On-target effect of FK866, a nicotinamide phosphoribosyl transferase inhibitor, by apoptosis-mediated death in chronic lymphocytic leukemia cells.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Disruption of SIRPα signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts.

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.

Stem cell gene expression programs influence clinical outcome in human leukemia.

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.

Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.

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.

Evolution of human BCR-ABL1 lymphoblastic leukaemia-initiating cells.

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.

Comment on "Tumor growth need not be driven by rare cancer stem cells".

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.

Multiple reduced-intensity conditioning regimens facilitate correction of Fabry mice after transplantation of transduced cells.

Hematopoietic cell transplantation can impact lysosomal storage disorders (LSDs) and will be enhanced by gene therapy. Transduced cells in LSDs often secrete the therapeutic hydrolase, which can be used by bystander cells. However, toxicity associated with myeloablative transplant preparative regimens limits many applications of this approach in gene therapy. We hypothesized that reduced-intensity (RI) conditioning regimens would allow stable engraftment of therapeutically transduced cells and allow correction of Fabry disease. We transplanted transduced cells into Fabry mice receiving eight different clinically relevant chemotherapy- and/or radiotherapy-based RI conditioning regimens generating modest and transient lymphoid/myeloid cell depletion. Two comprehensive transplantation Protocols were performed. Firstly, transplantation of 0.38 x 10(6) gene-modified stem/progenitor cells was nominally effective; none of the RI regimens led to stable alpha-galactosidase A (alpha-gal A) correction. Secondly, transduced cells were preselected for functional transgene expression and transplanted at a higher dose (0.72 x 10(6) cells). Each RI regimen yielded engraftment of functional transgene-positive cells through 180 days along with increased plasma alpha-gal A activity. Importantly, the RI regimens mediated broad organ enzyme correction and were not associated with immune responses against alpha-gal A. RI conditioning thus has an important role in gene therapy for LSDs; a variety of regimens can be effective in this context.

Bioluminescent imaging of a marking transgene and correction of Fabry mice by neonatal injection of recombinant lentiviral vectors.

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 alpha-galactosidase A. The injection procedure was well tolerated. We observed increased plasma levels of alpha-galactosidase A activity starting at our first plasma collection point (4 weeks). Levels of alpha-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.