RESUMEN
The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.
Asunto(s)
Hematopoyesis , Transducción de Señal , Ratones , Humanos , Animales , Proteínas Co-Represoras , Núcleo Celular , Genómica , Co-Represor 2 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/genéticaRESUMEN
Patients with acute lymphoblastic leukemia have experienced significantly improved outcomes due to the advent of chimeric antigen receptor (CAR) T cells and bispecific T-cell engagers, although a proportion of patients still relapse despite these advances. T-cell exhaustion has been recently suggested to be an important driver of relapse in these patients. Indeed, phenotypic exhaustion of CD4+ T cells is predictive of relapse and poor overall survival in B-cell acute lymphoblastic leukemia (B-ALL). Thus, therapies that counter T-cell exhaustion, such as immune checkpoint blockade, may improve leukemia immunosurveillance and prevent relapse. Here, we used a murine model of Ph+ B-ALL as well as human bone marrow biopsy samples to assess the fundamental nature of CD4+ T-cell exhaustion and the preclinical therapeutic potential for combining anti-PD-L1 based checkpoint blockade with tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein. Single-cell RNA-sequence analysis revealed that B-ALL induces a unique subset of CD4+ T cells with both cytotoxic and helper functions. Combination treatment with the tyrosine kinase inhibitor nilotinib and anti-PD-L1 dramatically improves long-term survival of leukemic mice. Depletion of CD4+ T cells prior to therapy completely abrogates the survival benefit, implicating CD4+ T cells as key drivers of the protective anti-leukemia immune response. Indeed, treatment with anti-PD-L1 leads to clonal expansion of leukemia-specific CD4+ T cells with the aforementioned helper/cytotoxic phenotype as well as reduced expression of exhaustion markers. These findings support efforts to use PD1/PD-L1 checkpoint blockade in clinical trials and highlight the importance of CD4+ T-cell dysfunction in limiting the endogenous anti-leukemia response.
Asunto(s)
Antineoplásicos , Leucemia de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Antígeno B7-H1 , Linfocitos T CD4-Positivos , Humanos , Ratones , Pirimidinas , RecurrenciaRESUMEN
Integration of external signals and B-lymphoid transcription factor activities organise B cell lineage commitment through alternating cycles of proliferation and differentiation, producing a diverse repertoire of mature B cells. We use single-cell transcriptomics/proteomics to identify differentially expressed gene networks across B cell development and correlate these networks with subtypes of B cell leukemia. Here we show unique transcriptional signatures that refine the pre-B cell expansion stages into pre-BCR-dependent and pre-BCR-independent proliferative phases. These changes correlate with reciprocal changes in expression of the transcription factor EBF1 and the RNA binding protein YBX3, that are defining features of the pre-BCR-dependent stage. Using pseudotime analysis, we further characterize the expression kinetics of different biological modalities across B cell development, including transcription factors, cytokines, chemokines, and their associated receptors. Our findings demonstrate the underlying heterogeneity of developing B cells and characterise developmental nodes linked to B cell transformation.
Asunto(s)
Linfocitos B/citología , Redes Reguladoras de Genes , Leucopoyesis/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proliferación Celular/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Pronóstico , Proteómica , Análisis de la Célula Individual , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
The transcription factors PAX5, IKZF1, and EBF1 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that compound heterozygous loss of multiple genes critical for B and T cell development drives transformation, including Pax5+/-xEbf1+/-, Pax5+/-xIkzf1+/-, and Ebf1+/-xIkzf1+/- mice for B-ALL, or Tcf7+/-xIkzf1+/- mice for T-ALL. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we performed a Sleeping Beauty (SB) transposon screen that identified cooperating partners including gain-of-function mutations in Stat5b (~65%) and Jak1 (~68%), or loss-of-function mutations in Cblb (61%) and Myb (32%). These findings underscore the role of JAK/STAT5B signaling in B cell transformation and demonstrate roles for loss-of-function mutations in Cblb and Myb in transformation. RNA-Seq studies demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/-xEbf1+/- leukemia cells with PDK1 inhibitors blocked proliferation in vitro. In addition, we identified a conserved transcriptional gene signature between human and murine leukemias characterized by upregulation of myeloid genes, most notably involving the GM-CSF pathway, that resemble a B cell/myeloid mixed-lineage leukemia. Thus, our findings identify multiple mechanisms that cooperate with defects in B cell transcription factors to generate either progenitor B cell or mixed B/myeloid-like leukemias.
Asunto(s)
Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Factores de Transcripción/genética , Transposasas/genética , Animales , Mutación con Ganancia de Función , Pruebas Genéticas , Humanos , Mutación con Pérdida de Función , Ratones , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Análisis de Secuencia de ARN , Transducción de Señal , Transactivadores/genéticaRESUMEN
B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Receptores de Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/metabolismo , Médula Ósea/inmunología , Médula Ósea/metabolismo , Diferenciación Celular/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Inmunoglobulina de Cadenas Ligeras Subrogadas/inmunología , Inmunoglobulina de Cadenas Ligeras Subrogadas/metabolismo , Hígado/embriología , Hígado/inmunología , Hígado/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Células Precursoras de Linfocitos B/genética , Receptores de Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/inmunología , Receptores de Interleucina-7/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Factor de Transcripción STAT5/metabolismoRESUMEN
The transcription factor STAT5 has a critical role in B cell acute lymphoblastic leukemia (B-ALL). How STAT5 mediates this effect is unclear. Here we found that activation of STAT5 worked together with defects in signaling components of the precursor to the B cell antigen receptor (pre-BCR), including defects in BLNK, BTK, PKCß, NF-κB1 and IKAROS, to initiate B-ALL. STAT5 antagonized the transcription factors NF-κB and IKAROS by opposing regulation of shared target genes. Super-enhancers showed enrichment for STAT5 binding and were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4 and IKAROS. Patients with a high ratio of active STAT5 to NF-κB or IKAROS had more-aggressive disease. Our studies indicate that an imbalance of two opposing transcriptional programs drives B-ALL and suggest that restoring the balance of these pathways might inhibit B-ALL.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Linfocitos B , Regulación Neoplásica de la Expresión Génica , Factor de Transcripción Ikaros/genética , Receptores de Células Precursoras de Linfocitos B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factor de Transcripción STAT5/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Inmunoprecipitación de Cromatina , Citometría de Flujo , Humanos , Factores Reguladores del Interferón/genética , Ratones , Reacción en Cadena de la Polimerasa Multiplex , Subunidad p50 de NF-kappa B/genética , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Proteína Quinasa C beta/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Tasa de Supervivencia , Transactivadores/genéticaRESUMEN
Naive CD8 T cells proliferate in response to TCR and CD28 signals, but require IL-12 or type I IFN to survive and develop optimal effector functions. Although murine CTL generated in vitro in response to IL-12 or IFN-α had comparable effector functions, IL-12-stimulated cells were significantly more effective in controlling tumor in an adoptive immunotherapy model. They maintained high numbers and function, whereas IFN-α-stimulated cells declined in number and became exhausted. Consistent with this, IFN-α-stimulated cells in the tumor expressed higher levels of programmed death 1 (PD-1) inhibitory receptor than did IL-12-stimulated cells. When blocking Ab specific for the PD-L1 ligand of PD-1 was administered, the efficacy of IFN-α-stimulated CTL became comparable with that of IL-12-stimulated cells. Thus, IL-12 and IFN-α differentially program CD8 T cells to re-express distinct levels of PD-1 upon re-encountering Ag, resulting in IL-12-stimulated cells being less susceptible to exhaustion in the face of sustained tumor Ag.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interferón Tipo I/metabolismo , Interferón-alfa/metabolismo , Interleucina-12/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Proliferación Celular , Activación de Linfocitos/inmunología , Ratones , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/biosíntesis , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The transcription factor Signal Tranducer and Activator of Transcription 5 (STAT5) plays an important role in many biological processes. To study STAT5 biology, several different constructs have been designed that render STAT5 constitutively active. These constructs have now been used to generate animal models that allow for targeted expression of constitutively active STAT5 including a model where STAT5 is expressed in developing B and T cells. Herein we briefly describe the design of constitutively active STAT5 constructs and recent advances in their use.
Asunto(s)
Biología/métodos , Factor de Transcripción STAT5/metabolismo , Animales , Ratones , Estructura Terciaria de Proteína , Factor de Transcripción STAT5/químicaRESUMEN
STAT5 plays a crucial role in B and T lymphocyte development. However, whether STAT5 primarily plays a role as a permissive factor, involved in lymphocyte survival, or an instructive factor, involved in lymphocyte differentiation, has been unclear. In addition, while STAT5 has been suggested to act as a transcriptional repressor, the mechanism by which it represses transcription was undefined. Recent reports have begun to shed new light on these roles for STAT5 in lymphocyte development, transcriptional repression, and leukemic transformation.
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Linfopoyesis , Factor de Transcripción STAT5/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Humanos , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Transcripción Genética/efectos de los fármacosRESUMEN
As STAT5 is critical for the differentiation, proliferation, and survival of progenitor B cells, this transcription factor may play a role in acute lymphoblastic leukemia (ALL). Here, we show increased expression of activated signal transducer and activator of transcription 5 (STAT5), which is correlated with poor prognosis, in ALL patient cells. Mutations in EBF1 and PAX5, genes critical for B cell development have also been identified in human ALL. To determine whether mutations in Ebf1 or Pax5 synergize with STAT5 activation to induce ALL, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice heterozygous for Ebf1 or Pax5. Haploinsufficiency of either Pax5 or Ebf1 synergized with Stat5b-CA to rapidly induce ALL in 100% of the mice. The leukemic cells displayed reduced expression of both Pax5 and Ebf1, but this had little effect on most EBF1 or PAX5 target genes. Only a subset of target genes was deregulated; this subset included a large percentage of potential tumor suppressor genes and oncogenes. Further, most of these genes appear to be jointly regulated by both EBF1 and PAX5. Our findings suggest a model whereby small perturbations in a self-reinforcing network of transcription factors critical for B cell development, specifically PAX5 and EBF1, cooperate with STAT5 activation to initiate ALL.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Mutación , Factor de Transcripción PAX5/genética , Factor de Transcripción STAT5/genética , Transactivadores/genética , Animales , Linfocitos B/citología , Proliferación Celular , Heterocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Interleucina-7/metabolismo , Factor de Transcripción STAT5/metabolismo , Resultado del Tratamiento , VDJ Recombinasas/metabolismoRESUMEN
The transcription factor signal transducer and activator of transcription 5 (STAT5) is activated by a number of cytokine and growth hormone receptors and plays a key role in the development and function of many organ systems. In this review, we focus on recent discoveries about the role of STAT5 in the development and function of B and T lymphocytes. Of particular interest is the growing appreciation for the function of STAT5 as a transcriptional repressor. Finally, we discuss recent discoveries about the role of STAT5 in transformation of B and T lymphocytes.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Factor de Transcripción STAT5/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Linfocitos B/citología , Humanos , Linfocitos T/citologíaRESUMEN
Activation-induced deaminase (AID) is expressed in activated B lymphocytes and initiates somatic hypermutation and class switch recombination. To determine if different stimuli affect the expression and function of AID, we monitored AID activity in murine B cells stimulated ex vivo with various ligands. AID was rapidly expressed at both the RNA and protein levels following stimulation with LPS, LPS plus IL-4, and anti-CD40 plus IL-4, but was delayed after stimulation with anti-IgM plus IL-4. By day 4, AID was expressed in all groups; however, cells stimulated with anti-IgM plus IL-4 did not undergo switch recombination. These cells expressed normal levels of gamma 1 germline transcripts, implying that the gamma 1 switch region was accessible. Furthermore, switching was suppressed by the addition of anti-IgM to cells stimulated with LPS plus IL-4 or anti-CD40 plus IL-4, even though AID was expressed. The lack of class switch recombination could be reversed by inhibition of phosphatidylinositol 3-kinase (PI3K). This suggests that activation through the B cell receptor induces PI3K, which interferes with the function of AID.