLL patients where higher TL1A levels were associated with early stage disease. T cells, monocytes and leukemic B cells have been identified as major sources of TL1A in CLL. The relevance of these findings has been sustained by functional data showing that exogenous TL1A reduces CLL proliferation induced by stimulation of the B cell receptor. Overall, these data document the expression of the TL1A/DR3 axis in early-stage CLL. They also identify a novel function for TL1A as a negative regulator of leukemic cell proliferation that may influence the CLL physiopathology and clinical outcome at an early-stage disease.
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