RESUMO
Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.
Assuntos
Medula Óssea , Síndromes Mielodisplásicas , Humanos , Camundongos , Animais , Medula Óssea/patologia , Síndromes Mielodisplásicas/genética , Células-Tronco Hematopoéticas/metabolismo , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Progressão da DoençaRESUMO
Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the ß-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leucemia Mieloide Aguda , Animais , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Proteína Meis1/genética , Proteína Meis1/metabolismo , Transdução de Sinais , Microambiente Tumoral/genéticaRESUMO
Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.
Assuntos
Vesículas Extracelulares , Células-Tronco Hematopoéticas , Medula Óssea , Células da Medula Óssea , HematopoeseRESUMO
Sharing reagents is of self-evident value in life science research, however, primary cell populations often do not cryopreserve well or can require extensive preparation by collaborators, making shipping difficult. Here we report an evaluation of different conditions for the storage shipment of mouse bone marrow (BM) cells that would best preserve the number, viability, and frequency of different hematopoietic lineages, as well as functionality of progenitor populations. Bones were either crushed to release BM cells or stored intact in one of three media: Phosphate buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS), Plasmalyte, or RPMI at 4°C. Cell numbers, viability, phenotype, and functionality were assessed 16 hours and 40 hours later and compared to freshly prepared samples. Whereas BM cells stored in suspension for 16 hours and BM cells kept in bone for 40 hours suffered major losses in cell number, hematopoietic lineages that were kept in the bone for 16 hours had only minor differences compared to fresh cells. With no significant differences among the different media used, intact long bones stored in media, Plasmalyte, or PBS 2% FBS for up to 16 hours provided a reasonable means of preserving bone marrow cell populations.
Assuntos
Medula Óssea , Meios de Cultura , Células-Tronco Hematopoéticas , Manejo de Espécimes/métodos , Preservação de Tecido/métodos , Animais , Bovinos , Sobrevivência Celular , CamundongosRESUMO
While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.