Enrichment of N-Cadherin and Tie2-bearing CD34/CD38/CD123 leukemic stem cells by chemotherapy-resistance

Acute myeloid leukemia (AML) arises from genetic changes at the level of stem cell, various mutations have been elucidated, including AML1–ETO fusion gene has been shown as the representative target of cellular transformation for LSCs originating from hematopoietic stem cells (HSCs) compartment. LSCs resemble HSCs with respect to self-renewal capacity and chemotherapy-resistance. However, LSCs possess specific cell-surface markers, they are proposed to reside within the CD34⁺/CD38⁻/CD123⁺ compartment. And the interaction mediated by adhesion molecules between LSCs and niche played a role in chemoresistance of LSCs. Therefore, study on the LSCs surface makers related to niche is helpful for the potential target therapy in the future. In this study, the proportions of CD34⁺/CD38⁻/CD123⁺ LSCs compartment co-expressing the three adhesion molecules, N-Cadherin, Tie2 and CD44, respectively, from AML patients before and after chemotherapy were analyzed. We demonstrated N-Cadherin and Tie2 positive CD34⁺/CD38⁻/CD123⁺ LSCs populations could be enriched by chemotherapy. Furthermore, AML1/ETO fusion signals and MDR1 expression were detected on the CD34⁺/CD38⁻/CD123⁺ LSCs populations expressing N-Cadherin and Tie2. Therefore, N-Cadherin and Tie2 are probably the potential markers for identification of LSCs.     

JmjC‐domain containing histone demethylase 1B‐mediated p15Ink4b suppression promotes the proliferation of leukemic progenitor cells through modulation of cell cycle progression in acute myeloid leukemia

The histone demethylase JHDM1B has been implicated in cell cycle regulation and tumorigenesis. In addition, it has been reported that JHDM1B is highly expressed in various human tumors, including leukemias. However, it is not clearly understood how JHDM1B contributes to acute myeloid leukemia (AML) cell proliferation. In this study, we investigated the cellular and molecular function of JHDM1B in AML cells. In AML cell lines and AML‐derived ALDH^hi (high aldehyde dehydrogenase activity)/CD34⁺ cells, the levels of JHDM1B mRNA were significantly higher than in normal ALDH^hi/CD34⁺ cells. Reduction of JHDM1B expression in AML cells inhibited cell proliferation compared to control cells, through induction of G1 cell cycle arrest, an increase in the p15^Ink4b mRNA and protein expression. JHDM1B mRNA was overexpressed in all 133 AML clinical specimens tested (n = 22, 57, 34, and 20 for M1, 2, 4, and 5 subtypes respectively). Compared to normal ALDH^hi/CD34⁺ cells, JHDM1B gene expression was 1.57‐ to 1.87‐fold higher in AML‐derived ALDH^hi/CD34⁺cells. Moreover, the JHDM1B protein was more strongly expressed in AML‐derived ALDH^hi/CD34⁺ cells from compared to normal ALDH^hi/CD34⁺ cells. In addition, depletion of JHDM1B reduced colony formation of AML‐derived ALDH^hi/CD34⁺ cells due to induction of p15^Ink4b expression through direct binding to p15^Ink4b promoter and loss of demethylation of H3K36me2. In summary, we found that JHDM1B mRNA is predominantly expressed in AML‐derived ALDH^hi/CD34⁺ cells, and that aberrant expression of JHDM1B induces AML cell proliferation through modulation of cell cycle progression. Thus, inhibition of JHDM1B expression represents an attractive target for AML therapy. © 2011 Wiley Periodicals, Inc.     

Irrespective of CD34 expression,lineage‐committed cell fraction reconstitutes and re‐establishes transformed Philadelphia chromosome‐positive leukemia in NOD / SCID / IL‐2Rγc−/− mice

(Cancer Sci 2010; 101: 631–638) Stem cells of acute myeloid leukemia (AML) have been identified as immunodeficient mouse‐repopulating cells with a Lin^?CD34⁺38^? phenotype similar to normal hematopoietic stem cells. To identify the leukemia‐propagating stem cell fraction of Philadelphia chromosome‐positive (Ph⁺) leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n = 3) or Ph⁺ acute lymphoblastic leukemia (n = 3) into NOD/SCID/IL‐2Rγc^?/? mice. Engrafted cells were almost identical to the original leukemia cells as to phenotypes, IGH rearrangements, and karyotypes. CD34⁺CD38^?CD19⁺, CD34⁺38⁺CD19⁺, and CD34^?CD38⁺CD19⁺ fractions could self‐renew and transfer the leukemia, whereas the CD34^?CD38⁺CD19⁺ fraction did not stably propagate in NOD/SCID mice. These findings suggest that leukemia‐repopulating cells in transformed Ph⁺ leukemia are included in a lineage‐committed but multilayered fraction, and that CD34⁺ leukemia cells potentially emerge from CD34^? populations.     

N-Cadherin and Tie2 positive CD34CD38CD123 leukemic stem cell populations can develop acute myeloid leukemia more effectively in NOD/SCID mice

Emerging studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). A lot of investigators have reported the identification of cell surface markers, such as CD123. Here, we report the identification of N-cadherin and Tie2 as LSCs markers. Inoculation of CD34⁺CD38⁻CD123⁺N-cadherin⁺ and CD34⁺CD38⁻CD123⁺ Tie2⁺ population can induce leukemia in NOD/SCID mice. The leukemic blast cells from the primary leukemic mice could also induce leukemia in the secondary transplantation. These findings suggested that N-cadherin and Tie2 were the important markers that can assist in leukemia development.     

The pan-Bcl2 Inhibitor AT101 Activates the Intrinsic Apoptotic Pathway and Causes DNA Damage in Acute Myeloid Leukemia Stem-Like Cells

Background

Leukemia stem cells (LSCs) are considered to be the cause of treatment failure and relapse in acute myeloid leukemia (AML). Overexpression of the Bcl-2 family of anti-apoptotic proteins such as Bcl-2, Bcl-xl, and Mcl-1 accounts for survival and self-renewal of LSCs. AT101 binds to the BH3 motif of all Bcl-2 family anti-apoptotic proteins and demonstrates anti-tumor activity in multiple types of tumor. Thus, we hypothesized that this agent might have the potential to deplete LSCs.

Objective

The present study aims to investigate if and by what mechanism AT101 is able to target AML stem-like cells.

Methods

As LSCs and hematopoietic stem cells (HSCs) are enriched in CD34⁺CD38^? populations, CD34⁺CD38^? cells from KG1α and Kasumi-1 cell lines as well as CD34⁺ blasts from AML patients were used as LSC models, while CD34⁺ cells from healthy donors were used as normal hematopoietic cells. Cell proliferation and apoptosis were assessed by a cell counting kit-8 (CCK8) assay and an Annexin V/PI assay using flow cytometry, respectively. Colony-forming units experiments were performed to monitor the stemness features of AML cells. Western blot and quantitative real-time polymerase chain reaction (qPCR) analysis were performed to examine the levels of proteins and mRNAs related to either the intrinsic apoptotic pathway or DNA damage response.

Results

AT101 inhibited proliferation and induced apoptosis in CD34⁺CD38^? KG1α and Kasumi-1 cells in a dose- and time-dependent manner. Exposure to AT101 for 24 h resulted in apoptosis in primary CD34⁺ AML blasts (EC₅₀ [concentration needed for a 50% maximal effect] = 2.45–76.00 μmol/L), while it only had a modest effect on normal CD34⁺ hematopoietic cells. Mechanistically, AT101 activated the intrinsic apoptotic pathway by inhibition of Bcl-2 anti-apoptotic proteins, reflected by a decrease in mitochondrial membrane potential. Moreover, AT101 caused DNA damage (e.g., increased γH2AX phosphorylation), which might also contribute to its anti-leukemic effects. Interestingly, the ex vivo efficacy of AT101 in primary AML samples significantly correlated to hyperleukocytosis and FLT3-ITD mutations. AT101 was also effective against CD34⁺ blasts isolated from elderly patients and patients who did not achieve complete remission after induction therapy.

Conclusions

AT101 effectively eliminates LSCs in vitro through the induction of DNA damage and activation of the intrinsic apoptotic pathway. AT101 is effective towards leukemic cells from patients with adverse prognostic factors, suggesting that AT101 could have the potential as an alternative salvage therapy for the treatment of relapsed and refractory AML.

     

Bcr‐Abl activates AURKA and AURKB in chronic myeloid leukemia cells via AKT signaling

This study explored molecular mechanisms by which Bcr‐Abl induced expression of Aurora kinase A and B (AURKA and AURKB) in chronic myeloid leukemia cells. Lentiviral transduction of Bcr‐Abl into either Ba/F3 or CD34⁺ hematopoietic stem/progenitor cells potently increased levels of AURKA and AURKB in association with phosphorylation of AKT and stimulated their proliferation. Bcr‐Abl‐mediated expression of AURKA and AURKB were decreased in CD34⁺ HSPCs when AKT was inactivated by an shRNA against AKT, suggesting that Bcr‐Abl induced expression of AURKA and AURKB via AKT signaling. MLN8237, an inhibitor of AURKA, significantly inhibited the proliferation of freshly isolated CD34⁺ CML cells in a dose‐dependent manner as measured by colony forming assay. Importantly, inhibition of AURKA in CD34⁺ leukemia cells freshly isolated from individuals with blast crisis of CML with Bcr‐Abl T315I mutant (n = 2) by MLN8237 significantly impaired the engraftment of these cells in severely immunocompromised mice and decreased the weight of spleens. Taken together, Bcr‐Abl induces expression of AURKA and AURKB at least in part via AKT. Inhibition of AURKA could be useful to overcome imatinib‐resistance mediated by Bcr‐Abl mutants.     

The rarity of ALDH+ cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH⁺ cells; <1.9%; ALDH‐rare AML), whereas 24 patients had relatively numerous ALDH⁺ cells (≥1.9%; ALDH‐numerous AML). In patients with ALDH‐rare AML, normal HSC could be separated by their CD34⁺ALDH⁺ phenotype, whereas LSC were exclusively detected among CD34⁺ALDH^? cells. For patients with ALDH‐numerous AML, the CD34⁺ALDH⁺ subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH⁺ cells from ALDH‐numerous AML were quiescent, refractory to ARA‐C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long‐term outcome were also characteristic for patients with ALDH‐numerous AML providing an additional risk‐stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.     

Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML)

Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34⁺/CD38⁻ and CD34⁺/CD38⁺ stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC₅₀ 0.05-0.5 μM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34⁺/CD38⁻ and CD34⁺/CD38⁺ stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34⁺/CD38⁻ and CD34⁺/CD38⁺ AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials.     

Leukemic Stem Cell (CD34+/CD38–/TIM3+) Frequency in Patients with Acute Myeloid Leukemia: Clinical Implications

This study aimed to address the prognostic relevance of CD34⁺/CD38^–/TIM3⁺ leukemic stem cell (LSC) frequency in patients with acute myeloid leukemia (AML) and its impact on patient outcome. We analyzed the expression of LSC markers (CD34⁺/CD38^–/TIM3⁺) using flow cytometry in bone marrow samples of 53 AML cases before and after induction chemotherapy. The LSC frequency at diagnosis was significantly higher compared with that postinduction (P < .001). Patients were categorized into high LSC expressers (≥ median) and low expressers (< median). Patients with AML with high number of LSCs at diagnosis had significantly lower induction of remission response (P = .0104), shorter disease-free survival, and shorter overall survival (P < .001 for both) compared with those with lower LSC count. Cox regression analysis revealed that LSC frequency at diagnosis is an independent prognostic factor in AML. Assessment of LSCs (CD34⁺/CD38^–/TIM3⁺) at diagnosis is recommended for refining of AML risk stratification.     

Altered intracellular distribution of daunorubicin in immature acute myeloid leukemia cells

We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34⁺ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34⁻ and CD34⁺ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34⁺ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34⁺ but not in CD34⁻ cells. However, DNR sequestration was not observed in normal CD34⁺ cells. Finally, our results show that DNR is sequestered in organelles in CD34⁺ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines. Int. J. Cancer 71:292-299, 1997. © 1997 Wiley-Liss, Inc.     

TWIST-1 promotes cell growth,drug resistance and progenitor clonogenic capacities in myeloid leukemia and is a novel poor prognostic factor in acute myeloid leukemia

Alterations of TWIST-1 expression are often seen in solid tumors and contribute to tumorigenesis and cancer progression. However, studies concerning its pathogenic role in leukemia are scarce. Our study shows that TWIST-1 is overexpressed in bone marrow mononuclear cells of patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). Gain-of-function and loss-of-function analyses demonstrate that TWIST-1 promotes cell growth, colony formation and drug resistance of AML and CML cell lines. Furthermore, TWIST-1 is aberrantly highly expressed in CD34⁺CD38⁻ leukemia stem cell candidates and its expression declines with differentiation. Down-modulation of TWIST-1 in myeloid leukemia CD34⁺ cells impairs their colony-forming capacity. Mechanistically, c-MPL, which is highly expressed in myeloid leukemia cells and associated with poor prognosis, is identified as a TWIST-1 coexpressed gene in myeloid leukemia patients and partially contributes to TWIST-1-mediated leukemogenic effects. Moreover, patients with higher TWIST-1 expression have shorter overall and event-free survival (OS and EFS) in AML. Multivariate analysis further demonstrates that TWIST-1 overexpression is a novel independent unfavourable predictor for both OS and EFS in AML. These data highlight TWIST-1 as a new candidate gene contributing to leukemogenesis of myeloid leukemia, and propose possible new avenues for improving risk and treatment stratification in AML.     

Small‐molecule Hedgehog inhibitor attenuates the leukemia‐initiation potential of acute myeloid leukemia cells

Aberrant activation of the Hedgehog signaling pathway has been implicated in the maintenance of leukemia stem cell populations in several model systems. PF‐04449913 (PF‐913) is a selective, small‐molecule inhibitor of Smoothened, a membrane protein that regulates the Hedgehog pathway. However, details of the proof‐of‐concept and mechanism of action of PF‐913 following administration to patients with acute myeloid leukemia (AML) are unclear. This study examined the role of the Hedgehog signaling pathway in AML cells, and evaluated the in vitro and in vivo effects of the Smoothened inhibitor PF‐913. In primary AML cells, activation of the Hedgehog signaling pathway was more pronounced in CD34⁺ cells than CD34^? cells. In vitro treatment with PF‐913 induced a decrease in the quiescent cell population accompanied by minimal cell death. In vivo treatment with PF‐913 attenuated the leukemia‐initiation potential of AML cells in a serial transplantation mouse model, while limiting reduction of tumor burden in a primary xenotransplant system. Comprehensive gene set enrichment analysis revealed that PF‐913 modulated self‐renewal signatures and cell cycle progression. Furthermore, PF‐913 sensitized AML cells to cytosine arabinoside, and abrogated resistance to cytosine arabinoside in AML cells cocultured with HS‐5 stromal cells. These findings imply that pharmacologic inhibition of Hedgehog signaling attenuates the leukemia‐initiation potential, and also enhanced AML therapy by sensitizing dormant leukemia stem cells to chemotherapy and overcoming resistance in the bone marrow microenvironment.     

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34 acute myeloid leukemia cells

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34⁺ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.     

Cancer stem/progenitor cells are highly enriched in CD133+CD44+ population in hepatocellular carcinoma

Both our previous study and other reports have suggested that CD133, originally classified as a hematopoietic stem cell marker, could be used for enrichment of cancer stem cells (CSCs) in human hepatocellular carcinoma (HCC). It was also noted that not all of CD133⁺ cells were representative of CSCs. Further identification and characterization of CSCs or tumor‐initiating cells in HCC are necessary to better understand hepatocarcinogenesis. In present study, we demonstrated that CSC phenotype could be precisely defined by co‐expression of CD133 and CD44 cell surface markers. CD133⁺CD44⁺ HCC cells showed stem cell properties, including extensive proliferation, self‐renewal, and differentiation into the bulk of cancer cells. In vivo xenograft experiments revealed that, actually, the highly tumorigenic capacity of CD133⁺ cells as previously described was primarily attributed to CD133⁺CD44⁺ cell subpopulation, instead of their CD133⁺CD44^? counterparts. Moreover, cells double‐positive for CD133 and CD44 exhibited preferential expression of some stem cell‐associated genes and were more resistant to chemotherapeutic agents due to the upregulation of ATP‐binding cassette (ABC) superfamily transporters, including ABCB1, ABCC1, and ABCG2, further supporting these cells as HCC cell origin. Our findings suggest that CD133⁺CD44⁺ cells might represent true cancer stem/progenitor cells in HCC, which could allow a better understanding of HCC initiation and progression, as well as establish a precise target for the development of more effective therapies.