RESUMO
Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2âBALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6âB10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.
Assuntos
Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Receptores Notch/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Proteínas de Ligação ao Cálcio , Doença Crônica , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/patologia , Transplante Homólogo/efeitos adversosRESUMO
Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.
Assuntos
Doença Enxerto-Hospedeiro/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Fator de Transcrição STAT3/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Animais , Bronquiolite Obliterante/tratamento farmacológico , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/fisiopatologia , Doença Crônica , Citocinas/biossíntese , Citocinas/genética , Avaliação Pré-Clínica de Medicamentos , Doença Enxerto-Hospedeiro/tratamento farmacológico , Humanos , Leucócitos Mononucleares/metabolismo , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/biossíntese , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-6/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6/genética , Fator de Transcrição STAT3/deficiência , Organismos Livres de Patógenos Específicos , Baço/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/transplante , Quinases Associadas a rho/fisiologiaRESUMO
Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification.
Assuntos
Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos , Feminino , Hematopoese/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/genética , Células Tumorais CultivadasRESUMO
Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell-specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8(+) T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.
Assuntos
Anticorpos Neutralizantes/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração/métodos , Imunidade/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Ligação ao Cálcio , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/antagonistas & inibidores , Receptores Notch/imunologia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Transplante HomólogoRESUMO
Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.
Assuntos
Doença Enxerto-Hospedeiro/tratamento farmacológico , Fatores Imunológicos/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Animais , Intervalo Livre de Doença , Avaliação Pré-Clínica de Medicamentos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Fatores Imunológicos/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Piperidinas , Pirazóis/uso terapêutico , Pirimidinas/uso terapêuticoRESUMO
Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs.