RESUMO
ABSTRACT: The T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.
Assuntos
Proliferação de Células , Leucemia Linfocítica Crônica de Células B , Proteínas com Domínio T , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Humanos , Camundongos , Linfócitos B/patologia , Linfócitos B/metabolismo , Linfócitos B/imunologia , Camundongos Knockout , Regulação Leucêmica da Expressão Gênica , NF-kappa B/metabolismoRESUMO
Although the genetic landscape of chronic lymphocytic leukemia (CLL) has been broadly profiled by large-scale sequencing studies performed over the past decade, the molecular basis of the transformation of CLL into aggressive lymphoma, or Richter syndrome (RS), has remained incompletely characterized. Recent advances in computational methods of clonal deconvolution, as well as extensive sample collection efforts in this rapidly progressive malignancy, have now enabled comprehensive analysis of paired CLL and RS samples and have led to multiple new studies investigating the genetic, transcriptomic, and epigenetic origins of RS. In parallel, new genetically engineered and xenograft mouse models have provided the opportunity for gleaning fresh biological and mechanistic insights into RS development and stepwise evolution from antecedent CLL. Altogether, these studies have defined RS driver lesions and CLL risk lesions and identified pathways dysregulated in transformation. Moreover, unique molecular subtypes of RS have been revealed, including a disease marked by profound genomic instability with chromothripsis/chromoplexy and whole genome duplication. Novel profiling approaches, including single-cell DNA and transcriptome sequencing of RS biopsy specimens and cell-free DNA profiling of patient plasma, demonstrate promise for the timely identification of RS clones and may translate to noninvasive identification and early diagnosis of RS. This review summarizes the recent scientific advances in RS and supports the integrated study of human genomics with mouse modeling to provide an advanced understanding of the biological underpinnings of transformation. These recent studies have major implications for much-needed novel therapeutic strategies for this still largely incurable malignancy.
Assuntos
Transformação Celular Neoplásica , Leucemia Linfocítica Crônica de Células B , Linfoma Difuso de Grandes Células B , Humanos , Animais , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Genoma Humano , Camundongos , Transformação Celular Neoplásica/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Rapid advances in large-scale next-generation sequencing studies of human samples have progressively defined the highly heterogeneous genetic landscape of chronic lymphocytic leukemia (CLL). At the same time, the numerous challenges posed by the difficulties in rapid manipulation of primary B cells and the paucity of CLL cell lines have limited the ability to interrogate the function of the discovered putative disease "drivers," defined in human sequencing studies through statistical inference. Mouse models represent a powerful tool to study mechanisms of normal and malignant B-cell biology and for preclinical testing of novel therapeutics. Advances in genetic engineering technologies, including the introduction of conditional knockin/knockout strategies, have opened new opportunities to model genetic lesions in a B-cell-restricted context. These new studies build on the experience of generating the MDR mice, the first example of a genetically faithful CLL model, which recapitulates the most common genomic aberration of human CLL: del(13q). In this review, we describe the application of mouse models to the studies of CLL pathogenesis and disease transformation from an indolent to a high-grade malignancy (ie, Richter syndrome [RS]) and treatment, with a focus on newly developed genetically inspired mouse lines modeling recurrent CLL genetic events. We discuss how these novel mouse models, analyzed using new genomic technologies, allow the dissection of mechanisms of disease evolution and response to therapy with greater depth than previously possible and provide important insight into human CLL and RS pathogenesis and therapeutic vulnerabilities. These models thereby provide valuable platforms for functional genomic analyses and treatment studies.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Animais , Regulação Neoplásica da Expressão Gênica , Genômica , Genética Humana , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Transplante de Neoplasias , Neoplasias ExperimentaisRESUMO
Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis.
Assuntos
Comunicação Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Leucemia Linfocítica Crônica de Células B/patologia , Microambiente Tumoral/genética , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Adesão Celular/genética , Quimiotaxia de Leucócito/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Baço/metabolismo , Baço/patologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
BCR signaling is a central pathogenetic pathway in chronic lymphocytic leukemia (CLL). Most CLL cells express BCRs of IgM and IgD isotypes, but the contribution of these isotypes to functional responses remains incompletely defined. We therefore investigated differences between IgM and IgD signaling in freshly isolated peripheral blood CLL cells and in CLL cells cultured with nurselike cells, a model that mimics the lymph node microenvironment. IgM signaling induced prolonged activation of ERK kinases and promoted CLL cell survival, CCL3 and CCL4 chemokine secretion, and downregulation of BCL6, the transcriptional repressor of CCL3 In contrast, IgD signaling induced activation of the cytoskeletal protein HS1, along with F-actin polymerization, which resulted in rapid receptor internalization and failure to support downstream responses, including CLL cell survival and chemokine secretion. IgM and IgD receptor downmodulation, HS1 and ERK activation, chemokine secretion, and BCL6 downregulation were also observed when CLL cells were cocultured with nurselike cells. The Bruton's tyrosine kinase inhibitor ibrutinib effectively inhibited both IgM and IgD isotype signaling. In conclusion, through a variety of functional readouts, we demonstrate very distinct outcomes of IgM and IgD isotype activation in CLL cells, providing novel insight into the regulation of BCR signaling in CLL.
Assuntos
Imunoglobulina D/metabolismo , Imunoglobulina M/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Sobrevivência Celular/imunologia , Células Cultivadas , Microambiente Celular/imunologia , Quimiocina CCL3/imunologia , Quimiocina CCL3/metabolismo , Quimiocina CCL4/imunologia , Quimiocina CCL4/metabolismo , Regulação da Expressão Gênica , Humanos , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Linfonodos/citologia , Linfonodos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/genética , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
Chronic Lymphocytic Leukemia (CLL) is a malignancy of mature B lymphocytes which are highly dependent on interactions with the tissue microenvironment for their survival and proliferation. Critical components of the microenvironment are monocyte-derived nurselike cells (NLCs), mesenchymal stromal cells, T cells and NK cells, which communicate with CLL cells through a complex network of adhesion molecules, chemokine receptors, tumor necrosis factor (TNF) family members, and soluble factors. (Auto-) antigens and/or autonomous mechanisms activate the B-cell receptor (BCR) and its downstream signaling cascade in secondary lymphatic tissues, playing a central pathogenetic role in CLL. Novel small molecule inhibitors, including the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and the phosphoinositide-3-kinase delta (PI3Kδ) inhibitor idelalisib, target BCR signaling and have become the most successful new therapeutics in this disease. We here review the cellular and molecular characteristics of CLL cells, and discuss the cellular components and key pathways involved in the cross-talk with their microenvironment. We also highlight the relevant novel treatment strategies, focusing on immunomodulatory agents and BCR signaling inhibitors and how these treatments disrupt CLL-microenvironment interactions. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.
Assuntos
Linfócitos B/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Microambiente Tumoral , Linfócitos B/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Modelos Biológicos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
HS1 (hematopoietic cell-specific Lyn substrate-1) is a cytoskeletal interactor in the B-cell receptor (BCR) signaling pathway whose phosphorylation correlates with prognosis in Chronic Lymphocytic Leukemia (CLL). The differentially phosphorylated sites and the kinases that regulate HS1 activity in CLL remain poorly understood. We demonstrate that HS1 activity is differentially regulated by LYN kinase that, in a subset of patients, phosphorylates HS1 on Tyrosine (Y)397, resulting in its activation. This correlates with increased cytoskeletal functionality in terms of migration, adhesion and F-actin polymerization. In these patients, LYN is also activated on Y396 residue and its inhibition with the tyrosine kinase inhibitor Dasatinib abrogates HS1-Y397 phosphorylation. This results in the reduction of HS1 activation along with that of cytoskeletal effector VAV1 and the downstream kinase ERK also in the presence of BCR and CXC chemokine receptor CXCR4 stimulation. Interestingly, targeting the LYN/HS1 axis in vitro leads to the concomitant reduction of cytoskeletal activity, BCR signaling and cell survival in the subset of patients with activated LYN/HS1. In a transplantable mouse model based on the EµTCL1 transgenic mouse, LYN/HS1 signaling inhibition interferes with CLL progression and lymphoid organ infiltration. Thus LYN/HS1 axis marks distinct signaling profiles and cytoskeletal-related features that may represent valuable targets for cytoskeleton-targeted therapeutic intervention in CLL.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas Sanguíneas/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Terapia de Alvo Molecular/métodos , Quinases da Família src/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dasatinibe , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Tiazóis/administração & dosagem , Tiazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.
Assuntos
Linfócitos B , Anergia Clonal/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Terapia de Alvo Molecular/métodos , Animais , Antineoplásicos/uso terapêutico , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Cadeias gama de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Tumorais CultivadasRESUMO
Murine models are indispensable tools for functional genomic studies and preclinical testing of novel therapeutic approaches. Mitochondrial single-cell assay for transposase-accessible chromatin using sequencing (mtscATAC-seq) enables the dissection of cellular heterogeneity and clonal dynamics by capturing chromatin accessibility, copy-number variations (CNV), and mitochondrial DNA (mtDNA) mutations, yet its applicability to murine studies remains unexplored. By leveraging mtscATAC-seq in novel chronic lymphocytic leukemia and Richter syndrome mouse models, we report the detection of mtDNA mutations, particularly in highly proliferative murine cells, alongside CNV and chromatin state changes indicative of clonal evolution upon secondary transplant. This study thus demonstrates the feasibility and utility of multi-modal single-cell and natural barcoding approaches to characterize murine cancer models. SIGNIFICANCE: mtDNA mutations can serve as natural barcodes to enable lineage tracing in murine cancer models, which can be used to provide new insights into disease biology and to identify therapeutic vulnerabilities.
Assuntos
DNA Mitocondrial , Neoplasias , Animais , Camundongos , DNA Mitocondrial/genética , Mitocôndrias/genética , Cromatina , Mutação , Neoplasias/genéticaRESUMO
Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders. For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,1 ten Hacken et al.,2 and ten Hacken et al.3.
Assuntos
Edição de Genes , Transtornos Linfoproliferativos , Camundongos , Animais , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Modelos Animais de Doenças , Mutação , Transtornos Linfoproliferativos/genéticaRESUMO
Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.
Assuntos
Leucemia Linfocítica Crônica de Células B , Linfoma Difuso de Grandes Células B , Linfoma não Hodgkin , Humanos , Animais , Camundongos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Fosfatidilinositol 3-Quinases/genética , Linfoma Difuso de Grandes Células B/genética , Linfócitos BRESUMO
The function of the intracellular protein hematopoietic cell-specific Lyn substrate-1 (HS1) in B lymphocytes is poorly defined. To investigate its role in migration, trafficking, and homing of leukemic B lymphocytes we have used B cells from HS1(-/-) mice, the HS1-silenced human chronic lymphocytic leukemia (CLL) MEC1 cell line and primary leukemic B cells from patients with CLL. We have used both in vitro and in vivo models and found that the lack of expression of HS1 causes several important functional effects. In vitro, we observed an impaired cytoskeletal remodeling that resulted in diminished cell migration, abnormal cell adhesion, and increased homotypic aggregation. In vivo, immunodeficient Rag2(-/-)γ(c)(-/-) mice injected with HS1-silenced CLL B cells showed a decreased organ infiltration with the notable exception of the bone marrow (BM). The leukemic-prone Eµ-TCL1 transgenic mice crossed with HS1-deficient mice were compared with Eµ-TCL1 mice and showed an earlier disease onset and a reduced survival. These findings show that HS1 is a central regulator of cytoskeleton remodeling that controls lymphocyte trafficking and homing and significantly influences the tissue invasion and infiltration in CLL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/patologia , Proteínas Sanguíneas/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/citologia , Proteínas Sanguíneas/genética , Medula Óssea/patologia , Adesão Celular , Linhagem Celular Tumoral , Citoesqueleto/patologia , Citoesqueleto/ultraestrutura , Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.
Assuntos
DNA Metiltransferase 3A/metabolismo , DNA Metiltransferase 3A/fisiologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Notch/metabolismo , Animais , Antibacterianos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , DNA Metiltransferase 3A/genética , Daptomicina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , RNA-Seq , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance.
Assuntos
Linfócitos B/patologia , Fator de Transcrição Ikaros/genética , Leucemia Linfocítica Crônica de Células B/genética , Mutação/genética , Transcrição Gênica/genética , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , NF-kappa B/genética , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais/genéticaRESUMO
Despite major improvements in treatment outcome with novel targeted therapies, such as the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, chronic lymphocytic leukemia (CLL) remains incurable in the majority of patients. Activation of PI3K, NF-κB, and/or MYC has been linked to residual disease and/or resistance in ibrutinib-treated patients. These pathways can be targeted by inhibitors of bromodomain and extra-terminal (BET) proteins. Here we report about the preclinical activity of GS-5829, a novel BET inhibitor, in CLL. GS-5829 inhibited CLL cell proliferation and induced leukemia cell apoptosis through deregulation of key signaling pathways, such as BLK, AKT, ERK1/2, and MYC. IκBα modulation indicates that GS-5829 also inhibited NF-κB signaling. GS-5829-induced apoptosis resulted from an imbalance between positive (BIM) and negative regulators (BCL-XL) of the intrinsic apoptosis pathway. The antileukemia activity of GS-5829 increased synergistically in combinations with B-cell receptor signaling inhibitors, the BTK inhibitor ibrutinib, the PI3Kδ inhibitor idelalisib, and the SYK inhibitor entospletinib. In cocultures that mimic the lymph node microenvironment, GS-5829 inhibited signaling pathways within nurselike cells and their growth, indicating that BET inhibitors also can target the supportive CLL microenvironment. Collectively, these data provide a rationale for the clinical evaluation of BET inhibitors in CLL.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
CRISPR-Cas9 gene editing has transformed our ability to rapidly interrogate the functional impact of somatic mutations in human cancers. Droplet-based technology enables the analysis of Cas9-introduced gene edits in thousands of single cells. Using this technology, we analyze Ba/F3 cells engineered to express single or multiplexed loss-of-function mutations recurrent in chronic lymphocytic leukemia. Our approach reliably quantifies mutational co-occurrences, zygosity status, and the occurrence of Cas9 edits at single-cell resolution.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Leucemia Linfocítica Crônica de Células B/genética , Mutação com Perda de Função , Análise de Célula Única/métodos , Animais , Feminino , Ensaios de Triagem em Larga Escala , Humanos , CamundongosRESUMO
The deletion of 11q (del(11q)) invariably comprises ATM gene in chronic lymphocytic leukemia (CLL). Concomitant mutations in this gene in the remaining allele have been identified in 1/3 of CLL cases harboring del(11q), being the biallelic loss of ATM associated with adverse prognosis. Although the introduction of targeted BCR inhibition has significantly favored the outcomes of del(11q) patients, responses of patients harboring ATM functional loss through biallelic inactivation are unexplored, and the development of resistances to targeted therapies have been increasingly reported, urging the need to explore novel therapeutic approaches. Here, we generated isogenic CLL cell lines harboring del(11q) and ATM mutations through CRISPR/Cas9-based gene-editing. With these models, we uncovered a novel therapeutic vulnerability of del(11q)/ATM-mutated cells to dual BCR and PARP inhibition. Ex vivo studies in the presence of stromal stimulation on 38 CLL primary samples confirmed a synergistic action of the combination of olaparib and ibrutinib in del(11q)/ATM-mutated CLL patients. In addition, we showed that ibrutinib produced a homologous recombination repair impairment through RAD51 dysregulation, finding a synergistic link of both drugs in the DNA damage repair pathway. Our data provide a preclinical rationale for the use of this combination in CLL patients with this high-risk cytogenetic abnormality.