Lifelong blood cell production is dependent on rare hematopoietic stem cells (HSCs) to perpetually replenish mature cells via a series of lineage-restricted intermediates. Investigating the molecular ...state of HSCs is contingent on the ability to purify HSCs away from transiently engrafting cells. We demonstrated that human HSCs remain infrequent, using current purification strategies based on Thy1 (CD90) expression. By tracking the expression of several adhesion molecules in HSC-enriched subsets, we revealed CD49f as a specific HSC marker. Single CD49f + cells were highly efficient in generating long-term multilineage grafts, and the loss of CD49f expression identified transiently engrafting multipotent progenitors (MPPs). The demarcation of human HSCs and MPPs will enable the investigation of the molecular determinants of HSCs, with a goal of developing stem cell—based therapeutics.
Chronic lymphocytic leukemia (CLL) remains incurable despite advances in therapy. In this study, we characterize the effect of nicotinamide phosphoribosyltransferase (NAMPT) inhibition by FK866 in ...primary CLL cells from patients with various clinical prognostic markers.
CLL cells were treated with FK866 to assess viability by Annexin V/PI staining. Functional analysis of FK866 included time- and concentration-dependent evaluation of cellular NAD, ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and apoptotic signaling. Chemosensitization potential by FK866 to fludarabine was also assessed. Prognostic markers were correlated with drug response.
FK866 induced CLL cell death by depleting cellular NAD content by day 1, followed by a drop in ATP on day 2. We observed loss of MMP, ROS increase, and induction of apoptotic signaling at day 3. On-target activity of FK866 was confirmed by NAD-mediated rescue of NAD and ATP loss, apoptotic signaling, and viability. The response to FK866 was independent of most prognostic markers. Higher doses were required with short lymphocyte doubling time and positive CD38 status, whereas CLL cells resistant to fludarabine in vitro and from patients with del17p13.1 were equally sensitive to FK866. FK866 did not upregulate the p53-target p21, nor did the p53 activator Nutlin improve FK866-mediated cell death. Furthermore, fludarabine and FK866 were synergistic at clinically relevant concentrations.
NAMPT inhibition by FK866 may be a potential treatment for CLL, including patients with del17p13.1 or other high-risk features. FK866 may complement standard agents to enhance their efficacy and/or allow dose reduction for improved tolerability.
Although tumor surveillance by T and B lymphocytes is well studied, the role of innate immune cells, in particular macrophages, is less clear. Moreover, the existence of subclonal genetic and ...functional diversity in some human cancers such as leukemia underscores the importance of defining tumor surveillance mechanisms that effectively target the disease-sustaining cancer stem cells in addition to bulk cells. In this study, we report that leukemia stem cell function in xenotransplant models of acute myeloid leukemia (AML) depends on SIRPα-mediated inhibition of macrophages through engagement with its ligand CD47. We generated mice expressing SIRPα variants with differential ability to bind human CD47 and demonstrated that macrophage-mediated phagocytosis and clearance of AML stem cells depend on absent SIRPα signaling. We obtained independent confirmation of the genetic restriction observed in our mouse models by using SIRPα-Fc fusion protein to disrupt SIRPα-CD47 engagement. Treatment with SIRPα-Fc enhanced phagocytosis of AML cells by both mouse and human macrophages and impaired leukemic engraftment in mice. Importantly, SIRPα-Fc treatment did not significantly enhance phagocytosis of normal hematopoietic targets. These findings support the development of therapeutics that antagonize SIRPα signaling to enhance macrophage-mediated elimination of AML.
Kelly et al. (Brevia, 20 July 2007, p. 337) questioned xenotransplant experiments supporting the cancer stem cell (CSC) hypothesis because they found a high frequency of leukemia-initiating cells ...(L-IC) in some transgenic mouse models. However, the CSC hypothesis depends on prospective purification of cells with tumor-initiating capacity, irrespective of frequency. Moreover, we found similar L-IC frequencies in genetically comparable leukemias using syngeneic or xenogeneic models.
Successful therapy for many inherited disorders could be improved if the intervention were initiated early. This is especially true for lysosomal storage disorders. Earlier intervention may allow ...metabolic correction to occur before lipid buildup has irreversible consequences and/or before the immune system mounts limiting responses. We have been developing gene therapy to treat lysosomal storage disorders, especially Fabry disease. We describe studies directed toward metabolic correction in neonatal animals mediated by recombinant lentiviral vectors. To develop this method, we first injected a marking lentiviral vector that engineers expression of luciferase into the temporal vein of recipient neonatal animals. The use of a cooled charged-coupled device camera allowed us to track transgene expression over time in live animals. We observed intense luciferase expression in many tissues, including the brain, that did not diminish over 24 weeks. Next, we injected neonatal Fabry mice a single time with a therapeutic lentiviral vector engineered to express human α-galactosidase A. The injection procedure was well tolerated. We observed increased plasma levels of α-galactosidase A activity starting at our first plasma collection point (4 weeks). Levels of α-galactosidase A activity were found to be significantly elevated in many tissues even after 28 weeks. No immune response was observed against the corrective transgene product. Increased levels of enzyme activity also led to significant reduction of globotriaosylceramide in the liver, spleen, and heart. This approach provides a method to treat lysosomal storage disorders and other disorders before destructive manifestations occur.
Xenograft studies indicate that some solid tumors and leukemias are organized as cellular hierarchies sustained by cancer stem cells (CSCs). Despite the promise of the CSC model, its relevance in ...humans remains uncertain. Here we show that acute myeloid leukemia (AML) follows a CSC model on the basis of sorting multiple populations from each of 16 primary human AML samples and identifying which contain leukemia stem cells (LSCs) using a sensitive xenograft assay. Analysis of gene expression from all functionally validated populations yielded an LSC-specific signature. Similarly, a hematopoietic stem cell (HSC) gene signature was established. Bioinformatic analysis identified a core transcriptional program shared by LSCs and HSCs, revealing the molecular machinery underlying 'stemness' properties. Both stem cell programs were highly significant independent predictors of patient survival and were found in existing prognostic signatures. Thus, determinants of stemness influence the clinical outcome of AML, establishing that LSCs are clinically relevant and not artifacts of xenotransplantation.
Many tumours are composed of genetically diverse cells; however, little is known about how diversity evolves or the impact that diversity has on functional properties. Here, using xenografting and ...DNA copy number alteration (CNA) profiling of human BCR-ABL1 lymphoblastic leukaemia, we demonstrate that genetic diversity occurs in functionally defined leukaemia-initiating cells and that many diagnostic patient samples contain multiple genetically distinct leukaemia-initiating cell subclones. Reconstructing the subclonal genetic ancestry of several samples by CNA profiling demonstrated a branching multi-clonal evolution model of leukaemogenesis, rather than linear succession. For some patient samples, the predominant diagnostic clone repopulated xenografts, whereas in others it was outcompeted by minor subclones. Reconstitution with the predominant diagnosis clone was associated with more aggressive growth properties in xenografts, deletion of CDKN2A and CDKN2B, and a trend towards poorer patient outcome. Our findings link clonal diversity with leukaemia-initiating-cell function and underscore the importance of developing therapies that eradicate all intratumoral subclones.
Hereditary xerocytosis (HX) is a rare form of hemolytic anemia with autosomal dominant inheritance in which iron loading is a prominent feature. The mutated gene causing HX has been identified as ...FAM38A, which codes for the PIEZO1 protein, a mechanosensitive ion channel 1.The phenotype and genotype of HX has been characterized in a large Canadian family with members spanning three generations and seven decades2. Affected family members demonstrate fully-compensated hemolytic anemia (average reticulocyte count 9.9%, hemoglobin 135g/L) and their red cells exhibit decreased levels of osmotic fragility. Despite elevated reticulocyte counts and elevated unconjugated bilirubin levels, serum lactate dehydrogenase levels are normal, suggesting that little if any of the erythropoiesis is ‘ineffective'. Affected family members accumulate iron with age, with average ferritin levels for adults of 478 μg/L. The mechanism behind the iron loading in HX is not known. It is now recognized that in these forms of anemia, hepcidin levels are inappropriately low for the degree of iron store, implying the presence of a mediator produced by the hematopoietic progenitors that acts on the liver to suppress hepcidin production. One pathway that appears important in regulating hepcidin synthesis is the bone morphogenetic protein (BMP)-SMAD signaling cascade. The importance of BMP6 is evident from studies using gene knockout mice. Likewise, liver-targeted knockdown of SMAD4 impairs production of hepcidin and resulted in iron overload in the mice 3. BMP6 may act in an autocrine fashion, and its secretion by hepatocytes is upregulated in the presence of elevated iron levels.
Another proposed pathway for hepcidin regulation – speculated to be involved in the erythropoietic regulation of iron – involves another cytokine also from the TGF-β superfamily. Growth differentiation factor 15 (GDF15) is expressed in high levels in placenta tissue and in smaller quantities in the liver, lungs and kidneys. It is also secreted by erythroblasts, at least in culture. Plasma levels of GDF15 are greatly increased in thalassemia and correlate with markers of erythroid mass such as the soluble transferrin receptor. Serum from thalassemic patients suppresses hepcidin mRNA expression by cultured hepatocytes, an effect partially recapitulated by recombinant GDF15, suggesting that the cytokine requires a co-factor for full hepcidin inhibition 4.
We evaluated the level of hepcidin, EPOand ferritin along with GDF15 in 29 affected individuals from a single kindred with HX, and a similar number of age matched unaffected family members to explore the putative erythropoietic regulator of iron absorption in a homogeneous genetic context. We find that ferritin level positively predicts hepcidin level (p<.001) and age negatively predicts hepcidin level. After adjustment for age and ferritin, GDF15 does negatively predict hepcidin level(p=.046 in the final fully-adjusted model). However, in a regression model adjusting for ferritin, age and GDF15, xerocytosis still predicts hepcidin level, with lower hepcidin among the affected family members (p<.001).
These results suggest that GDF15 may be one mediator of hepcidin suppression and iron loading in hereditary xerocytosis. However, its effect is insufficient to explain the full iron-loading propensity.'
1. Zarychanski, R., et al., Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis. Blood, 2012. 120(9): p. 1908-15.
2. Houston, B.L., et al., Refinement of the hereditary xerocytosis locus on chromosome 16q in a large Canadian kindred. Blood Cells Mol Dis, 2011. 47(4): p. 226-31.
3. Corradini, E., et al., Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice. Hepatology, 2011. 54(1): p. 273-84.
4. Tanno, T., et al., High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med, 2007. 13(9): p. 1096-101.
No relevant conflicts of interest to declare.
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