RESUMEN
Growth factor independence 1 (GFI1) is a transcriptional repressor protein that plays an essential role in the differentiation of myeloid and lymphoid progenitors. We and other groups have shown that GFI1 has a dose-dependent role in the initiation, progression, and prognosis of acute myeloid leukaemia (AML) patients by inducing epigenetic changes. We now demonstrate a novel role for dose-dependent GFI1 expression in regulating metabolism in haematopoietic progenitor and leukaemic cells. Using in-vitro and ex-vivo murine models of MLL::AF9-induced human AML and extra-cellular flux assays, we now demonstrate that a lower GFI1 expression enhances oxidative phosphorylation rate via upregulation of the FOXO1- MYC axis. Our findings underscore the significance of therapeutic exploitation in GFI1-low-expressing leukaemia cells by targeting oxidative phosphorylation and glutamine metabolism.
Asunto(s)
Leucemia Mieloide Aguda , Factores de Transcripción , Humanos , Ratones , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Diferenciación Celular , Pronóstico , Epigénesis Genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
WT1 is a true chameleon, both acting as an oncogene and tumor suppressor. As its exact role in leukemogenesis is still ambiguous, research with model systems representing natural conditions surrounding the genetic alterations in WT1 is necessary. In a cohort of 59 leukemia/lymphoma cell lines, we showed aberrant expression for WT1 mRNA, which does not always translate into protein levels. We also analyzed the expression pattern of the four major WT1 protein isoforms in the cell lines and primary AML blasts with/without WT1 mutations and demonstrated that the presence of mutations does not influence these patterns. By introduction of key intronic and exonic sequences of WT1 into a lentiviral expression vector, we developed a unique tool that can stably overexpress the four WT1 isoforms at their naturally occurring tissue-dependent ratio. To develop better cellular model systems for WT1, we sequenced large parts of its gene locus and also other important myeloid risk factor genes and revealed previously unknown alterations. Functionally, inhibition of the nonsense-mediated mRNA decay machinery revealed that under natural conditions, the mutated WT1 alleles go through a robust degradation. These results offer new insights and model systems regarding the characteristics of WT1 in leukemia and lymphoma.
RESUMEN
The great clinical success of chimeric antigen receptor (CAR) T cells has unlocked new levels of immunotherapy for hematological malignancies. Genetically modifying natural killer (NK) cells as alternative CAR immune effector cells is also highly promising, as NK cells can be transplanted across HLA barriers without causing graft-versus-host disease. Therefore, off-the-shelf usage of CAR NK cell products might allow to widely expand the clinical indications and to limit the costs of treatment per patient. However, in contrast to T cells, manufacturing suitable CAR NK cell products is challenging, as standard techniques for genetically engineering NK cells are still being defined. In this study, we have established optimal lentiviral transduction of primary human NK cells by systematically testing different internal promoters for lentiviral CAR vectors and comparing lentiviral pseudotypes and viral entry enhancers. We have additionally modified CAR constructs recognizing standard target antigens for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) therapy-CD19, CD33, and CD123-to harbor a CD34-derived hinge region that allows efficient detection of transduced NK cells in vitro and in vivo and also facilitates CD34 microbead-assisted selection of CAR NK cell products to >95% purity for potential clinical usage. Importantly, as most leukemic blasts are a priori immunogenic for activated primary human NK cells, we developed an in vitro system that blocks the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46, and NKp80 on these cells and therefore allows systematic testing of the specific killing of CAR NK cells against ALL and AML cell lines and primary AML blasts. Finally, we evaluated in an ALL xenotransplantation model in NOD/SCID-gamma (NSG) mice whether human CD19 CAR NK cells directed against the CD19+ blasts are relying on soluble or membrane-bound IL15 production for NK cell persistence and also in vivo leukemia control. Hence, our study provides important insights into the generation of pure and highly active allogeneic CAR NK cells, thereby advancing adoptive cellular immunotherapy with CAR NK cells for human malignancies further.
Asunto(s)
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Línea Celular Tumoral , Ingeniería Genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/terapia , Humanos , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMEN
Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.
Asunto(s)
Leucemia Mieloide Aguda/etiología , Síndromes Mielodisplásicos/etiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras/fisiología , Animales , Epigenómica , Proteína Forkhead Box O1/metabolismo , Eliminación de Gen , Heterocigoto , Homocigoto , Humanos , Ratones , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit+ cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción/metabolismo , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones Endogámicos C57BL , Ensayo de Tumor de Célula Madre , Regulación hacia ArribaRESUMEN
The growth of malignant cells is not only driven by cell-intrinsic factors, but also by the surrounding stroma. Monocytes/Macrophages play an important role in the onset and progression of solid cancers. However, little is known about their role in the development of acute myeloid leukemia, a malignant disease characterized by an aberrant development of the myeloid compartment of the hematopoietic system. It is also unclear which factors are responsible for changing the status of macrophage polarization, thus supporting the growth of malignant cells instead of inhibiting it. We report herein that acute myeloid leukemia leads to the invasion of acute myeloid leukemia-associated macrophages into the bone marrow and spleen of leukemic patients and mice. In different leukemic mouse models, these macrophages support the in vitro expansion of acute myeloid leukemia cell lines better than macrophages from non-leukemic mice. The grade of macrophage infiltration correlates in vivo with the survival of the mice. We found that the transcriptional repressor Growth factor independence 1 is crucial in the process of macrophage polarization, since its absence impedes macrophage polarization towards a leukemia supporting state and favors an anti-tumor state both in vitro and in vivo These results not only suggest that acute myeloid leukemia-associated macrophages play an important role in the progression of acute myeloid leukemia, but also implicate Growth factor independence 1 as a pivotal factor in macrophage polarization. These data may provide new insights and opportunities for novel therapies for acute myeloid leukemia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Leucemia Mieloide Aguda/patología , Macrófagos/patología , Factores de Transcripción/fisiología , Animales , Médula Ósea/patología , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Ratones Transgénicos , Bazo/patologíaRESUMEN
Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted.