ABSTRACT
REV-ERB is a member of the nuclear receptor superfamily of transcription factors involved in the regulation of many physiological processes, from circadian rhythm, to immune function and metabolism. Accordingly, REV-ERB has been considered as a promising, but difficult drug target for the treatment of numerous diseases. Here, we concisely review current understanding of the function of REV-ERB, modulation by endogenous factors and synthetic ligands, and the involvement of REV-ERB in select human diseases. Particular focus is placed on the medicinal chemistry of synthetic REV-ERB ligands, which demonstrates the need for higher quality ligands to aid in robust validation of this exciting target.
Subject(s)
Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Circadian Rhythm/physiology , Heme/chemistry , Humans , Immune System Diseases/metabolism , Immune System Diseases/pathology , Ligands , Nuclear Receptor Subfamily 1, Group D, Member 1/agonists , Nuclear Receptor Subfamily 1, Group D, Member 1/antagonists & inhibitors , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Repressor Proteins/agonists , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The majority of acute myeloid leukemia (AML) patients have a poor response to conventional chemotherapy. The survival of chemoresistant cells is thought to depend on leukemia-bone marrow (BM) microenvironment interactions, which are not well understood. The CXCL12/CXCR4 axis has been proposed to support AML growth but was not studied at the single AML cell level. We recently showed that T-cell acute lymphoblastic leukemia (T-ALL) cells are highly motile in the BM; however, the characteristics of AML cell migration within the BM remain undefined. Here, we characterize the in vivo migratory behavior of AML cells and their response to chemotherapy and CXCR4 antagonism, using high-resolution 2-photon and confocal intravital microscopy of mouse calvarium BM and the well-established MLL-AF9-driven AML mouse model. We used the Notch1-driven T-ALL model as a benchmark comparison and AMD3100 for CXCR4 antagonism experiments. We show that AML cells are migratory, and in contrast with T-ALL, chemoresistant AML cells become less motile. Moreover, and in contrast with T-ALL, the in vivo exploratory behavior of expanding and chemoresistant AML cells is unaffected by AMD3100. These results expand our understanding of AML cells-BM microenvironment interactions, highlighting unique traits of leukemia of different lineages.
Subject(s)
Cell Movement , Chemokine CXCL12/metabolism , Heterocyclic Compounds/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Receptors, CXCR4/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Benzylamines , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Line, Tumor , Cyclams , Drug Resistance, Neoplasm/drug effects , Heterocyclic Compounds/metabolism , Intravital Microscopy , Leukemia, Myeloid, Acute/metabolism , Mice , Microscopy, Confocal , Microscopy, Fluorescence, Multiphoton , Tumor MicroenvironmentABSTRACT
Natural killer (NK) cells are lymphocytes that play an important role at clearing virally infected or cancer cells. Their potential and role in cancer immunotherapy have generated great interest, given the promising results of NK cell adoptive transfer clinical trials. The remaining challenge to bring emerging NK cell immunotherapies to the clinic is to enhance the production of large numbers of functionally competent NK cells ex vivo. Here, we describe two in vitro NK cell development assays using hematopoietic progenitor cells (HPCs), one for human NK cells and one for mouse NK cells. These protocols describe two robust methods that can be utilized for investigation of NK cell development and function.
Subject(s)
Hematopoietic Stem Cells , Killer Cells, Natural , Adoptive Transfer , Humans , ImmunotherapyABSTRACT
Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.
Subject(s)
Hematopoiesis/physiology , Hematopoietic Stem Cells/physiology , Malaria/physiopathology , Stem Cell Niche/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Bone Marrow Cells/physiology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Endothelial Cells/physiology , Gene Expression Profiling/methods , Hematopoiesis/drug effects , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Malaria/parasitology , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/physiology , Mice, Inbred C57BL , Mice, Knockout , Osteoblasts/cytology , Osteoblasts/metabolism , Osteoblasts/physiology , Parathyroid Hormone/pharmacology , Plasmodium berghei/physiology , Reactive Oxygen Species/metabolism , Stem Cell Niche/geneticsABSTRACT
Understanding cellular metabolism holds immense potential for developing new classes of therapeutics that target metabolic pathways in cancer. Metabolic pathways are altered in bulk neoplastic cells in comparison to normal tissues. However, carcinoma cells within tumors are heterogeneous, and tumor-initiating cells (TICs) are important therapeutic targets that have remained metabolically uncharacterized. To understand their metabolic alterations, we performed metabolomics and metabolite tracing analyses, which revealed that TICs have highly elevated methionine cycle activity and transmethylation rates that are driven by MAT2A. High methionine cycle activity causes methionine consumption to far outstrip its regeneration, leading to addiction to exogenous methionine. Pharmacological inhibition of the methionine cycle, even transiently, is sufficient to cripple the tumor-initiating capability of these cells. Methionine cycle flux specifically influences the epigenetic state of cancer cells and drives tumor initiation. Methionine cycle enzymes are also enriched in other tumor types, and MAT2A expression impinges upon the sensitivity of certain cancer cells to therapeutic inhibition.
Subject(s)
Methionine/metabolism , Neoplastic Stem Cells/metabolism , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Differentiation , Cell Line, Tumor , Female , Gene Knockdown Techniques , Glycine Dehydrogenase (Decarboxylating)/antagonists & inhibitors , Glycine Dehydrogenase (Decarboxylating)/genetics , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Metabolic Networks and Pathways , Metabolomics , Methionine Adenosyltransferase/antagonists & inhibitors , Methionine Adenosyltransferase/metabolism , Mice , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , S-Adenosylmethionine/metabolismABSTRACT
In the version of this article originally published, there is an error in Fig. 5a. Originally, 'MAT2A' appeared between 'Methionine' and 'Homocysteine'. 'MAT2A' should have been 'MTR'. The error has been corrected in the PDF and HTML versions of this article.
ABSTRACT
Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.
Subject(s)
Hematopoietic Stem Cells/pathology , Leukemia, Myeloid, Acute/pathology , Stem Cell Niche , Animals , Bone Marrow/blood supply , Bone Marrow/pathology , Cell Count , Hematopoiesis , Humans , Intravital Microscopy , Mice, Inbred C57BL , Spleen/pathology , Stromal Cells/pathology , Time Factors , Tumor MicroenvironmentABSTRACT
Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.
Subject(s)
Cell Shape , Cyclin A2/metabolism , Erythrocytes/cytology , Erythrocytes/metabolism , Animals , Bone Marrow Cells/metabolism , Bromodeoxyuridine/metabolism , Cell Count , Cell Cycle , Cell Nucleus/metabolism , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p27/metabolism , DNA Damage , Erythroid Cells/cytology , Erythroid Cells/metabolism , Erythropoiesis , Green Fluorescent Proteins/metabolism , Integrases/metabolism , Mice, Inbred C57BL , Phenotype , Promoter Regions, Genetic/genetics , Real-Time Polymerase Chain Reaction , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolismABSTRACT
Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.