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1.
J Biol Chem ; 289(31): 21605-16, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24952944

RESUMO

ETS family transcription factors regulate diverse genes through binding at cognate DNA sites that overlap substantially in sequence. The DNA-binding domains of ETS proteins (ETS domains) are highly conserved structurally yet share limited amino acid homology. To define the mechanistic implications of sequence diversity within the ETS family, we characterized the thermodynamics and kinetics of DNA site recognition by the ETS domains of Ets-1 and PU.1, which represent the extremes in amino acid divergence among ETS proteins. Even though the two ETS domains bind their optimal sites with similar affinities under physiologic conditions, their nature of site recognition differs strikingly in terms of the role of hydration and counter ion release. The data suggest two distinct mechanisms wherein Ets-1 follows a "dry" mechanism that rapidly parses sites through electrostatic interactions and direct protein-DNA contacts, whereas PU.1 utilizes hydration to interrogate sequence-specific sites and form a long-lived complex relative to the Ets-1 counterpart. The kinetic persistence of the high affinity PU.1 · DNA complex may be relevant to an emerging role of PU.1, but not Ets-1, as a pioneer transcription factor in vivo. In addition, PU.1 activity is critical to the development and function of macrophages and lymphocytes, which present osmotically variable environments, and hydration-dependent specificity may represent an important regulatory mechanism in vivo, a hypothesis that finds support in gene expression profiles of primary murine macrophages.


Assuntos
DNA/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Técnicas Biossensoriais , Calorimetria , Clonagem Molecular , DNA/química , Cinética , Dados de Sequência Molecular , Pressão Osmótica , Conformação Proteica , Proteína Proto-Oncogênica c-ets-1/química , Proteínas Proto-Oncogênicas/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Termodinâmica , Transativadores/química , Água/química
2.
bioRxiv ; 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38766031

RESUMO

Hematopoietic multipotent progenitors (MPPs) regulate blood cell production to appropriately meet the biological demands of the human body. Human MPPs remain ill-defined whereas mouse MPPs have been well characterized with distinct immunophenotypes and lineage potencies. Using multiomic single cell analyses and complementary functional assays, we identified new human MPPs and oligopotent progenitor populations within Lin-CD34+CD38dim/lo adult bone marrow with distinct biomolecular and functional properties. These populations were prospectively isolated based on expression of CD69, CLL1, and CD2 in addition to classical markers like CD90 and CD45RA. We show that within the canonical Lin-CD34+CD38dim/loCD90CD45RA-MPP population, there is a CD69+ MPP with long-term engraftment and multilineage differentiation potential, a CLL1+ myeloid-biased MPP, and a CLL1-CD69-erythroid-biased MPP. We also show that the canonical Lin-CD34+CD38dim/loCD90-CD45RA+ LMPP population can be separated into a CD2+ LMPP with lymphoid and myeloid potential, a CD2-LMPP with high lymphoid potential, and a CLL1+ GMP with minimal lymphoid potential. We used these new HSPC profiles to study human and mouse bone marrow cells and observe limited cell type specific homology between humans and mice and cell type specific changes associated with aging. By identifying and functionally characterizing new adult MPP sub-populations, we provide an updated reference and framework for future studies in human hematopoiesis.

3.
Blood Cancer Discov ; : OF1-OF18, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38261864

RESUMO

Rare preleukemic hematopoietic stem cells (pHSC) harboring only the initiating mutations can be detected at the time of acute myeloid leukemia (AML) diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSC). We confirm that IDH1-driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in the complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wild-type HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent the development and relapse of leukemia. SIGNIFICANCE: A high burden of pHSCs is associated with worse overall survival in AML. Using single-cell sequencing, metabolic assessment, and gene-edited human models, we find human pHSCs with IDH1 mutations to be metabolically vulnerable and sensitive to eradication by complex I inhibition. See related commentary by Steensma.

4.
Cancer Cell ; 42(1): 119-134.e12, 2024 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-38194912

RESUMO

The period between "successful" treatment of localized breast cancer and the onset of distant metastasis can last many years, representing an unexploited window to eradicate disseminated disease and prevent metastases. We find that the source of recurrence-disseminated tumor cells (DTCs) -evade endogenous immunity directed against tumor neoantigens. Although DTCs downregulate major histocompatibility complex I, this does not preclude recognition by conventional T cells. Instead, the scarcity of interactions between two relatively rare populations-DTCs and endogenous antigen-specific T cells-underlies DTC persistence. This scarcity is overcome by any one of three immunotherapies that increase the number of tumor-specific T cells: T cell-based vaccination, or adoptive transfer of T cell receptor or chimeric antigen receptor T cells. Each approach achieves robust DTC elimination, motivating discovery of MHC-restricted and -unrestricted DTC antigens that can be targeted with T cell-based immunotherapies to eliminate the reservoir of metastasis-initiating cells in patients.


Assuntos
Neoplasias da Mama , Linfócitos T , Humanos , Feminino , Evasão da Resposta Imune , Transferência Adotiva , Neoplasias da Mama/terapia , Imunoterapia
5.
Blood Cancer Discov ; 2023 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-38091010

RESUMO

Rare preleukemic hematopoietic stem cells (pHSCs) harboring only the initiating mutations can be detected at the time of AML diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene-editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSCs). We confirm that IDH1 driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC Class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wildtype HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent development and relapse of leukemia.

6.
Cancer Cell ; 41(2): 356-372.e10, 2023 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-36706760

RESUMO

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.


Assuntos
Antineoplásicos , Neoplasias , Camundongos , Animais , Humanos , Neutrófilos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Leucotrieno B4/metabolismo , Leucotrieno B4/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
7.
Cancer Discov ; 13(5): 1164-1185, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-36856575

RESUMO

Therapeutic cancer vaccination seeks to elicit activation of tumor-reactive T cells capable of recognizing tumor-associated antigens (TAA) and eradicating malignant cells. Here, we present a cancer vaccination approach utilizing myeloid-lineage reprogramming to directly convert cancer cells into tumor-reprogrammed antigen-presenting cells (TR-APC). Using syngeneic murine leukemia models, we demonstrate that TR-APCs acquire both myeloid phenotype and function, process and present endogenous TAAs, and potently stimulate TAA-specific CD4+ and CD8+ T cells. In vivo TR-APC induction elicits clonal expansion of cancer-specific T cells, establishes cancer-specific immune memory, and ultimately promotes leukemia eradication. We further show that both hematologic cancers and solid tumors, including sarcomas and carcinomas, are amenable to myeloid-lineage reprogramming into TR-APCs. Finally, we demonstrate the clinical applicability of this approach by generating TR-APCs from primary clinical specimens and stimulating autologous patient-derived T cells. Thus, TR-APCs represent a cancer vaccination therapeutic strategy with broad implications for clinical immuno-oncology. SIGNIFICANCE: Despite recent advances, the clinical benefit provided by cancer vaccination remains limited. We present a cancer vaccination approach leveraging myeloid-lineage reprogramming of cancer cells into APCs, which subsequently activate anticancer immunity through presentation of self-derived cancer antigens. Both hematologic and solid malignancies derive significant therapeutic benefit from reprogramming-based immunotherapy. This article is highlighted in the In This Issue feature, p. 1027.


Assuntos
Vacinas Anticâncer , Leucemia , Neoplasias , Animais , Camundongos , Células Apresentadoras de Antígenos , Neoplasias/terapia , Antígenos de Neoplasias , Imunoterapia
8.
Nat Cancer ; 3(8): 976-993, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35817829

RESUMO

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.


Assuntos
Gangliosídeos , Neuroblastoma , Anticorpos Monoclonais , Criança , Humanos , Imunoterapia , Recidiva Local de Neoplasia/induzido quimicamente , Neuroblastoma/tratamento farmacológico
9.
Nat Med ; 28(2): 333-344, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35027753

RESUMO

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.


Assuntos
Neoplasias Ósseas , Antígeno CD47 , Animais , Linhagem Celular Tumoral , Humanos , Imunoterapia , Camundongos , Recidiva Local de Neoplasia , Fagocitose , Microambiente Tumoral
10.
Front Immunol ; 12: 624284, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717133

RESUMO

Natural killer (NK) cells are effector cells of the innate immune system involved in defense against virus-infected and transformed cells. The effector function of NK cells is linked to their ability to migrate to sites of inflammation or damage. Therefore, understanding the factors regulating NK cell migration is of substantial interest. Here, we show that in the absence of aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, NK cells have reduced capacity to migrate and infiltrate tumors in vivo. Analysis of differentially expressed genes revealed that ankyrin repeat and SOCS Box containing 2 (Asb2) expression was dramatically decreased in Ahr-/- NK cells and that AhR ligands modulated its expression. Further, AhR directly regulated the promoter region of the Asb2 gene. Similar to what was observed with murine Ahr-/- NK cells, ASB2 knockdown inhibited the migration of human NK cells. Activation of AHR by its agonist FICZ induced ASB2-dependent filamin A degradation in NK cells; conversely, knockdown of endogenous ASB2 inhibited filamin A degradation. Reduction of filamin A increased the migration of primary NK cells and restored the invasion capacity of AHR-deficient NK cells. Our study introduces AHR as a new regulator of NK cell migration, through an AHR-ASB2-filamin A axis and provides insight into a potential therapeutic target for NK cell-based immunotherapies.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular , Filaminas/metabolismo , Células Matadoras Naturais/enzimologia , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Tumoral , Humanos , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/enzimologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores de Hidrocarboneto Arílico/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Proteínas Supressoras da Sinalização de Citocina/genética , Microambiente Tumoral , Ubiquitinação
11.
Matrix Biol ; 96: 69-86, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33290836

RESUMO

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.


Assuntos
Células Dendríticas/citologia , Rejeição de Enxerto/prevenção & controle , Ácido Hialurônico/biossíntese , Himecromona/administração & dosagem , Linfócitos T Reguladores/citologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto/imunologia , Transplante de Coração/efeitos adversos , Humanos , Himecromona/farmacologia , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Camundongos , Transplante de Pâncreas/efeitos adversos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Transplante Homólogo
12.
Clin Cancer Res ; 25(8): 2560-2574, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30655315

RESUMO

PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. RESULTS: B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos B7/imunologia , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/metabolismo , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos B7/antagonistas & inibidores , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Imunoterapia Adotiva/métodos , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
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