The ETS-domain transcription factors divide into subfamilies based on protein similarities, DNA-binding sequences, and interaction with cofactors. They are regulated by extracellular clues and ...contribute to cellular processes, including proliferation and transformation.
genes are targeted through genomic rearrangements in oncogenesis. The
gene is inactivated by point mutations in human myeloid malignancies. We identified a recurrent somatic mutation (Q226E) in
in Waldenström macroglobulinemia, a B-cell lymphoproliferative disorder. It affects the DNA-binding affinity of the protein and allows the mutant protein to more frequently bind and activate promoter regions with respect to wild-type protein. Mutant SPI1 binding at promoters activates gene sets typically promoted by other ETS factors, resulting in enhanced proliferation and decreased terminal B-cell differentiation in model cell lines and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration of DNA binding leading to cellular proliferation and differentiation arrest. SIGNIFICANCE: The demonstration that a somatic point mutation tips the balance of genome-binding pattern provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.
.
Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate ...into plasma cells
and
. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers particularly solid tumors (3-5) can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare for a review of the few reported cases, see Ref. (6). The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (V
and D
gene replacement), BCR editing, and somatic mutations-events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements clonality assessment, V(D)J sequencing, and somatic hypermutation analysis in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same V
and J
genes but different D
genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.
Despite improving outcomes, 40% of patients with newly diagnosed multiple myeloma treated with regimens containing daratumumab, a CD38-targeted monoclonal antibody, progress prematurely. By ...integrating tumor whole-genome and microenvironment single-cell RNA sequencing from upfront phase 2 trials using carfilzomib, lenalidomide and dexamethasone with daratumumab ( NCT03290950 ), we show how distinct genomic drivers including high APOBEC mutational activity, IKZF3 and RPL5 deletions and 8q gain affect clinical outcomes. Furthermore, evaluation of paired bone marrow profiles, taken before and after eight cycles of carfilzomib, lenalidomide and dexamethasone with daratumumab, shows that numbers of natural killer cells before treatment, high T cell receptor diversity before treatment, the disappearance of sustained immune activation (that is, B cells and T cells) and monocyte expansion over time are all predictive of sustained minimal residual disease negativity. Overall, this study provides strong evidence of a complex interplay between tumor cells and the immune microenvironment that is predictive of clinical outcome and depth of treatment response in patients with newly diagnosed multiple myeloma treated with highly effective combinations containing anti-CD38 antibodies.
Abstract
Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center (GC) B cells. Despite progress in the treatment of DLBCL, approximately 40% of patients relapse or ...are refractory to the treatment, which usually leads to fatality. DLBCLs are characterized by aberrant DNA methylation and this feature correlates with poor clinical outcome. We and others have shown that deregulated epigenetic mechanisms contribute to lymphoma formation. Particularly, we have demonstrated that TET2, an enzyme that converts methylcytosine (mC) into hydroxymethylcytosine (hmC) and is mutated in ~10% of DLBCLs, is a B-cell tumor suppressor. GC-specific deletion of TET2 (Cg1Cre/Tet2-/-) resulted in accelerated lymphomagenesis in DLBCL mouse models driven by BCL6 overexpression, with 100% Cg1Cre/Tet2-/-;ImBcl6 mice developing lymphoma at 7 months compared to only 50% in ImBcl6 control mice. In addition, TET2 deletion in hematopoietic stem cells (VavCre/Tet2-/-) induced GC B-cell hyperplasia (B220+GL7+CD95+ cells; 10% VavCre/Tet2-/- vs 5% VavCre/Tet2+/+), promoting malignant transformation. Further analysis of the GC reaction revealed that TET2-deficient GC B cells displayed anomalous patterns of DNA methylation. GC B cells from VavCre/Tet2-/- mice presented 1) focal loss of hmC—using hMeDIPseq—with 25,000 differentially hydroxymethylated regions (DHMR) lost compared to VavCre/Tet2+/+ GC B cells and 2) increased mC—using RRBS—with almost 11,000 differentially methylated cytosines (DMCs), 84% hypermethylated, compared to VavCre/Tet2+/+ GC B cells. TET2-mediated reduction of hmC and hypermethylation affected enhancers and promoters, respectively, of genes mediating GC exit and terminal differentiation of GC B cells, especially those regulated at enhancers by the opposing functions of CREBBP and HDAC3. We are currently investigating the potential cooperative role between TET2-mediated hmC and CREBBP-mediated H3K27Ac, supported by reduced H3K27Ac at enhancers activated by CREBBP in VavCre/Tet2-/- GC B cells and mutual exclusion between TET2-mutant and CREBBP-mutant primary DLBCL. RNA sequencing analysis revealed that the genes epigenetically regulated by TET2 were aberrantly repressed in VavCre/Tet2+/+ GC B cells, explaining the observed GC hyperplasia in TET2-deficient GC B cells since these genes control the differentiation of GC B cells into plasma cells. Importantly, TET2-mutant DLBCL primary samples display a similar repressive transcriptional signature associated with GC B-cell terminal differentiation. Our data show how TET2-induced epigenetic changes contribute to lymphoma development and highlight the multilayered nature of the epigenome, which can be therapeutically exploited. We are evaluating the therapeutic potential in TET2-mutant DLBCL of a combinatorial therapy consisting of DNA methylation inhibitors (DNMTi), to revert hypermethylation at promoters, plus specific HDAC3 inhibitors, to compensate for the loss of hmC at enhancers.
Citation Format: Pilar M. Dominguez, Wojciech Rosikiewicz, Xiaowen Chen, Hussein Ghamlouch, Said Aoufouchi, Olivier A. Bernard, Ari M. Melnick, Sheng Li, Ricky W. Johnstone. TET2 deficiency alters the epigenome of germinal center B cells, contributing to lymphoma formation abstract. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-05.
Abstract Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible ...activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie -deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.
Summary
In B cells, B‐cell receptor (BCR) immunoglobulin revision is a common route for modifying unwanted antibody specificities via a mechanism called VH replacement. This in vivo process, mostly ...affecting heavy‐chain rearrangement, involves the replacement of all or part of a previously rearranged IGHV gene with another germline IGHV gene located upstream. Two different mechanisms of IGHV replacement have been reported: type 1, involving the recombination activating genes complex and requiring a framework region 3 internal recombination signal; and type 2, involving an unidentified mechanism different from that of type 1. In the case of light‐chain loci, BCR immunoglobulin editing ensures that a second V‐J rearrangement occurs. This helps to maintain tolerance, by generating a novel BCR with a new antigenic specificity. We report that human B cells can, surprisingly, undergo type 2 replacement associated with κ light‐chain rearrangements. The de novo IGKV‐IGKJ products result from the partial replacement of a previously rearranged IGKV gene by a new germline IGKV gene, in‐frame and without deletion or addition of nucleotides. There are wrcy/rgyw motifs at the ‘IGKV donor–IGKV recipient chimera junction’ as described for type 2 IGHV replacement, but activation‐induced cytidine deaminase (AID) expression was not detected. This unusual mechanism of homologous recombination seems to be a variant of gene conversion‐like recombination, which does not require AID. The recombination phenomenon described here provides new insight into immunoglobulin locus recombination and BCR immunoglobulin repertoire diversity.
Recent research has shown that chronic lymphocytic leukemia (CLL) B-cells display a strong tendency to differentiate into antibody-secreting cells (ASCs) and thus may be amenable to differentiation ...therapy. However, the effect of this differentiation on factors associated with CLL pathogenesis has not been reported. In the present study, purified CLL B-cells were stimulated to differentiate into ASCs by phorbol myristate acetate or CpG oligodeoxynucleotide, in combination with CD40 ligand and cytokines in a two-step, seven-day culture system. We investigated (i) changes in the immunophenotypic, molecular, functional, morphological features associated with terminal differentiation into ASCs, (ii) the expression of factors involved in CLL pathogenesis, and (iii) the expression of pro- and anti-apoptotic proteins in the differentiated cells. Our results show that differentiated CLL B-cells are able to display the transcriptional program of ASCs. Differentiation leads to depletion of the malignant program and deregulation of the apoptosis/survival balance. Analysis of apoptosis and the cell cycle showed that differentiation is associated with low cell viability and a low rate of cell cycle entry. Our findings shed new light on the potential for differentiation therapy as a part of treatment strategies for CLL.
Although hepatocellular carcinoma (HCC) is one of the most common malignancies and constitutes the third leading cause of cancer-related deaths, the underlying molecular mechanisms are not fully ...understood. In the present study, we demonstrate for the first time that hepatocytes express signalling lymphocytic activation molecule family member 3 (SLAMF3/CD229) but not other SLAMF members. We provide evidence to show that SLAMF3 is involved in the control of hepatocyte proliferation and in hepatocellular carcinogenesis. SLAMF3 expression is significantly lower in primary human HCC samples and HCC cell lines than in human healthy primary hepatocytes. In HCC cell lines, the restoration of high levels of SLAMF3 expression inhibited cell proliferation and migration and enhanced apoptosis. Furthermore, SLAMF3 expression was associated with inhibition of HCC xenograft progression in the nude mouse model. The restoration of SLAMF3 expression levels also decreased the phosphorylation of MAPK ERK1/2, JNK and mTOR. In samples from resected HCC patients, SLAMF3 expression levels were significantly lower in tumorous tissues than in peritumoral tissues. Our results identify SLAMF3 as a specific marker of normal hepatocytes and provide evidence for its potential role in the control of proliferation of HCC cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study, we investigated the capacity of chronic lymphocytic leukemia (CLL) B cells to undergo terminal differentiation into Ig‐secreting plasma cells in T cell‐independent and T cell‐dependent ...responses. We used a two‐step model involving stimulation with phorbol myristate acetate (PMA) and CD40L, together with cytokines (PMA/c and CD40L/c), for 7 days. We describe immunophenotypic modifications, changes in the levels of mRNA and protein for transcription factors and morphological and functional events occurring during the differentiation of CLL B cells into antibody‐secreting cells (ASCs). The induction of differentiation differed significantly between the CD40L/c and PMA/c culture systems. The PMA/c culture system allowed CLL B cells to differentiate into IgM‐secreting cells with an immunophenotype and molecular profile resembling those of preplasmablasts. By contrast, CD40L/c‐stimulated cells had a phenotype and morphology similar to those of activated B cells and resembling those of the CLL B cells residing in the lymph node and bone marrow. These data suggest that the CLL B cells are not frozen permanently at a stage of differentiation and are able to differentiate into ASCs as appropriate stimulation are provided. The data presented here raise questions about the molecular processes and stimulation required for CLL B‐cell differentiation and about the inability of CD40 ligand to induce differentiation of the CLL B cells.
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