The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions
. It has been particularly challenging to study dynamic HSC behaviour, given that the ...visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow
. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.
Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. ...Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.
CAR-T therapy is a promising, novel treatment modality for B-cell malignancies and yet many patients relapse through a variety of means, including loss of CAR-T cells and antigen escape. To ...investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a modular guide RNA library. We identified IFNγR/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo; intriguingly, loss of this pathway yielded the opposite effect in vitro (sensitized leukemia to CAR-T cells). Transcriptional characterization of this model demonstrated upregulation of these pathways in tumors relapsed after CAR-T treatment, and functional studies showed a surprising role for natural killer (NK) cells in engaging this resistance program. Finally, examination of data from B-ALL patients treated with CAR-T revealed an association between poor outcomes and increased expression of JAK/STAT and MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with the in vivo tumor microenvironment, including NK cells, induces expression of an adaptive, therapy-induced, T-cell resistance program in tumor cells.
Whole chromosome losses resulting in near-haploid karyotypes are found in a rare subgroup of treatment-refractory acute lymphoblastic leukemia. To systematically dissect the unique physiology and ...uncover susceptibilities that can be exploited in near-haploid leukemia, we leveraged single-cell RNA-Seq and computational inference of cell cycle stages to pinpoint key differences between near-haploid and diploid leukemia cells. Combining cell cycle stage-specific differential expression with gene essentiality scores from a genome-wide CRISPR-Cas9-mediated knockout screen, we identified the homologous recombination pathway component RAD51B as an essential gene in near-haploid leukemia. DNA damage analyses revealed significantly increased sensitivity of RAD51-mediated repair to RAD51B loss in the G2/M stage of near-haploid cells, suggesting a unique role of RAD51B in the homologous recombination pathway. Elevated G2/M and G1/S checkpoint signaling was part of a RAD51B signature expression program in response to chemotherapy in a xenograft model of human near-haploid B-ALL, and RAD51B and its associated programs were overexpressed in a large panel of near-haploid B-ALL patients. These data highlight a unique genetic dependency on DNA repair machinery in near-haploid leukemia and demarcate RAD51B as a promising candidate for targeted therapy in this treatment-resistant disease.
Stem cell (SC) activity fluctuates throughout an organism's lifetime to maintain homeostatic conditions in all tissues. As animals develop and age, their organs must remodel and regenerate themselves ...in response to environmental and physiological demands. Recently, the highly conserved Hippo signaling pathway, discovered in Drosophila melanogaster , has been implicated as a key regulator of organ size control across species. Deregulation is associated with substantial overgrowth phenotypes and eventual onset of cancer in various tissues. Importantly, emerging evidence suggests that the Hippo pathway can modulate its effects on tissue size by the direct regulation of SC proliferation and maintenance. These findings provide an attractive model for how this pathway might communicate physiological needs for growth to tissue-specific SC pools. In this review, we summarize the current and emerging data linking Hippo signaling to SC function.
The tumor microenvironment has recently been shown to play decisive roles in chemotherapeutic response. In this issue of Cell, Yu et al. add to these findings by identifying the bacterium ...Fusobacterium nucleatum as a previously unrecognized chemoresistance mediator in colorectal cancer, thereby establishing the microbiota as a potential therapeutic target.
The tumor microenvironment has recently been shown to play decisive roles in chemotherapeutic response. In this issue of Cell, Yu et al. add to these findings by identifying the bacterium Fusobacterium nucleatum as a previously unrecognized chemoresistance mediator in colorectal cancer, thereby establishing the microbiota as a potential therapeutic target.
Targeting MTHFD2 in acute myeloid leukemia Pikman, Yana; Puissant, Alexandre; Alexe, Gabriela ...
The Journal of experimental medicine,
06/2016, Letnik:
213, Številka:
7
Journal Article
Recenzirano
Odprti dostop
Drugs targeting metabolism have formed the backbone of therapy for some cancers. We sought to identify new such targets in acute myeloid leukemia (AML). The one-carbon folate pathway, specifically ...methylenetetrahydrofolate dehydrogenase-cyclohydrolase 2 (MTHFD2), emerged as a top candidate in our analyses. MTHFD2 is the most differentially expressed metabolic enzyme in cancer versus normal cells. Knockdown of MTHFD2 in AML cells decreased growth, induced differentiation, and impaired colony formation in primary AML blasts. In human xenograft and MLL-AF9 mouse leukemia models, MTHFD2 suppression decreased leukemia burden and prolonged survival. Based upon primary patient AML data and functional genomic screening, we determined that FLT3-ITD is a biomarker of response to MTHFD2 suppression. Mechanistically, MYC regulates the expression of MTHFD2, and MTHFD2 knockdown suppresses the TCA cycle. This study supports the therapeutic targeting of MTHFD2 in AML.
Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe ...that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.
Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.
Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.
It is currently thought that life-long blood cell production is driven by the action of a small number of multipotent haematopoietic stem cells. Evidence supporting this view has been largely ...acquired through the use of functional assays involving transplantation. However, whether these mechanisms also govern native non-transplant haematopoiesis is entirely unclear. Here we have established a novel experimental model in mice where cells can be uniquely and genetically labelled in situ to address this question. Using this approach, we have performed longitudinal analyses of clonal dynamics in adult mice that reveal unprecedented features of native haematopoiesis. In contrast to what occurs following transplantation, steady-state blood production is maintained by the successive recruitment of thousands of clones, each with a minimal contribution to mature progeny. Our results demonstrate that a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells, are the main drivers of steady-state haematopoiesis during most of adulthood. Our results also have implications for understanding the cellular origin of haematopoietic disease.
Fungi can thrive in strawberries, mainly Botrytis cinerea, which causes relevant fruit losses. Moreover, thermal preservation results in phytonutrient losses. This study evaluated the effects of high ...hydrostatic pressure (HHP, 0, 400, and 600 MPa/5 min) processing with potassium sorbate (PS) or avocado seed acetogenins (ASA) on the nutritional, microbiological, and physicochemical properties of strawberry puree inoculated with Botrytis cinerea. Sole inoculation reduced L-ascorbic acid (L-AA, 84%) and total ascorbic acid (TAA, 38%). Instrumental color Hue angles were reduced from 35.0 to 29.4°, likewise the contents of pelargonidin−3-glucoside (25%) and ellagic acid (38%). Both HHP-PS treated samples contained no viable conidia and high TAA; the no-preservative and HHP-ASA (661 µg/mL, 400 MPa/5 min) purees contained high TAA but had viable conidia, which can induce further phytonutrient losses. HHP and antifungal additives maintained ellagic acid, increased pelargonidin−3-glucoside contents, and inactivated conidia, strengthening the value of combined non-thermal technologies for the preservation of strawberry puree
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