RESUMO
ABSTRACT: We have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, an inhibitor of integrin α4ß1, plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice. Mobilization with WU-106 + AMD3100 in SCD mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of the prime editing vector together with in vivo selection resulted in â¼23% correction of the SCD mutation in the bone marrow and peripheral blood cells of SCD mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing frequencies were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.
Assuntos
Anemia Falciforme , Terapia Genética , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas , Animais , Humanos , Camundongos , Anemia Falciforme/terapia , Anemia Falciforme/genética , Benzilaminas , Ciclamos/farmacologia , Ciclamos/uso terapêutico , Modelos Animais de Doenças , Edição de Genes , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos/uso terapêuticoRESUMO
Allogeneic hematopoietic cell transplantation (allo-HCT) is a highly effective, well-established treatment for patients with various hematologic malignancies and non-malignant diseases. The therapeutic benefits of allo-HCT are mediated by alloreactive T cells in donor grafts. However, there is a significant risk of graft-versus-host disease (GvHD), in which the donor T cells recognize recipient cells as foreign and attack healthy organs in addition to malignancies. We previously demonstrated that targeting JAK1/JAK2, mediators of interferon-gamma receptor (IFNGR) and IL-6 receptor signaling, in donor T cells using baricitinib and ruxolitinib results in a significant reduction in GvHD after allo-HCT. Furthermore, we showed that balanced inhibition of JAK1/JAK2 while sparing JAK3 is important for the optimal prevention of GvHD. Thus, we have generated novel JAK1/JAK2 inhibitors, termed WU derivatives, by modifying baricitinib. Our results show that WU derivatives have the potential to mitigate GvHD by upregulating regulatory T cells and immune reconstitution while reducing the frequencies of antigen-presenting cells (APCs) and CD80 expression on these APCs in our preclinical mouse model of allo-HCT. In addition, WU derivatives effectively downregulated CXCR3 and T-bet in primary murine T cells. In summary, we have generated novel JAK inhibitors that could serve as alternatives to baricitinib or ruxolitinib.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Pirazóis , Transplante Homólogo , Animais , Camundongos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Azetidinas/farmacologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/prevenção & controle , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 2/antagonistas & inibidores , Inibidores de Janus Quinases/farmacologia , Camundongos Endogâmicos C57BL , Purinas/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited treatment options and poor survival rates. About half of MPNST cases are associated with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome. Overexpression of TYK2 occurs in the majority of MPNST, implicating TYK2 as a therapeutic target. EXPERIMENTAL DESIGN: The effects of pharmacologic TYK2 inhibition on MPNST cell proliferation and survival were examined using IncuCyte live cell assays in vitro, and downstream actions were analyzed using RNA-sequencing (RNA-seq), qPCR arrays, and validation of protein changes with the WES automated Western system. Inhibition of TYK2 alone and in combination with MEK inhibition was evaluated in vivo using both murine and human MPNST cell lines, as well as MPNST PDX. RESULTS: Pharmacologic inhibition of TYK2 dose-dependently decreased proliferation and induced apoptosis over time. RNA-seq pathway analysis on TYK2 inhibitor-treated MPNST demonstrated decreased expression of cell cycle, mitotic, and glycolysis pathways. TYK2 inhibition resulted in upregulation of the MEK/ERK pathway gene expression, by both RNA-seq and qPCR array, as well as increased pERK1/2 levels by the WES Western system. The compensatory response was tested with dual treatment with TYK2 and MEK inhibitors, which synergistically decreased proliferation and increased apoptosis in vitro. Finally, combination therapy was shown to inhibit growth of MPNST in multiple in vivo models. CONCLUSIONS: These data provide the preclinical rationale for the development of a phase I clinical trial of deucravacitinib and mirdametinib in NF1-assosciated MPNST.
Assuntos
Neoplasias de Bainha Neural , Neurofibromatose 1 , Neurofibrossarcoma , Humanos , Camundongos , Animais , Neurofibromatose 1/tratamento farmacológico , Neurofibromatose 1/genética , Linhagem Celular , Apoptose , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias de Bainha Neural/tratamento farmacológico , Neoplasias de Bainha Neural/genética , Neoplasias de Bainha Neural/metabolismo , Linhagem Celular Tumoral , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , TYK2 Quinase/farmacologiaRESUMO
Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADCs) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation, and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.
Assuntos
Azetidinas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Imunoconjugados/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Inibidores de Janus Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Aloenxertos , Animais , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Efeito Enxerto vs Leucemia/genética , Efeito Enxerto vs Leucemia/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Janus Quinase 1/genética , Janus Quinase 1/imunologia , Janus Quinase 2/genética , Janus Quinase 2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Allogeneic hematopoietic transplantation (allo-HCT) is a curative therapy for a variety of hematologic malignancies, primarily through immune-mediated clearance of malignant cells. This graft-versus-leukemia (GvL) effect is mediated by alloreactive donor T-cells against recipient malignant cells. Unfortunately, graft versus host disease is a potentially lethal complication of this procedure, also mediated by alloreactive donor T-cells against recipient normal tissues. Graft-versus-host disease (GVHD) remains a key contributor to nonrelapse mortality and long-term morbidity in patients undergoing allo-HCT. Reducing GVHD without interfering with - or ideally while enhancing - GvL, would improve outcomes and increase patient eligibility for allo-HCT. The JAK/STAT signaling pathway acts downstream of over 50 cytokines and is central to a wide variety of inflammatory pathways. These pathways play a role in the development and maintenance of GVHD throughout the disease process and within T-cells, B-cells, macrophages, neutrophils, and natural killer cells. Agents targeting JAK/STAT signaling pathways have shown clinical efficacy and gained US Food and Drug Administration approval for numerous diseases. Here, we review the preclinical and clinical evidence for the role of JAK/STAT signaling in the development and maintenance of GVHD and the utility of blocking agents at preventing and treating GVHD.
Assuntos
Suscetibilidade a Doenças , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Integrina alfa4beta1/antagonistas & inibidores , Terapia de Alvo Molecular , Animais , Biomarcadores , Modelos Animais de Doenças , Antígenos H-2/genética , Antígenos H-2/imunologia , Transplante de Células-Tronco Hematopoéticas , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante HomólogoRESUMO
Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.
Assuntos
Medula Óssea/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Integrina alfa4beta1 , Receptores de Interleucina-8B , Aloenxertos , Animais , Granulócitos/metabolismo , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
The integrin alpha(v)beta(3), vitronectin receptor, is expressed in a number of cell types and has been shown to mediate adhesion of osteoclasts to bone matrix, vascular smooth muscle cell migration, and angiogenesis. We recently disclosed the discovery of a tripeptide Arg-Gly-Asp (RGD) mimic, which has been shown to be a potent inhibitor of the integrin alpha(v)beta(3) and has excellent anti-angiogenic properties including its suppression of tumor growth in animal models. In other investigations involving RGD mimics, only compounds containing the S-isomers of the beta-amino acids have been shown to be potent. We were surprised to find the potencies of analogs containing enantiomerically pure S-isomers of beta-amino acids which were only marginally better than the corresponding racemic mixtures. We therefore synthesized RGD mimics containing R-isomers of beta-amino acids and found them to be relatively potent inhibitors of alpha(v)beta(3). One of the compounds was examined in tumor models in mice and has been shown to significantly reduce the rate of growth and the size of tumors.
Assuntos
Aminoácidos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Mimetismo Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Aminoácidos/síntese química , Animais , Antineoplásicos/farmacocinética , Neoplasias do Colo , Hipercalcemia/induzido quimicamente , Isomerismo , Melanoma , Camundongos , Camundongos Endogâmicos , Oligopeptídeos/farmacocinética , Neoplasias Cutâneas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Integrins expressed on endothelial cells modulate cell migration and survival during angiogenesis. Integrins expressed on carcinoma cells potentiate metastasis by facilitating invasion and movement across blood vessels. We describe the activities of two synthetic low-molecular-weight peptidomimetics of the ligand amino acid sequence arg-gly-asp (RGD) in integrin-based functional assays in vitro. We also evaluate efficacy and potential mechanisms of action in models of both spontaneous and experimental metastasis. Broad-spectrum potency against the family of alpha v subunit-containing integrins was observed, with significantly less potency against alpha5beta1 and alpha(IIb)beta3. Both endothelial and tumor cell migration mediated by alpha(v)beta3 was inhibited, whereas proliferation of endothelial cells but not tumor cells was diminished. Continuous infusion of compound by minipumps or oral administration twice daily significantly reduced metastatic tumor burden in the lungs of mice despite no reduction in growth of 435/HAL primary tumors, and only a slight reduction in tumor cells detected in circulating blood. Delaying treatment in this model until after extensive dissemination of tumor cells to the lungs had occurred, and after primary tumor resection, still produced significant efficacy. Conversely, administration of the agent for only the first 18 h after tumor-cell inoculation into the tail vein also resulted in decreased metastases observed after several weeks. These data suggest these compounds or their relatives have potential to interfere with both early and late steps of metastasis involving tumor and endothelial cell functions. Furthermore, the metastatic process can be effectively inhibited independently of primary tumor growth using integrin antagonists.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Carcinoma Ductal/secundário , Dipeptídeos/uso terapêutico , Integrina alfaVbeta3/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Proteínas de Neoplasias/antagonistas & inibidores , Compostos Orgânicos/uso terapêutico , Pirimidinas/uso terapêutico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/patologia , Carcinoma Ductal/prevenção & controle , Carcinoma Ductal/cirurgia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacologia , Esquema de Medicação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Feminino , Humanos , Bombas de Infusão Implantáveis , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Neoplásicas Circulantes , Oligopeptídeos , Compostos Orgânicos/administração & dosagem , Compostos Orgânicos/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many viruses and bacterial pathogens are capable of exploiting host cell surface integrins during their replication cycles. The ligands for many integrins contain an arginine-glycine-aspartic acid (RGD) amino acid sequence that is essential for protein-protein interaction. Human adenovirus particles contain this sequence in the penton base protein, and previous studies support a role for this RGD in integrin-dependent internalization of the virus by the cell. As synthetic peptidomimetics of RGD have been shown in other experimental systems to be antagonists of the activities of specific integrins both in vitro and in vivo, we sought to determine whether these small molecules are antagonists of adenovirus infection. Such compounds inhibited viral infection of cultured cells with similar rank order potency to that determined in assays utilizing purified extracellular matrix proteins as integrin ligands. The maximal level of inhibition achieved with the peptidomimetics was comparable to that of RGD-containing peptides, whereas no significant effects were apparent with an RGE-containing peptide. An engineered adenovirus having a mutated RGD sequence in the penton base was not susceptible to the inhibition. The results obtained with these synthetic antagonists, which have varied structures and potencies, suggest that integrins interact with adenoviral RGD in a manner similar to that of other protein ligands such as vitronectin. Furthermore, the results confirm the role of RGD in the replication cycle, and suggest peptidomimetic compounds may be useful antimicrobial agents in the treatment of a variety of diseases.