Normal Human Hematopoietic Stem Cells Research Articles

Abstract PL03-02: Some cancers follow a stem cell model, while other cancers have common tumorigenic cells with little hierarchical organization

Abstract In our studies of hematopoietic malignancies, acute (Nature 441:475) and chronic myeloid leukemia (Cancer Cell 16:137) appear to follow a cancer stem cell model. In both cases, leukemogenic cells are rare, phenotypically distinct from the vast majority of other leukemia cells, and hierarchically organized. However, tumorigenic capacity is a common attribute of cells from a variety of other human and mouse cancers we have studied, including melanoma. Many cancers are not driven by rare cancer stem cells. We recently showed that the optimization of xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID IL2Rγnull mice, can increase the detection of tumorigenic cells in some cancers by orders-of-magnitude (Nature 456:593). This indicated that tumorigenic cells are common in some human cancers, and that to accurately estimate the frequency of cancer cells with tumorigenic potential it is critical to optimize assay conditions. In this regard, the characterization of human cancer stem cells is no different from the characterization of normal human hematopoietic stem cells, which also depended on the use of the most permissive possible xenotransplantation assay conditions, including the use of highly immunocompromised NOD/SCID IL2Rγnull mice, rather than NOD/SCID mice (which retain natural killer and other innate immune cells that reject some transplanted human cells). Optimization of xenotransplantation assay conditions revealed that cells with tumorigenic potential are common in human melanoma (at least 25% of cells from stage 3 and 4 melanomas have the potential to form tumors after transplantation), suggesting that melanoma growth and progression are unlikely to be driven by rare cancer stem cells. We have also been unable to find any markers that can distinguish tumorigenic from non-tumorigenic melanoma cells, suggesting that melanoma lacks the obvious hierarchical organization observed in other cancers that follow a stem cell model, such as leukemia. Despite observing extensive phenotypic heterogeneity among melanoma cells, we have been unable to detect any differences in tumorigenic capacity among melanoma cells from multiple patients that differed in the expression of ABCB5, CD133, CD166, L1-CAM, CD49f, A2B5, p75, CD44, CD54, CD29, MCAM, HNK1, CD49b, CD49d, E-Cadherin, N-Cadherin, or c-kit. When transplanted into NOD/SCID IL2Rγnull (NOG) mice, only 10 melanoma cells from either the positive or negative fractions of each of these markers readily formed tumors. We therefore remain unable to find any evidence that human melanoma cells are segregated into a hierarchy of intrinsically distinct tumorigenic and non-tumorigenic fractions. Moreover, all fractions of melanoma cells appear to be able to recapitulate the phenotypic diversity of the tumors from which they are drawn. For example, irrespective of whether melanomas arose from CD133+ or CD133- melanoma cells, they contained similar proportions of CD133+ and CD133- cells (Cell 138:822). This suggests that the capacity to recapitulate the phenotypic diversity of primary tumors is not necessarily unique to cancer stem cells but rather that many cells can form phenotypically diverse progeny in certain cancers. Finally, we observed significant differences in the growth rates of tumors from different patients, but these differences were not associated with differences in the frequency of cells with tumorigenic potential. This suggests there are likely to be biologically and clinically important differences among melanoma cells that are not explained by the cancer stem cell model. The available data suggest that many melanoma cells are capable of contributing to disease progression and that it will not be possible to cure melanoma by targeting rare populations of cancer stem cells. Preliminary studies of additional mouse and human cancers suggest that melanoma is not unusual in having common tumorigenic cells. Overall, some cancers appear to be driven by minority populations of cancer stem cells, while other cancers appear to have common tumorigenic cells with little evidence of hierarchical organization. It will be critical to determine which cancers follow a stem cell model and which do not so that efforts to identify and target stem cells can be appropriately focused. Citation Format: Elsa Quintana, Mark Shackleton, Sean J. Morrison. Some cancers follow a stem cell model, while other cancers have common tumorigenic cells with little hierarchical organization [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr PL03-02

Abstract 4334: Tracking of leukemic stem cells from frozen and fresh samples in a mouse model of human AML using bioluminescent imaging

Abstract The aim of this study was to develop and use in vivo bioluminescent imaging to track luciferase-labeled acute myelogenous leukemia (AML) stem cells in immunedeficient mice using AML blood samples collected from patients and subsequently frozen. The ability to utilize frozen pools as viable reservoirs for AML stem cells will allow the freezing back of samples from the clinic for later use as well as the use of previously frozen samples for engraftment and therapeutic studies. Using thawed, isolated cells, we assessed AML stem cell engraftment and quantitatively compared it to fresh AML stem cell engraftment in a xenogeneic transplant model using in vivo bioluminescent imaging. Frozen AML stem cell engraftment was also compared to both fresh and frozen normal human hematopoietic stem cell (HSC) engraftment. Human hematopoietic stem cells (CD34+CD38-) were isolated from both normal frozen cord blood and from peripheral blood of AML patients using modified Miltenyi Biotec (Auburn, CA) magnetic bead isolation kits. Isolated cells were transduced with a lentiviral vector (HIV-luc) pseudotyped with the VSV envelope able to express the firefly luciferase (fluc) gene in human HSC. Using a Caliper Life Sciences/Xenogen IVIS 200 optical imaging system, cells were assayed for fluc expression 48 hours after transduction. Transduced HSC and AML cells (6 × 104 each) were transplanted intravenously into sublethally irradiated adult NOD/SCID/IL2Rγnull mice and mice were imaged serially from day 1 up to day 160 post transplantation. Observed bioluminescent signal and patterns from thawed AML stem cell engraftment are similar to our results obtained using fresh AML stem cells in mice. Luminescent signal from peripheral blood and spleen engraftment of frozen AML stem cells was detectable within one day post-transplantation. Both fresh and frozen AML stem cell populations exhibited diffuse whole body signal, indicating luciferase-labeled stem cells in the peripheral blood, similar to a hallmark of the disease in humans. The presence of blast cells consistent with AML disease was observed by blood smears taken from mice engrafted with frozen AML stem cells. To our knowledge, we have demonstrated for the first time that frozen AML stem cells can be efficiently labeled with a luciferase enzyme for dynamic tracking in vivo. This work has resulted in a useful animal model that will enable us to elucidate the engraftment patterns of AML cells in living animals, learn the dynamics of the interplay of different stem cell populations, and provide information on the efficacy of new treatments in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4334.

Purinergic Signaling Differentially Modulates Normal and Leukemic Hematopoiesis.

Abstract 1436Poster Board I-459Extracellular nucleotides adenosine and uridine triphosphates (ATP and UTP, respectively) are emerging as ubiquitous molecules involved in a wide variety of biological responses and their biological effects are mediated by specific plasma membrane receptors, P2 receptors (P2R). Previously, we showed that extracellular nucleotides stimulate the proliferation and engraftment potential of normal human hematopoietic stem cells. In this study, we assessed whether P2R are expressed on acute myeloblastic leukemia (AML) cells and whether their engagement modulates leukemic cell functions. We show that AML cells express several functional P2R subtypes at the molecular and protein level. Stimulation of AML cells by extracellular nucleotides induced intracellular Ca2+ concentration increases. Furthermore, gene expression profiling revealed that leukemic cells stimulated with ATP underwent a down-regulation of genes involved in cell proliferation and migration whereas those involved in cell cycle inhibition were strongly up-regulated. At the functional level, the clonogenic efficiency of leukemic blasts and CD34+CD38− leukemic stem/progenitor cells was significantly inhibited by the addition of ATP and, to a higher extent, by the stable analogs INS415 and INS973. We also observed a pronounced inhibitory effect of triphosphate nucleotides on spontaneous migration of AML cells and in response to CXCL12.In vivo, xenotransplant experiments demonstrated that the homing and the engraftment capacity of human AML blasts and leukemic stem cells to NOD/SCID/IL-2RGamma-Null mice was significantly inhibited by pre-treatment with ATP, UTP and INS415 and INS973 analogues. Thus, our data show that purinergic signaling modulates leukemic cells in a opposite way than normal cells. Characterization of purinergic signalling in leukemia may help the better understading of the mechanism of neoplastic transformation and tumor progression. DisclosuresDi Virgilio: F. Di Virgilio serves as a consultant for Cordex Pharma Inc. (USA) involved in the development of “P2 receptor-based drugs”.: Consultancy; F. Di Virgilio serves as a consultant for Affectis Pharmaceuticals AG (Germany), involved in the development of “P2 receptor-based drugs”.: Consultancy.

CD47-Sirpα Interaction Modulates Homing and Engraftment of Human Acute Myeloid Leukemia Stem Cells in Mice.

Abstract 476 Introduction:A large body of work has shown that acute myeloid leukemia (AML) clones are hierarchically organized and maintained by leukemia initiating cells (AML-LSC). However, little is known about molecular regulators that govern AML-LSC fate. Using the non-obese diabetic (NOD)–severe combined immunodeficiency (SCID) xenotransplantation model, our group recently found that CD47-SIRPα protein interaction is essential for repopulation of normal hematopoietic stem cells (HSC) in mice (Takenaka et al, Nat Immunol 2007). The NOD background conferred the best support for human engraftment, whereas mice with other polymorphisms of Sirpa could not be engrafted (i.e. NOD.NOR-Idd13.SCID). CD47 on human AML contributes to pathogenesis by inhibiting phagocytosis of leukemia cells through CD47-SIRPα interaction (Jaiswal et al, Cell 2009). CD47 is increased on human AML LSCs compared to normal HSCs, and high levels of CD47 are associated with poor patient outcome (Majeti et al, Cell 2009). This indicates that CD47 may function as an important molecular regulator of AML-LSC fate through communication with SIRPα protein expressed on cells of the innate immune system. Results:Consistent with published results, we observed increased CD47 expression in primary AML cells compared to normal cord blood cells. We next investigated the functional relevance of CD47 for AML-LSCs by xenografting primary human AMLs into NOD.SCID and NOD.NOR-Idd13.SCID (Idd) mice. Following intravenous (i.v.) transplantation, none of three primary human AML samples could engraft Idd mice while robust engraftment in NOD.SCID mice was observed, consistent with our previous data using normal human HSCs. When the same samples were transplanted intrafemorally (i.f.), 2 of 6 Idd mice showed engraftment in the injected femur but no engraftment in other bones or the spleen; evidence of the latter is linked to stem cell function. To assess the role of innate immunity, we pre-treated mice with antibody against murine CD122 which depletes host natural killer (NK) cells and macrophages. Engraftment in the injected femur was observed in NOD.SCID mice (43/43) for all 10 AML samples tested, and interestingly in 31 of 42 Idd mice (8/10 AML samples tested), with similar engraftment levels (Idd 37.4±6% vs NOD.SCID 53.8±5%, p=0.33). As expected, NOD.SCID mice supported migration to non-injected bones (38/43 mice, 10/10 AML samples tested). In contrast to results obtained in the absence of anti-CD122 pre-treatment, engraftment in non-injected bones was now detectable in 8 of 42 Idd mice (2/10 AML samples tested), pointing to improved migration of AML-LSCs following depletion of NK cells and macrophages. However, the engraftment level in non-injected bones was significantly lower in Idd compared to NOD.SCID mice (2.4±4% vs 63.2±6%, p=0.001). Moreover, AML-LSCs were unable to repopulate the spleens of Idd mice. Homing assays revealed decreased homing to BM and spleen 16 hours following i.v. injection in Idd compared to NOD.SCID mice. Conclusion:Our results support the hypothesis that CD47-SIRPα interaction is critical for engraftment of AML-LSCs. Attenuation of CD47-SIRPα interaction (as in Idd mice) leads to decreased engraftment ability of AML-LSCs. Enhancement of engraftment in the injected femur and in some cases migration to other bones in Idd mice following anti-CD122 treatment is likely mediated through interference with cells of the innate immune system. Interruption of CD47-SIRPα signaling through targeting of either CD47 or SIRPα provides a potential therapeutic approach for eradication of AML-LSCs. Disclosures:Dick:Roche: Research Funding; CSL Ltd: Research Funding.

Dysregulated gene expression networks in human acute myelogenous leukemia stem cells

We performed the first genome-wide expression analysis directly comparing the expression profile of highly enriched normal human hematopoietic stem cells (HSC) and leukemic stem cells (LSC) from patients with acute myeloid leukemia (AML). Comparing the expression signature of normal HSC to that of LSC, we identified 3,005 differentially expressed genes. Using 2 independent analyses, we identified multiple pathways that are aberrantly regulated in leukemic stem cells compared with normal HSC. Several pathways, including Wnt signaling, MAP Kinase signaling, and Adherens Junction, are well known for their role in cancer development and stem cell biology. Other pathways have not been previously implicated in the regulation of cancer stem cell functions, including Ribosome and T Cell Receptor Signaling pathway. This study demonstrates that combining global gene expression analysis with detailed annotated pathway resources applied to highly enriched normal and malignant stem cell populations, can yield an understanding of the critical pathways regulating cancer stem cells.

SHP-2 Knockdown Results in Profound Inhibition of Human CD34+ Hematopoietic Stem/Progenitor Cell Survival, Proliferation and Differentiation in Response to Growth Factor Stimulation

SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.

Expression of S100 proteins in normal human hematopoietic stem cells and in AML

7072 Background: In the quest to reduce mortality and morbidity from cancer, there is continued effort to identify novel biomarkers to aid in the early detection and the accurate prediction of tumo...

The Novel Akt Inhibitor Perifosine Induces Apoptosis, Cell Cycle Arrest and Synergizes with Chemotherapeutic Drugs in Acute Myelogenous Leukemia Cells by a JNK Dependent Mechanism - A Novel Therapeutic Approach for Leukemia Displaying Elevated Akt Signaling.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). In the following study, we have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents which target the plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV4-11 cell lines, as well as primary leukemia cells. The drug blocked AML cells in G2/M phase of the cell cycle and, at longer incubation times (24 hr), decreased survival and induced cell death by apoptosis, as demonstrated by MTT assays and flow cytometric analysis of annexin V-FITC/propidium iodide stained samples. The IC50 for perifosine with THP-1 cells was 15.8 microM, while MV4-11 cells were more sensitive to the drug, as the IC50 was 2.7 microM. In THP-1 cells, perifosine caused complete Akt dephosphorylation on Ser 473 and Thr 308, without affecting total Akt expression levels. Perifosine treatment also resulted in dephosphorylation of the Akt downstream target p70S6 kinase. Perifosine did not affect phosphorylation and activity of the Akt upstream kinase, PDK1 nor altered formation or equilibrium of mTORC1 and mTORC2 complexes. Perifosine induced activation of apical caspases-2, -8, -9, and of executioner caspases-3, -6-, and -7, as well as cleavage of PARP and Bid. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on Fas/FasL interactions, as blocking antibodies to either Fas or FasL significantly reduced perifosine mediated cytotoxicity. Perifosine also activated JNK, and a JNK selective inhibitor (SP600125) markedly reduced perifosine-dependent apoptosis. In MV4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine was cytotoxic for AML blasts with activated Akt (IC50 range: 5.6–7.8 microM). Perifosine also reduced the clonogenic activity of CD34+ cells from AML patients displaying constitutive Akt upregulation, but not the clonogenic activity of CD34+ cells from AML patients without Akt activation, or from healthy donors. The inhibitor markedly increased blast cell sensitivity to etoposide. Taken together, our findings indicate that perifosine, either alone or in combination with existing drugs, is a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K/Akt survival pathway. Furthermore this novel Akt inhibitor perifosine appeared not to affect normal human hematopoietic stem cells, further documenting its therapeutic potential.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

A Novel Assay for Development of Chromosomal Lesions in Human CD34+ Cells Following Exposure to Ionizing Radiation Reveals Increased Susceptibility To Acquire Unstable Chromosomal Lesions in CML CD34+ Cells.

Acquisition of chromosomal lesions likely plays an important role in pathogenesis of primary and therapy-related leukemia, as well as in disease evolution in myeloproliferative and myelodysplastic disorders. However the process of acquisition of chromosomal lesions in hematopoietic stem cells (HSC) is not well understood. Development of persistent chromosomal abnormalities may involve several factors including acquisition of DNA lesions, repair of DNA damage, sensing of nonrepaired or misrepaired lesions and activation of cell cycle checkpoint and apoptotic pathways, and clonal growth advantage conferred by the lesions. In the current study we assessed the frequency, nature and kinetics of chromosomal lesions following exposure to genotoxic agents in normal human HSC and determined whether these were altered in CML, a prototypic HSC malignancy associated with genetic instability and acquisition of new chromosomal abnormalities during disease progression. CD34+ cells were selected from 4 normal donors and 4 newly diagnosed, untreated chronic phase CML patients. Cells were exposed to increasing doses of γ-radiation, cultured with growth factors and metaphase spreads assessed for development of chromosomal lesions. Chromosome painting was performed using chromosomes 1, 3, 5, 7, 11 and 21 probes, representing 32% of genomic DNA, with >100 metaphases scored per dose per time. Radiation exposure resulted in induction of chromosomal lesions in normal CD34+ cells in a dose-dependent manner. Chromosomal lesions were not seen in cells cultured without radiation exposure. The frequency of aberrant metaphases after 72 h culture (shown by cell cycle analysis to represent the first cell division for normal CD34+ cells) was 2.7% with 0.5 Gy, 4.8% with 1.0 Gy, 9.9% with 2.0 Gy and 23.5% with 4.0 Gy exposures. Chromosome aberrations observed at first division included both stable (translocations, insertions) and unstable (excess fragments, dicentrics) lesions. Subsequent results are shown for exposure to 2.0 Gy. The frequency of aberrant metaphases dropped to 5.3% at 144 h (3–4 cell divisions); only stable lesions persisted at this time. In contrast to normal CD34+ cells, first cell division in CML CD34+ cells was seen at 24 h, at which time 15.3% of the metaphases showed aberrations, whereas 11.2% of metaphases showed aberrations after 72 h (3–4 divisions) and 11.2% after 144 h (6–7 divisions). A striking difference between normal and CML cells was persistence of unstable aberrations after several cell divisions in the latter (42.9% unstable lesions present even after 6–7 divisions). These observations suggest impaired ability to sense and eliminate cells with chromosomal lesions or continued generation of such lesions after initial radiation exposure in CML cells. In conclusion we have developed a novel chromosome painting based assay for evaluation of acquisition of chromosomal lesions in primitive hematopoietic cells. We have demonstrated an inherent chromosomal instability that may contribute to clonal evolution and disease progression in CML CD34+ cells. This assay will provide a useful platform for: i) assessment of mechanisms underlying development of chromosomal lesions in response to DNA damage; and ii) assessing susceptibility to genotoxic agents, and allow improved understanding of pathogenesis and disease evolution in myeloid malignancies.

The Tyrosine Kinase Inhibitor TK258 Differentially Targets Proliferating Human Hematopoietic Progenitor Cells but Does Not Eliminate the Quiescent Fraction.

NOD-SCID mouse models have shown that, as with normal hematopoietic stem cells, leukemic stem cells (LSC) exist in a quiescent state and are thus capable of sustaining disease in vivo. Targeted therapy of LSC may therefore have a significant impact on disease eradication, relapse rates and toxicity reduction. Inhibition of FLT3 and c-kit is a current therapeutic strategy for AML but the efficacy of this approach on quiescent cells is unknown. Furthermore, although both FLT3 and c-kit play crucial roles in the development of normal human hematopoietic progenitor (HPC) and stem cells, the hematopoietic consequences of FLT3 and c-kit inhibition remain undefined. Here we show that the FLT3, c-kit and PDGF directed tyrosine kinase inhibitor TK258 specifically targets slowly dividing human HPCs, but not rapidly proliferating HPCs or quiescent cells displaying an immature phenotype. For normal CD34+ cells, treatment of short-term cultures with TK258 resulted in a dose dependent inhibition of cell expansion. Moreover, treatment of normal CD34+ cells with TK258 within the clinical dose range [0.2μM to 1.5μM] inhibited myeloid colony growth but had no effect on erythroid colony output, suggesting a differential effect on lineage-committed progenitors. To further elucidate the effect of TK258 on dividing and also quiescent CD34+ cells, normal CD34+ cells were labelled with a non-toxic fluorescent cell membrane dye, PKH26, prior to TK258 treatment in vitro. Coupled with flow cytometry, this dye enabled the discrimination of progenitor cells with different proliferative capacities. Thus, using this technique, we defined a CD34+/CD133+ quiescent population of cells that retained the same PKH fluorescence intensity as from day 0 of culture. TK258 treatment in culture generated a significant increase in the CD34+/CD133+ quiescent fraction (15.8% treated versus 5.9% untreated of total viable cells). In contrast with untreated cells this fraction maintained c-kit expression, suggesting maintenance of an immature population by TK258. Total viable cell recoveries were higher in the TK258 treated quiescent fraction (24% treated versus 13.5% untreated, p=0.037). Rapidly dividing progenitors were unaffected by TK258, while progenitors with a slower division rate showed significant sensitivity to the compound. The anti-proliferative effect of TK258 was mediated via G0/G1 cell cycle arrest, but not by apoptosis of CD34+ cells. We assessed the functional read-out of TK258 pre-treated progenitors in CFU assays, by sorting cells on the basis of their PKH26 fluorescence. These assays demonstrated a higher total colony output and diverse colony type for TK258 pre-treated ‘quiescent' cells compared to untreated controls, in keeping with the preservation of a more immature population by TK258. Rapidly proliferating populations displayed a lower CFU potential, attributable to a reduction in myeloid output, which was not significantly altered by TK258 pre-treatment. CAFC assays and gene expression profiling of PKH26-sorted subsets are in progress. Taken together, these data demonstrate that TK258 has significant inhibitory effects on normal human hematopoietic progenitor cells in vitro and that the sensitivity of progenitor cells to the compound can be predicted from their proliferative index. Importantly, immature hematopoietic quiescent cells are preserved, which may have implications for the activity of tyrosine kinase inhibitors on non-cycling human leukemia cells.

Differential Expression of SLAM Family Receptor Markers in Normal Human Hematopoietic Stem Cells and Their Malignant Counterpart in MDS and AML.

Members of the signaling lymphocytic activation molecule (SLAM) family, including CD150, CD48 and CD244, were shown to precisely distinguish more committed lineage restricted progenitor cells from pluripotent and multipotent murine hematopoietic stem cells (HSC; Kiel et al; 2005 Cell). Similar SLAM profiles may also be present on HSC subsets in humans. We hypothesized that these SLAM markers may be indicators not only of stem cell potential in normal hematopoiesis but also distinguish a subset of the most immature malignant precursors of leukemia. In agreement with the concept of a “cancer stem cell,” the presence of leukemic stem cell population may be an indicator of important clinical and biological properties. We first tested the distribution of CD150, CD48 and CD244 antigens on human CD34+ cells derived from 7 control individuals using 4-color flow cytometry. CD34+ cells were measured in the blast gate based on side scatter and CD45 expression. Within CD34+ blasts, expression of CD48, CD150, and CD244 was detected on 16.71%±9.69, 6.53%±2.93, and 26.92%±6.95 of cells respectively. Subsequently, we investigated SLAM expression in 9 immature leukemic cell lines, including KG-1, K562, U937, HEL, HL60, MKN-95, NB-4, Kasumi and UT7, and found increased expression of SLAM markers in KG-1 (CD48+, CD150+, CD244+) and Kasumi (CD48−, CD150−, CD244+). Consequently, none of the leukemic cells showed pluripotent/multipotent SLAM profiles. We then compared the SLAM marker expression on blasts from patients with AML and MDS with that of CD34+ cells from normal controls. We studied a total of 28 patients: 11 MDS (2 low grade, 5 advanced MDS, 3 MDS/MPD overlap) and 10 AML (FAB: 3 M1, 2 M2, 1 M3, 2 M4/M4E0 and 2 M6). In our cohort, 8/10 AML patients expressed one of the three SLAM markers; 6/10 were CD150−CD48−CD244+ (63.57%±6.96) and 2/10 were CD150+CD48−CD244−(46%±10.96) suggestive of the presence of either pluripotent or multipotent leukemic stem cell phenotype. In the MDS cohort, 8/11 patients expressed one of three SLAM markers, 7/11 were CD150−CD48−CD244+ (41.21% ± 8.9) and 1/11 were CD150+CD48−CD244− (1.26%±0.59) again consistent with a profile derived from either pluripotent or multipotent stem cells. None of the MDS and AML patients had either co-expression of CD244 and CD48 or increased expression of CD48 alone. Two of the M1 type AML patients with CD150−CD48−CD244+ phenotype received prior chemotherapy and achieved complete remission on bone marrow biopsy and flow cytometry using traditional blast markers. In some, we conclude that the SLAM receptor markers may be associated with certain types of leukemic blasts and may be useful in the identification of leukemic stem cell population in both MDS and AML.

Anti-Apoptotic MCL-1 Plays a Critical Role in Maintenance of Normal and Malignant Human Hematopoietic Stem Cells through FLT3 Signaling.

Mcl-1 (myeloid cell leukaemia-1) is an anti-apoptotic member of the Bcl-2 family protein, originally identified through the analysis of differentiating myeloid cells. We have recently reported that disruption of murine MCL-1 in adult murine hematopoiesis resulted in a complete deficiency of hematopoietic cells in the bone marrow, and that MCL-1 expression was activated in response to activation of c-Kit tyrosine kinase receptor signaling (Science 307;1101, 2005). Here we propose that human MCL-1 also plays a critical role in maintenance of normal and malignant human hematopoiesis. The analysis of FACS-sorted human HSCs and myeloid progenitors demonstrated that, as in case of murine hematopoiesis, HSCs expressed hMCL-1 at the highest level, and that its expression gradually downregulated in committed myeloid progenitors such as CMPs and GMPs. We also found that unlike murine long-term HSCs that express c-Kit but not FLT3 receptor tyrosine kinase, human long-term HSCs express both of these receptors. Ligation of FLT3 receptor in purified human HSCs immediately induced the expression of MCL-1, suggesting that FLT3 supports human HSC survival through stimulating MCL-1 expression. We then evaluated whether hMCL-1 is expressed in leukemic stem cells. Thirty patients with acute myeloid leukemia (AML) were enrolled in this study. Within the immature CD34+ blast population, the CD34+CD38− fraction expressed higher levels of MCL-1 than those in more differentiated CD34+CD38+ fraction. In most cases, CD34+CD38− AML cells expressed 2 to 10-fold higher levels of MCL-1 transcripts as compared to normal HSCs. Interestingly, the group of samples expressed highest levels of MCL-1 constituted mostly of FLT3/internal tandem duplications (ITDs) positive AML, suggesting that constitutive activation of FLT3 signaling by FLT3 mutations might induce overexpression of MCL-1. A high level expression of MCL-1 was also found in MOLM-13 and MV4-11, FLT3/ITD positive AML cell lines. Treatment of MOLM-13 and MV4-11 with the small molecule tyrosine kinase inhibitor PKC 412, resulted in induction of apoptosis, which was associated with decreased expression of MCL-1 transcripts and proteins. Based on these data, MCL-1 might play a critical role in maintenance of normal and malignant HSCs in human at least through FLT3 signaling. Our data also suggest that constitutive activation of FLT3 signaling by FLT3 mutation might contribute to leukemic transformation through enforcing survival of AML stem cells.

Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.

Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1

AbstractComplement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34+ cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell–derived factor 1 (SDF-1)–dependent chemotaxis of human CD34+ cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1–dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)–mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1+ cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF–CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

33 The role of B-catenin in chronic myelogenous leukemic compared with normal human hematopoietic stem cell and myeloid progenitor expansion

33 The role of B-catenin in chronic myelogenous leukemic compared with normal human hematopoietic stem cell and myeloid progenitor expansion

Activity of Acetyl-Ser-Asp-Lys-Pro (AcSDKP) on Human Hematopoietic Progenitor Cells in Short-Term and Long-Term Bone Marrow Cultures

The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent inhibitor of hematopoietic stem cell proliferation. We examined the effects of AcSDKP on the production of granulocyte-macrophage colony-forming cells (CFU-GM) and high proliferative potential colony-forming cells (HPP-CFC) in human long-term bone marrow (LTBM) cultures and CFU-GM and erythroid burst-forming cells (BFU-e) in short-term liquid cultures. The addition of AcSDKP in short-term bone marrow cultures resulted in a maximum depression of the total number of progenitor cells as well as the number of progenitor cells entering cell cycle following culture with 10(-12) to 10(-14) M AcSDKP and 10(-14) M AcSDKP when exogenous cytokines (GM-CSF, IL-3, or SCF) were added. AcSDKP was added daily to LTBM cultures at various concentrations (10(-8) M to 10(-16) M) for up to 5 weeks. In these LTBM culture studies, AcSDKP inhibited the entry of nonadherent progenitor cells into S phase and decreased the number of nonadherent progenitor cells with peak activity at 10(-12) M. In contrast, AcSDKP had no effect on the number of adherent CFU-GM, HPP-CFC, or cellularity per culture or percent of adherent progenitor cells in S phase. These studies indicate that the concentration of the tetrapeptide is critical to the activity of AcSDKP on human hematopoietic progenitor cells. Furthermore, we report that the presence of cytokines or stromal cells also affects the response of progenitor cells to AcSDKP. These results will aid in determining kinetic properties of AcSDKP for the development of clinical protocols to protect normal human hematopoietic stem and progenitor cells following cycle-specific chemotherapy agents.

A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

AbstractHuman C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

In vitro modulation of the toxicity associated with the use of zidovudine on normal murine, human, and murine retrovirus-infected hematopoietic progenitor stem cells with basic fibroblast growth factor and synergistic activity with interleukin-1.

The antiviral drug used in the treatment of acquired immunodeficiency syndrome, zidovudine, has proved effective in ameliorating the morbidity and mortality associated with human immunodeficiency virus infection. However, associated with zidovudine is the development of severe bone marrow toxicity manifested by anemia, neutropenia, and occasionally thrombocytopenia. We report the results of studies that demonstrate the ability of basic fibroblast growth factor (B-FGF) to reduce zidovudine toxicity to several classes of hematopoietic progenitors (granulocyte-macrophage, CFU-GM; megakaryocyte. CFU-Meg; and erythroid, BFU-E) from normal murine, human, and murine retrovirus-infected bone marrow cells when cocultured with zidovudine in vitro. Optimal response to B-FGF was observed at a dose concentration of 10 ng/ml. The specificity of B-FGF was demonstrated in the presence of protamine sulfate, an effective inhibitor of B-FGF mitogenic activity. In addition, synergistic activity of B-FGF on zidovudine-induced hematopoietic stem cell toxicity was observed in the presence of interleukin 1 (IL-1) (30 ng/ml). These studies demonstrate that B-FGF is capable of reducing the hematopoietic toxicity associated with zidovudine and that such an effect can be amplified in the presence of IL-1.