Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells.

Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ~ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert(-/-) HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert(-/-) HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation.

Ontogeny stage-independent and high-level clonal expansion in vitro of mouse hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor.

Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin(-)Sca-1(+)c-kit(+) cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd-transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.

Loss-of-function Additional sex combs like 1 mutations disrupt hematopoiesis but do not cause severe myelodysplasia or leukemia.

The Additional sex combs like 1 (Asxl1) gene is 1 of 3 mammalian homologs of the Additional sex combs (Asx) gene of Drosophila. Asx is unusual because it is required to maintain both activation and silencing of Hox genes in flies and mice. Asxl proteins are characterized by an amino terminal homology domain, by interaction domains for nuclear receptors, and by a C-terminal plant homeodomain protein-protein interaction domain. A recent study of patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) revealed a high incidence of truncation mutations that would delete the PHD domain of ASXL1. Here, we show that Asxl1 is expressed in all hematopoietic cell fractions analyzed. Asxl1 knockout mice exhibit defects in frequency of differentiation of lymphoid and myeloid progenitors, but not in multipotent progenitors. We do not detect effects on hematopoietic stem cells, or in peripheral blood. Notably, we do not detect severe myelodysplastic phenotypes or leukemia in this loss-of-function model. We conclude that Asxl1 is needed for normal hematopoiesis. The mild phenotypes observed may be because other Asxl genes have redundant function with Asxl1, or alternatively, MDS or oncogenic phenotypes may result from gain-of-function Asxl mutations caused by genomic amplification, gene fusion, or truncation of Asxl1.

Modeling the functional heterogeneity of leukemia stem cells: role of STAT5 in leukemia stem cell self-renewal.

Although the cancer stem cell (CSC) concept implies that CSCs are rare, recent reports suggest that CSCs may be frequent in some cancers. We hypothesized that the proportion of leukemia stem cells would vary as a function of the number of dysregulated pathways. Constitutive expression of MN1 served as a 1-oncogene model, and coexpression of MN1 and a HOX gene served as a 2-oncogene model. Leukemia-initiating cell (LIC) number and in vitro expansion potential of LICs were functionally assessed by limiting dilution analyses. LIC expansion potential was 132-fold increased in the 2- compared with the 1-oncogene model, although phenotypically, both leukemias were similar. The 2-oncogene model was characterized by granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity and activated STAT/ERK signaling. GM-CSF hypersensitivity of the 2-oncogene model (MN1/HOXA9) was lost in Stat5b(-/-) cells, and the LIC expansion potential was reduced by 86- and 28-fold in Stat5b(-/-) and Stat1(-/-) cells, respectively. Interestingly, in 201 acute myeloid leukemia (AML) patients, coexpression of MN1 and HOXA9 was restricted to patients with the poorest prognosis and was associated with highly active STAT signaling. Our data demonstrate the functional heterogeneity of LICs and show that STAT signaling is critical for leukemia stem cell self-renewal in MN1- and HOXA9-expressing leukemias.

Expression of the insulin-like growth factor I receptor and urokinase plasminogen activator in breast cancer is associated with poor survival: potential for intervention with 17-allylamino geldanamycin.

Urokinase plasminogen activator (uPA) expression in breast cancer is associated with relapse and a reduction in disease-specific survival. Thus, efforts are under way to identify uPA inhibitors. By screening a chemical library of >1000 compounds, 17-allyaminogeldanamycin (17AAG) was identified as a potent inhibitor of uPA by the National Cancer Institute and is now in Phase I clinical trials. At this time, it remains unclear how 17AAG blocks uPA; one possibility is through disruption of the insulin-like growth factor I receptor (IGF-IR) pathway. This would be consistent with studies from our laboratory showing that activation of IGF-IR results in the induction of uPA protein. In the study described herein, we observed that IGF-IR and uPA were highly expressed in 87 and 55% of breast cancer by screening tumor tissue microarrays representing 930 cases. A significant proportion (52.1% = 354 of 680 cases, P < 0.0001) of the patients had tumors expressing both proteins. uPA alone (P = 0.033) or in combination with IGF-IR (P = 0.0104) was indicative of decreased disease-specific survival. Next, we demonstrated that treating MDA-MB-231 cells with increasing concentrations of 17AAG resulted in IGF-IR degradation (IC(50) = 1.0 micro M) and blocked signal transduction through the Akt and mitogen-activated protein kinase pathways. Finally, we found that 17AAG had a robust inhibitory effect on the production of uPA mRNAand protein in the presence of IGF-I. Thus, our study raises the possibility that 17AAG could prove to be an effective therapeutic agent for a large number of breast cancer patients by inhibiting the IGF-IR and ultimately uPA.