The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.
Abstract Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including TP53 gene inactivation. While TP53 gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, TP53 mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic TP53 mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had TP53 germ-line variants. While del(17p) with or without TP53 mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, TP53 mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. TP53 mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.
<p>PDF file - 289K, Unsupervised analyses in cMBL and Rai0-CLL cases. (A) Gene and (B) miRNA expression profiling analyses by hierarchical clustering. Information about IGHV mutational status ('+' = M, '-' = UM), CD38, ZAP-70, chromosome 12 trisomy, chromosome 17, 11, and 13 deletions ('+' = positive, '-' = negative) are included alongside the patient ID. In the legend bar: turquoise indicates Rai0-CLL and yellow cMBL samples, respectively. The color scale bar represents the relative gene expression changes normalized by the standard deviation, and the color changes in each row represent gene expression relative to the mean across the samples.</p>
Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.
Abstract The racemic compound indobufen and its (+)-and (—)-enantiomers have been compared for their effects on blood platelet function and rat carrageenan pleurisy. The antiplatelet properties were studied in-vitro in human platelets by measuring the inhibition of platelet aggregation and generation of serum thromboxane (Tx) B2. In-vivo, the antiplatelet and anti-inflammatory properties were studied in rats by measuring the inhibition of serum TxB2, the amount of 6-keto-PGF1α in pleural exudate and pleural exudate volume. In all tests the (+)-enantiomer was slightly more potent than the racemate, while the (—)-enantiomer was far less potent. In the same rats, treatment with the lowest doses of the compounds giving 90% inhibition of serum thromboxane B2 generation was associated with occasional macroscopic lesions of the gastric mucosa.
Vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic (VEXAS) syndrome, first identified by Beck et al.,1 is an acquired monogenic autoinflammatory disorder characterized by multisystemic relapsing inflammation, often accompanied by multilineage cytopenia, venous thromboembolism, myelodysplastic syndrome and monoclonal gammopathy. VEXAS is caused by post-zygotic mutations of the ubiquitin-like modifier activating enzyme 1 (UBA1) gene within haematopoietic stem cells, resulting in the disruption of the ubiquitin–proteasome pathway.1, 2 Recent research indicates that these aberrations ultimately cause abnormal myeloid cell cycling and increased cytokine production by monocytes.3, 4 Nevertheless, the pathophysiology of VEXAS remains only partially understood, preventing the identification of actionable therapeutic targets. Consistently with these knowledge gaps, few therapeutic options are currently available for the management of VEXAS. Indeed, while high-dose steroids are beneficial during disease flares, their long-term utilization is discouraged by their side effects. On the other hand, conventional disease-modifying anti-rheumatic drugs are scarcely useful for the management of inflammatory symptoms.2, 5, 6 Recent retrospective studies have identified Janus kinase inhibitors (JAKi) as effective therapeutic agents for patients with VEXAS.6 Of these, ruxolitinib, a dual inhibitor of JAK1 and JAK2, is associated with higher response rates compared to other JAKi.6 However, the mechanisms by which patients with VEXAS respond more to ruxolitinib than other JAKi are still unclear. Addressing this knowledge gap is critical for discovering the specific actionable targets in VEXAS, possibly allowing for the optimization of current therapeutic strategies. To dissect the molecular characteristics of response to dual JAK inhibition, we charted the transcriptomic response to therapy in one patient with VEXAS treated with ruxolitinib. The patient, a 63-year-old Caucasian man, was diagnosed with VEXAS by genetic testing in April 2023 following an extensive diagnostic work-up for unexplained inflammatory symptoms, empirically treated with corticosteroids. VEXAS coexisted with WHO-defined myelodysplastic syndrome with low blasts7 and immunoglobulin M monoclonal gammopathy of undetermined significance (MGUS). Azacytidine was initiated in mid-May 2023, with contemporary steroid tapering. By early June, the patient required hospitalization due to high remittent fever, diffuse purpuric rash and enanthem. The biopsy of a purpuric skin lesion revealed dermic neutrophil and lympho-monocytic infiltrates positive for myeloperoxidase, confirming the diagnosis of VEXAS flare. Following initial treatment with intravenous steroids, ruxolitinib was initiated as steroid-sparing agent, resulting in complete clinical remission within 2 weeks (Figure 1A). Ruxolitinib was later discontinued due to worsening pancytopenia, necessitating red cell transfusions. Azacytidine was then resumed, showing good tolerance and a sustained response. To comprehensively profile with high temporal resolution of the response to ruxolitinib, we performed serial blood RNA-sequencing (RNA-seq) coupled with bulk and single-cell RNA-seq (scRNA-seq) of VEXAS skin lesions (Figure 1A). Peripheral blood samples were obtained at multiple time points during treatment with ruxolitinib throughout patient's hospitalization and discharge. Specifically, blood samples were collected on day 0 before ruxolitinib initiation and on days 1, 3, 5, 6, and 11 after treatment started. Formalin-fixed, paraffin-embedded skin biopsies obtained per clinical practice were collected before starting steroid therapy (2 days before ruxolitinib initiation, day −2) and on day 5 after ruxolitinib initiation for bulk RNA-seq. Additionally, a fresh skin tissue sample was collected on day 5 for scRNA-seq. A detailed description of the materials and methods, along with the ethics statement, is provided in the Supporting Information. To verify whether blood RNA-seq was informative of disease activity, we first assessed a transcriptomic VEXAS-specific signature identified by Kosmider et al. and comprising IL-1α, IL-1β, IL-18, TGF-α, IL-7, LGALS3, S100A8 and S100A9.4 Treatment with ruxolitinib was associated with a prompt downregulation of this signature (Figure 1B), in parallel with the progressive resolution of symptoms. Indeed, the VEXAS signature positively correlated with several laboratory markers of inflammatory activity assessed per clinical practice, including red cell distribution width, neutrophil percentage and absolute count, C-reactive protein, ferritin and lactate dehydrogenase. Conversely, it was negatively associated with monocyte percentage and absolute count, mean corpuscular haemoglobin concentration, as well as lymphocyte percentage and absolute count (Figure 1C; Table S1). To shed light on the molecular determinants of response to JAK inhibition, we next profiled the transcriptional activity of the JAK–signal transducer and activator of transcription proteins (STAT) signalling pathway, observing a progressive downregulation of the JAK–STAT signature following ruxolitinib initiation (Figure S1). Notably, when considering the individual transcripts of JAK1 and JAK2, we observed a marked downregulation of JAK2, while JAK1 expression did not change over time (Figure 1C). JAK2 was positively associated with the VEXAS signature, as well as with interferon signalling, a key inflammatory pathway downstream of JAK-STAT. JAK2 expression was also associated with plasma cell response, indicative of MGUS activity. JAK1 displayed an opposite trend compared to JAK2 (Figure 1D). Considering bulk RNA-seq performed on VEXAS skin lesions, the transcriptomic changes occurring after treatment initiation recapitulated those observed in blood, with the downregulation of VEXAS and interferon signatures, coupled with JAK2, but not JAK1, transcript decrease (Figure 2A). Immune cell deconvolution revealed a concomitant shift from a pro-inflammatory environment to a tissue repair status, with the complete loss of neutrophils and monocytes and a switch of macrophages from an M1 pro-inflammatory phenotype towards an M2 anti-inflammatory one (Figure 2B), consistent with clinical improvement. scRNA-seq analysis performed on fresh skin tissue collected on day 5 further confirmed the differential expression of JAK1 and JAK2 genes after treatment initiation (Figure 2C). Notably, both genes were mainly expressed on clusters of fibroblasts, vascular endothelial cells and pericytes. Indeed, JAK-STAT signalling is known to modulate endothelial permeability, inducing a pro-adhesive and pro-coagulant switch in inflammatory conditions.8 Overall, our results indicate that JAK2 is a critical actionable target in patients with VEXAS, which has been suggested in clinical reports but never described from a molecular standpoint.6 Despite the efficacy of ruxolitinib, its discontinuation due to pancytopenia in the above-described case underscores the need for careful management of its haematological side effects. Our findings provide a strong pathophysiological basis to investigate the efficacy of selective anti-JAK2 molecules in patients with VEXAS, potentially reducing the rate of therapy-related side effects while preserving the efficacy in inducing and maintaining clinical remission. Conceptualization: GZ, LF and AlB, methodology: GZ, LF and MD, data collection: NG, AnB, BC, MS, AC, MD, IL, TV, GR, MD, IS and AG, data analysis: LF, GZ, FR, MD, MC and AC, software: LF, formal analysis: AC, MD, FR, LF, AlB and GZ, writing and review: all authors, visualization: LF, GZ and FR, supervision: AlB, LF and GZ, project administration: MD and GZ and funding acquisition: AlB and GZ. This work was supported by Associazione Italiana Linfoma, Leucemie e Mieloma Multiplo (AIL to AlB), Alleanza Contro il Cancro (ACC), Fondazione AIRC – Associazione Italiana per la Ricerca contro il Cancro (IG21761 to GZ), Research Projects of National Interest (Italian Ministry of University and Research, ID: M4.C2.1.1 to GZ), CURIOSITY driven research funds (University of Genoa to GZ) and unrestricted donations from the Benvenuto and Marra families. GZ wishes to thank Dr. P. Blandini for his ever-insightful criticisms of the technical aspects of the present work. GZ declares consultancy fees from Menarini Stemline and holds the co-ownership of Immunomica Ltd. The remaining authors have no conflict of interest to disclose. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
Abstract Background B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5 + and CD5 − B cells from the spleen and peripheral blood (PB). Methods Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5 + and CD5 − cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. Results CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5 + B (0.57%) cells compared to CD5 − B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. Conclusions CBS-IG receptors appear to represent a part of the “public” BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.
Abstract This study describes a CD5 + B cell that differs from the majority of the CD5 + B cells from human tonsils. This cell, isolated from in vivo activated B cells, expressed activation markers and featured a CD23 – , IgM high , IgD low surface phenotype, responded to T cell‐independent type‐2 antigens in vitro , and was detected in the subepithelial (SE) areas, the tonsil equivalent of the splenic marginal zone (MZ). Most of the cells utilized unmutated Ig VH genes, although cells with mutated genes also were found, a finding confirmed by single‐cell studies. Mutated sequences were more frequent in suspensions enriched for CD27 + cells. Repeated VDJ gene sequences were observed in different molecular clones from the same cell suspension, suggesting in situ expansion. These CD5 + B cells seem to share features with previously characterized tonsil CD5 – SE B cells and differ from the majority of tonsil CD5 + B cells, which have the surface phenotype of follicular mantle B cells, lack activation markers, do not respond to T cell‐independent antigens, and utilize unmutated VH genes. These data are discussed considering the present views on the origin of B cell subset populations and the relationships between MZ and B1 cells.
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated IGHV genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.
