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.     

The Expression Pattern of CD33 Antigen Can Differentiate Leukemic from Normal Progenitor Cells in Acute Myeloid Leukemia

Leukemic stem cells (LSC) in acute myeloid leukemia (AML), defined by CD34 and CD38 antigens also express CD33 similar to normal hematopoietic stem cells. Residual LSC are believed to be responsible for relapse in AML after chemotherapy. Leukemic progenitor cell compartments were defined by CD34 and CD38 expression by flow cytometry in 61 new cases of AML. In each of four compartments thus defined, CD34+CD38−, CD34+CD38+, CD34−CD38− and CD34−CD38+, the pattern and intensity of expression of CD33 were studied in comparison to similar progenitor cell compartments in normal bone marrow and peripheral blood stem cell harvests. Post induction bone marrow samples from 10/61 cases were studied for aberrant CD33 expression. The intensity and pattern of expression of CD33 in AML progenitor cells were significantly different compared to normal progenitor cells. In two cases who were in morphological remission post induction, aberrant CD33 expressing progenitor cells were detectable at a frequency of 1.6 and 0.5 % respectively in the bone marrow. Aberrant CD33 expression in bone marrow LSC identified as CD34+CD38− cells in the CD45 dim/low side scatter region on flow cytometry may be useful as minimal residual disease marker after AML therapy. The method involves the use of a limited number of reagents and can be applied to all cases of AML.     

Preferential induction of apoptosis for primary human leukemic stem cells

Acute myelogenous leukemia (AML) is typically a disease of stem progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G(0) cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor kappaB (IkappaBalpha) demonstrate that inhibition of nuclear factor kappaB (NF-kappaB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.     

Prognostic significance of HOXB4 in de novo acute myeloid leukemia

As research into hematopoiesis advances, new factors associated with hematopoietic stem cell (HSC) activity have been discovered, and the contribution of HSC factors to hematopoiesis is now actively being investigated. Since the involvement of stem cells is considered to be important in hematological malignancies as well as normal hematopoiesis, we examined the expression of newly defined HSC factors including HOXB4, TCFEC, HMGB-1, FOS, and SPI-1 in the bone marrow (BM) from de novo acute myeloid leukemia (AML) patients. Expression levels of mRNA extracted from frozen specimens of AML patients and normal controls were measured by real-time polymerase chain reaction (PCR). Among the HSC factors, HOXB4 exhibited significantly higher expression in the BM of AML as compared with normal controls. Immunostaining for HOXB4 revealed that the HOXB4-positive cell ratios correlated well with the expression levels of mRNA for HOXB4 in AML BM. Based on the HOXB4-positive cell ratio, AML patients were divided into two groups (cases with higher ratios and lower ratios). The group with higher HOXB4-positive cell ratios had better prognoses as compared with the group with lower ratios. Multivariate analysis confirmed the HOXB4-positivity as an independent predictor of overall survival of AML patients. Lastly, mRNA expression levels for HOXB4 were inversely correlated with the expression levels of at least two genes, including ABCB1, which is known to be a multidrug-resistance gene. Our study distinguishes a subgroup of AML with higher HOXB4 expression and better prognosis, and this might be reflected by relative drug sensitivity in leukemic cells.     

The ETO domain is necessary for the developmental abnormalities associated with AML1-ETO expression in the hematopoietic stem cell compartment in vivo

Translocation of the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO) is a recurrent cytogenetic abnormality associated with approximately 12% of acute myelogenous leukemia (AML) cases. To understand the contribution of the t(8;21) to AML, we transduced purified hematopoietic stem cells (HSC) with a retroviral vector that coexpressed AML1-ETO or just the AML1 portion (AML1d) of the translocation along with a green fluorescent protein reporter gene. Animals reconstituted with AML1-ETO-expressing cells exhibited many of the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21), although the animals did not develop acute leukemia. We noted a gradual increase in primitive myeloblasts that accounted for approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. In addition, accumulation of late stage metamyelocytes was observed in bone marrow along with an increase in immature eosinophil myelocytes that showed abnormal basophilic granulation. There was also a gradual increase in both the frequency and absolute number of AML1-ETO-expressing HSC so that by 10 months posttransplant, there were 29-fold greater HSC numbers than in transplant-matched control mice. These phenotypes were not observed in animals reconstituted with cells expressing only the DNA-binding domain of AML1, suggesting that the ETO domain is necessary to establish the developmental abnormalities associated with AML1-ETO expression in HSC.     

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34⁺CD38⁻ stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34⁺CD38⁺ progenitors were reduced. Residual normal CD34⁺ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.     

Changes in integrin expression are associated with altered homing properties of Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor

OBJECTIVE: Although migration of hematopoietic stem cells (HSC) is essential for normal hematopoiesis and successful hematopoietic cell transplantation, little is known about the mechanisms that underlie this movement. We have sought to characterize the factors that regulate HSC migration by analyzing changes in expression of particular adhesion receptors associated with cyclophosphamide/granulocyte colony-stimulating factor (Cy/G-CSF)-induced HSC mobilization. METHODS: Expression by Lineage(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) HSC of members of the beta1 integrin family of adhesion molecules was assessed in untreated or Cy/G-CSF-treated mice by multiparameter flow cytometry. In parallel, the in vivo homing properties of normal and mobilized HSC were compared following intravenous transfer of fluorescently marked HSC. RESULTS: Normal adult HSC express high levels of several beta1 integrin family members. Following Cy/G treatment, bone marrow HSC selectively downregulate alpha 2 integrin expression and upregulate alpha 5 expression. HSC found in the blood following Cy/G-CSF treatment express significantly lower levels of multiple integrins than their bone marrow and/or splenic counterparts. Changes in integrin expression by blood-borne HSC correlate with a 50% decrease in their ability to home to the bone marrow in short-term assays, and with previously observed defects in competitive engraftment by these HSC. Similar reductions in bone marrow (BM) homing are observed for BM HSC treated with alpha 4 integrin function blocking mAb prior to injection. Modulation of integrin expression induced by mobilization was not associated with cell-cycle progression. CONCLUSION: Changes in integrin expression and function are associated with HSC mobilization and likely significantly affect the engraftment potential of hematopoietic stem cells.     

CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia

Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.     

Characterization of Thy-1 (CDw90) expression in CD34+ acute leukemia

Thy-1 (CDw90) is a phosphatidylinositol-anchored cell surface molecule which, when coexpressed with CD34 in normal human bone marrow, identifies a population of immature cells that includes putative hematopoietic stem cells. To date, the characterization of Thy-1 expression has been confined largely to normal tissues and cell lines. In this study, we evaluated the frequency and intensity of Thy-1 expression as defined by reactivity with the anti-Thy-1 antibody 5E10 in 38 cases of CD34+ acute leukemia (21 acute myelogenous leukemia [AML], 8 chronic myelogenous leukemia [CML] in blast crisis, and 9 acute lymphoblastic leukemia [ALL]). In 34 of 38 cases (89%) the CD34+ cells lacked expression of the Thy-1 antigen. High-density Thy-1 expression was found in 1 case of CML in lymphoid blast crisis, and low- density Thy-1 expression was identified on a portion of the leukemic cells in 2 cases of AML with myelodysplastic features, and 1 case of CML in myeloid blast crisis, suggesting a possible correlation between Thy-1 expression and certain instances of stem cell disorders such as CML and AML with dysplastic features. In contrast, the dissociation of Thy-1 and CD34 expression in the majority of acute leukemias studied suggests that the development of these leukemias occurs at a later stage than the hematopoietic stem cell. Characterization of Thy-1 expression in acute leukemia may eventually provide insights into the origin of the disease. In addition, separation of leukemic blasts from normal stem cells based on Thy-1 expression may prove useful in assessing residual disease, as well as in excluding leukemic blasts from stem cell preparations destined for autologous bone marrow or peripheral stem cell transplantation.     

TIM-3 as a novel therapeutic target for eradicating acute myelogenous leukemia stem cells

Acute myelogenous leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), which represent the ultimate therapeutic target for AML. Recent studies have identified several AML LSC-specific surface antigens as candidate targets of therapeutic molecules. T cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML, with the exception of acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). In xenograft models reconstituted with human AML LSCs or HSCs, an anti-human TIM-3 mouse IgG2a antibody with cytotoxic activities eradicates AML LSCs in vivo, but does not affect normal human hematopoiesis. Thus, TIM-3 is a promising therapeutic target for the eradication of AML LSCs.     

Stem cell therapy. Biology of hematopoietic stem cells

In recent years much progress has been made in the understanding of the biology of hematopoietic stem cells (HSC) and their involvement in normal blood cell development. Using immunophenotyping it is possible, to enrich HSC, however, so far we are not able to positively select HSC. For the identification, characterization and quantification of HSC it is necessary to use functional assay systems, such as xenotransplantation models. HSC from bone marrow, peripheral blood and in some cases also cord blood have been used for years in transplantation settings especially in patients with leukemia. A better understanding of the mechanisms underlying stem cell regulation as well as stem cell self renewal would have clinical implications e. g. for clinical transplantation strategies. A number of hematological diseases such as chronic myeloid leukemia originates from a malignant transformed HSC. A better understanding of the biology of normal as well as malignant HSC is therefore crucial not only for a better understanding of the disease, but also for the development of strategies aiming at the discrimination of normal and malignant stem cell candidates and the development of therapies targeting the leukemic stem cell.     

Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia

The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.     

Stem cell factor, interleukin-3, and interleukin-6 promote retroviral-mediated gene transfer into murine hematopoietic stem cells.

The efficiency of retroviral-mediated gene transfer into hematopoietic stem cells (HSC) is dependent on the survival and self-renewal of HSC in vitro during retroviral infection. We have examined the effect of prestimulation of bone marrow with various cytokines, including the product of the Steel gene, Steel factor or stem cell factor (SCF) (the ligand for the c-kit receptor) on the efficiency of retroviral transduction of the human adenosine deaminase (hADA) cDNA into murine HSC. Bone marrow cells were prestimulated for 48 hours with hematopoietic growth factors, then cocultivated with the packaging cell line producing the ZipPGK-ADA simplified retrovirus for an additional 48 hours with continued growth factor exposure. Nonadherant cells from these cocultures were injected into lethally irradiated recipients. The content of day 12 colony-forming unit-spleen (CFU-S12) in SCF/interleukin 6 (IL-6)-prestimulated and cocultured bone marrow was more than threefold greater than that of IL-3/IL-6-prestimulated bone marrow cells. All mice receiving bone marrow cells infected with the PGK-ADA virus after prestimulation with IL-3/IL-6 or SCF/IL-6 demonstrated hADA expression in the peripheral blood after full hematopoietic reconstitution. While all recipients of IL-3/IL-6-prestimulated bone marrow expressed hADA at 4 months posttransplant, in three independent experiments examining a total of 33 mice, in most recipients of SCF/IL-6-prestimulated and infected bone marrow cells, the expression of human enzyme was higher than IL-3/IL-6 mice. Southern blot analysis of DNA from hematopoietic tissues from these same mice prepared at least 4 months posttransplantation also demonstrated a higher infection efficiency of HSC as measured by proviral integration patterns and genome copy number analysis. These results suggest that the higher level of hADA expression seen in mice receiving marrow prestimulated with SCF/IL-6 before retroviral infection is due to more efficient infection of reconstituting HSC. Other growth factor combinations were also studied; however, prestimulation with SCF/IL-6 or IL-3/IL-6 appeared optimal. Using retroviral-mediated gene transfer and viral integration patterns, Steel factor (SCF) in combination with IL-6 appears to increase the survival and self-renewal of reconstituting hematopoietic stem cells and proves useful in effecting expression of foreign genes in transplant recipients. Such pretreatment may also be useful in the application of retroviral transfer methods to human cells.     

A small molecule screening strategy with validation on human leukemia stem cells uncovers the therapeutic efficacy of kinetin riboside

Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34(+)CD38(-) AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias.     

Long-term persistence of host cells detected by X-chromosome gene-based assay in patients undergoing gender-mismatched hematopoietic stem cell transplantation

Using our recently developed human androgen receptor (HUMARA) gene-based chimerism assay, long-term chimerism was investigated in female patients who underwent hematopoietic stem cell transplantation (HCT) with cells from male donors. After restriction digestion of samples, we detected a small number of female-derived cells within a large population of male-derived cells, with a sensitivity from 0.1% to 0.05%. Chimerism was examined in four patients with myeloid malignancies: two patients with acute myeloid leukemia (AML) from myelodysplastic syndrome (MDS), and one patient each with AML M3 and AML M4. All patients underwent myeloablative conditioning regimens and exhibited good clinical results during a median follow-up period of 6.6 years (range, 3.4-7.5 years). Female-derived cells were detected throughout the entire follow-up period in all bone marrow samples, but they became undetectable in the peripheral blood samples of 3 patients. Moreover, the HUMARA band pattern suggests that these residual host cells were normal cells. This study confirms the usefulness of the HUMARA gene-based assay, which showed that patients undergoing HCT frequently show mixed chimerism (MC) for a long period, especially in bone marrow, although the possibility of contamination by host stromal cells cannot be excluded.     

The IL1-IL1RAP axis plays an important role in the inflammatory leukemic niche that favors acute myeloid leukemia proliferation over normal hematopoiesis

Upregulation of the plasma membrane receptor IL1RAP in acute myeloid leukemia (AML) has been reported but its role in the context of the leukemic bone marrow niche is unclear. Here, we studied the signaling events downstream of IL1RAP in relation to leukemogenesis and normal hematopoiesis. High IL1RAP expression was associated with a leukemic GMPlike state, and knockdown of IL1RAP in AML reduced colony-forming capacity. Stimulation with IL1b resulted in the induction of multiple chemokines and an inflammatory secretome via the p38 MAPK and NFkB signaling pathways in IL1RAP-expressing AML cells, but IL1b-induced signaling was dispensable for AML cell proliferation and NFkB-driven survival. IL1RAP was also expressed in stromal cells where IL1b induced expression of inflammatory chemokines and cytokines as well. Intriguingly, the IL1b-induced inflammatory secretome of IL1RAP-expressing AML cells grown on a stromal layer of mesenchymal stem cells affected normal hematopoiesis including hematopoietic stem/progenitor cells while AML cell proliferation was not affected. The addition of Anakinra, an Food and Drug Aministration-approved IL1 receptor antagonist, could reverse this effect. Therefore, blocking the IL1-IL1RAP signaling axis might be a good therapeutic approach to reduce inflammation in the bone marrow niche and thereby promote normal hematopoietic recovery over AML proliferation after chemotherapy.