Nanoparticle‐Mediated Delivery of Micheliolide Analogs to Eliminate Leukemic Stem Cells in the Bone Marrow

2021 ◽  
pp. 2100100
Author(s):  
Marian A. Ackun‐Farmmer ◽  
Hanan Alwaseem ◽  
Michele Counts ◽  
Andrew Bortz ◽  
Simone Giovani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Leukemic Stem Cells

scholarly journals The quiescent fraction of chronic myeloid leukemic stem cells depends on BMPR1B, Stat3 and BMP4-niche signals to persist in patients in remission

Haematologica ◽  
2020 ◽  
Vol 106 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Sandrine Jeanpierre ◽  
Kawtar Arizkane ◽  
Supat Thongjuea ◽  
Elodie Grockowiak ◽  
Kevin Geistlich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Stromal Cells ◽  
Kinase Inhibitor ◽  
Bmp Signaling ◽  
Myelogenous Leukemia ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Chronic myelogenous leukemia arises from the transformation of hematopoietic stem cells by the BCR-ABL oncogene. Though transformed cells are predominantly BCR-ABL-dependent and sensitive to tyrosine kinase inhibitor treatment, some BMPR1B+ leukemic stem cells are treatment-insensitive and rely, among others, on the bone morphogenetic protein (BMP) pathway for their survival via a BMP4 autocrine loop. Here, we further studied the involvement of BMP signaling in favoring residual leukemic stem cell persistence in the bone marrow of patients having achieved remission under treatment. We demonstrate by single-cell RNA-Seq analysis that a sub-fraction of surviving BMPR1B+ leukemic stem cells are co-enriched in BMP signaling, quiescence and stem cell signatures, without modulation of the canonical BMP target genes, but enrichment in actors of the Jak2/Stat3 signaling pathway. Indeed, based on a new model of persisting CD34+CD38- leukemic stem cells, we show that BMPR1B+ cells display co-activated Smad1/5/8 and Stat3 pathways. Interestingly, we reveal that only the BMPR1B+ cells adhering to stromal cells display a quiescent status. Surprisingly, this quiescence is induced by treatment, while non-adherent BMPR1B+ cells treated with tyrosine kinase inhibitors continued to proliferate. The subsequent targeting of BMPR1B and Jak2 pathways decreased quiescent leukemic stem cells by promoting their cell cycle re-entry and differentiation. Moreover, while Jak2-inhibitors alone increased BMP4 production by mesenchymal cells, the addition of the newly described BMPR1B inhibitor (E6201) impaired BMP4-mediated production by stromal cells. Altogether, our data demonstrate that targeting both BMPR1B and Jak2/Stat3 efficiently impacts persisting and dormant leukemic stem cells hidden in their bone marrow microenvironment.

Loss of P-Selectin Promotes the Function of Aged Hematopoietic and Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1577-1577
Author(s):  
Yaoyu Chen ◽  
Sullivan Con ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
Cong Peng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Post Transplant ◽  
Marrow Cells

Abstract Abstract 1577 Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between the blood cells and their microenvironment. Adhesion molecules like P-selectin are essential to hematopoiesis, and their dysregulation has been implicated in leukemogenesis. We have previously shown a role for P-selectin in chronic myeloid leukemia and demonstrated that in its absence the disease process accelerates. Recently, there has also been speculation that P-selectin may play a role in the aging hematopoietic stem cells (HSCs), as its expression in upregulated as a mouse ages. In this study, we show that the loss of P-selectin function dysregulates the balance of stem cells and progenitors and that these differences become more pronounced with age. We compared the percentages of HSCs, long-term (LT)-HSCs, short-term (ST)-HSCs, multipotent progenitors (MPPs), CMPs, GMPs and MEPs in bone marrow by flow cytometry between wild type (WT) and Selp-/- mice. An age-dependent LT-HSC expansion was observed in WT mice. However, this expansion was prevented by the loss of Selp as observed in Selp-/-mice. Further, we demonstrate that with age LT-HSCs in particular express more elevated levels of P-selectin. LT-HSCs and ST-HSC/MPPs were isolated from the bone marrow of young (2 months old) and old (15 months old) WT mice and examined P-selectin expression by FACS. A significant increase in P-selectin expression was observed in LT-HSCs of old mice, and this increase was not observed in the ST-HSC+MPP subpopulations. We also show that the loss of P-selectin gene has profound effects of stem cell function, altering the capacity of these cells to home. Despite impaired homing capacity, stem cells lacking P-selectin possess a competitive advantage over their wild type counterparts. Using a stem cell competition assay, HSCs derived from Selp-/- mice (CD45.2+) and WT control mice (CD45.2+GFP+) were mixed in 1:1 ratio and transplanted into irradiated WT recipients (CD45.1). The initial findings were potentially indicative of the ability of cells derived from GFP mice to more efficiently home and engraft. Despite this initial advantage, cells derived from Selp-/- eventually exhibited a competitive and statistically significant advantage over the cells derived from GFP mice. At 30 days post-transplant, 49.9±1.4% of the CD45.2 subpopulation was GFP+. At 86 days post-transplant, 25.7±3.3 % of the CD45.2 cells derived from the peripheral blood were GFP+. Similarly, 23.0±3.7% of the CD45.2 cells derived from the bone marrow of these mice were GFP+. Indeed, we demonstrate that recipients of P-selectin deficient bone marrow cells more efficiently repopulate the bone marrow than controls and that this advantage extends and expands in the long-term. Finally, we demonstrate that recipients of leukemic cells lacking P-selectin develop a more accelerated form of leukemia accompanied by significant increases in stem and progenitor cells. Bone marrow cells from donor WT and Selp-/- mice were infected with retrovirus expressing BCR-ABL-GFP, and irradiated WT recipients were transplanted with 2×105 of these transduced donor cells. At 14 days post-transplant, recipient mice from each of the groups were sacrificed, and bone marrow cells were harvested and analyzed by flow cytometry. Recipients of leukemic Selp-/- cells possessed 3.5-fold more LSCs than recipients of wild-type cells. There were 3.1-fold more LT-LSCs and 3.8-fold more ST-LSCs and MPPs in recipients of Selp-/- cells than WT cells. In addition, recipients of leukemic Selp-/- cells possessed significantly more CMP (16.9-fold) and MEP (4.5-fold) cells. Because P-selectin expression increases with age on LT-HSCs, we sought to determine the role that age plays in CML development and progression. Bone marrow cells derived from 15-month-old donor Selp-/- and WT mice were transduced with BCR-ABL, respectively, followed by transplantation of the transduced cells into recipient mice. All recipients of BCR-ABL transduced Selp-/- cells died by 23 days after induction of CML and had a median survival of 19 days, whereas recipients of the transduced WT cells survived significantly longer. This pro-leukemic role for cells lacking P-selectin expression is leukemic stem cell-specific rather than stromal cell-specific and supports an essential role for P-selectin on leukemic stem cells. Disclosures: No relevant conflicts of interest to declare.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1882-1882
Author(s):  
Shawnya J. Michaels ◽  
Ngoc DeSouza ◽  
Yi Shan ◽  
Shaoguang Li ◽  
Saira Bates
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Solvent Control ◽  
Equity Ownership ◽  
Marrow Cells

Abstract Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed. The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs). Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells. To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner. The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive. Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure). To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table). We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. Figure Figure. Table Table. Disclosures Michaels: Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2653-2653 ◽  
Author(s):  
Yuichiro Nakata ◽  
Norimasa Yamasaki ◽  
Takeshi Ueda ◽  
Kenichiro Ikeda ◽  
Akiko Nagamachi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Epigenetic Mark ◽  
M2 Macrophage ◽  
Leukemic Stem Cells ◽  
Cell Potential ◽  
Hematopoietic Stem

Abstract Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive. To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs. We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. Disclosures No relevant conflicts of interest to declare.

Berberine inhibits SDF-1-induced AML cells and leukemic stem cells migration via regulation of SDF-1 level in bone marrow stromal cells

2008 ◽  
Vol 62 (9) ◽  
pp. 573-578 ◽  
Author(s):  
Huiyu Li ◽  
Linlin Guo ◽  
Shenghua Jie ◽  
Wei Liu ◽  
Jiang Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Stem Cells

scholarly journals The Role of Notch in Endothelial Cell-Mediated Protection of AML Precursors from Targeted Therapy

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3947-3947
Author(s):  
Quy Le ◽  
Brandon Hadland ◽  
Soheil Meshinchi ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Notch Signaling ◽  
Liquid Culture ◽  
Critical Role ◽  
Common Mutation ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibitory Antibodies

Abstract Background: AML is an aggressive hematologic malignancy that remains difficult to treat. A common mutation found in AML is FLT3-ITD, occurring in 15% of childhood AML. Although chemotherapy has successfully induced remission, patients with a high FLT3 ITD:WT allelic ratio (FLT3-AR) exhibit a high relapse rate, requiring hematopoietic stem cell transplantation to increase the chance of long-term remission. In this study, we demonstrate the requirement of ECs for survival of FLT3-ITD progenitors from primary pediatric AML specimens in the presence of AC220, a potent and selective inhibitor of FLT3. We further show that the Notch pathway plays a role in EC-mediated protection amongst patient samples with high FLT3-AR, suggesting the potential therapeutic use of Notch blockade in the treatment of this high-risk subset. Results: To determine whether ECs confer protection to FLT3-ITD progenitors, we quantified the number of CFC present after 2 weeks of liquid culture or EC co-culture with AC220 (added at days 0, 3 and 7) from four AML specimens with high FLT3-AR (≥1). We used PCR to determine the presence of FLT3-ITD in individual CFC. We found that the numbers of FLT3-ITD CFC (p=0.007) and FLT3-WT CFC (p=0.044) were reduced in liquid culture compared to EC co-culture, suggesting that ECs mediate the survival of FLT3-ITD hematopoietic progenitors against the therapeutic treatment of AC220. Previously, we demonstrated that ECs are critical for the growth and expansion of hematopoietic stem cells, which is dependent on the activation of Notch signaling. We asked whether Notch plays a role in EC-mediated protection of AML progenitors against AC220, using RNA-seq analysis on three FLT3-ITD-harboring AML. Among the significantly altered genes (FDR<0.05), we found an enrichment of Notch target genes that were expressed at significantly higher levels in AC220-treated cells compared to DMSO-treated cells, including HES1, HES4, NRARP, CDKN1A, CCND1, andGATA3, suggesting that Notch signaling may facilitate EC-mediated protection against AC220. Next, we assessed the effect of inhibiting Notch signaling on AML progenitor survival during AC220 treatment in EC co-culture, using inhibitory antibodies specific to the Negative Regulatory Region (NRR) of both Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2; kindly provided by Chris Siebel, Genentech). We co-cultured bone marrow cells from eight patient specimens with low FLT3-AR (<1) and five patient specimens with high FLT3-AR (≥1), with ECs and briefly treated the co-cultures with Notch inhibitory antibodies or IgG1 antibody for 3 days. AC220 was added to the cultures at days 0, 3 and 7. We assessed CFC numbers present after 2 weeks of culture. Patient samples with low FLT3-AR did not exhibit changes in the numbers of FLT3-ITD CFC (p = 0.735) and FLT3-WT CFC (p = 0.489) in response to Notch inhibition relative to IgG1 control. In contrast, patient samples with high FLT3-AR showed reduction in the number of FLT3-ITD CFC (p=0.019) but the number of FLT3-WT CFC remained unaffected (p=0.874). These results suggest a critical role for Notch in EC-mediated protection in AML with high FLT3-AR. Conclusion: Our studies suggest that inhibiting Notch signaling may have therapeutic potential for overcoming drug resistance induced by the tumor microenvironment in a subset of AML with high FLT3-AR. We have previously shown that a high FLT3-AR is associated with the presence of FLT3-ITD in the least mature hematopoietic subset (CD34+ CD33- precursors), which is thought to contain leukemic stem cells, and this association is correlated with poorer outcome. Additionally, AML cells that give rise to CFC after long-term co-culture with bone marrow stroma or ECs are derived from the CD34+CD33- AML precursors. Ongoing studies aim to determine whether Notch signaling plays a role in the survival of AML CD34+CD33- cells with the goal of eliminating leukemic stem cells responsible for relapse. Disclosures No relevant conflicts of interest to declare.

MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 62-62
Author(s):  
Brian J.P. Huntly ◽  
Hirokazu Shigematzu ◽  
Kenji Deguchi ◽  
Benjamin Lee ◽  
Shinichi Mizuno ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mononuclear Cells ◽  
Hierarchical Organization ◽  
Leukemic Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Acute Myeloid ◽  
Myeloid Progenitors

Abstract The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype. In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype. These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

BMI-1 Overexpression in 32Dcl3 Cells Suppresses Granulocytic Differentiation and Ameliorates Their Growth under IL-3 Diminished Conditions.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4211-4211
Author(s):  
Asahi Hishida ◽  
Kazuhito Yamamoto ◽  
Masashi Sawa ◽  
Tomoki Naoe
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Real Time Pcr ◽  
Bone Marrow Cells ◽  
Polycomb Group ◽  
The Self ◽  
Leukemic Stem Cells ◽  
Granulocytic Differentiation ◽  
Self Renewal ◽  
Mtt Assays

Abstract The BMI-1 gene is one of the polycomb group (PcG) genes that have been shown to play essential roles in the self-renewal of both normal and leukemic stem cells. BMI-1 is reported to be highly expressed in primitive cells and its expression level decreases as bone marrow cells differentiate. We hypothesized that overexpression of BMI-1 might modulate the growth, differentiation and survival of 32D cells, and examined this hypothesis by generating BMI-1 overexpressing 32D cells. 32D cells were infected with MIG-BMI-1-IRES-EGFP vector or MIG vector, and the GFP positive rates of BMI-1 overexpressing 32D cells gradually increased under IL-3 diminished conditions, and the consecutive MTT assays using cells sorted by GFP positivity confirmed this finding, suggesting that BMI-1 could confer growth advantages on 32D cells under IL-3 diminished conditions. Exposure of the GFP sorted 32D cells to G-CSF revealed that BMI-1 overexpression suppressed the granulocytic differentiation of 32D cells by G-CSF. The expressions of CD11b and Gr-1 were both suppressed by the overexpression of BMI-1, and real-time PCR analyses indicated that the expression levels of MPO was remarkably suppressed, while those of C/EBPα were not significantly changed. Our experiments revealed that BMI-1 delayed granulocytic differentiation and ameliorated the growth of 32D cells under IL-3 diminished conditions.

Selectins and Their Ligands Are Required for Homing and Engraftment of BCR-ABL+ Leukemia-Initiating Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 697-697 ◽  
Author(s):  
Daniela S. Krause ◽  
Ulrich H. von Andrian ◽  
Richard A. Van Etten
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Expression Levels ◽  
Selectin Ligand ◽  
Beta 1 Integrin

Abstract Autologous hematopoietic stem cell (HSC) transplantation is a feasible form of treatment for many types of leukemias and lymphomas, including chronic myeloid leukemia (CML). Malignant cells contaminating the graft, however, can engraft and lead to relapse of the original disease. Previous studies have demonstrated that BCR-ABL+ leukemic progenitors have defects in the adhesive function of beta-1 integrins and in their response to the chemokine SDF-1alpha, pathways that are critical for homing and engraftment of normal HSC. We hypothesized that BCR-ABL-expressing leukemic stem cells differ from normal HSC in their homing and engraftment properties. Using a retroviral transduction/transplantation model of CML and donor/recipient mice with mutations in adhesion molecules, we investigated the role of specific adhesion pathways in the engraftment of CML-like leukemia. We found no difference in the expression levels of integrins, LFA-1, and CXCR4 between normal and BCR-ABL+ c-Kit+ Lin- cells, but lower expression levels of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. In transplantation experiments, VCAM-1, the principal bone marrow ligand for beta-1 integrin, was not required in the bone marrow endothelium of the recipient for efficient engraftment of CML-like disease, confirming that progenitors capable of initiating CML-like leukemia upon transplantation are independent of the beta-1 integrin pathway for engraftment. Likewise, recipient P-selectin was also not required for the engraftment of CML-like leukemia. By contrast, deficiency of PSGL-1 in the leukemic cells or of E-selectin in the recipient significantly reduced engraftment by BCR-ABL-expressing stem cells, as assessed by Southern blot quantitation of proviral clone frequency. The requirement for recipient E-selectin could be bypassed by direct intrafemoral injection of BCR-ABL-expressing cells, leading to polyclonal leukemia. BCR-ABL-expressing cells that were deficient for the selectin ligand-synthesizing enzymes Core-2 or Fucosyltransferases IV and VII also exhibited decreased engraftment and increased disease latency. Treatment of BCR-ABL-transduced cells with neuraminidase, which destroys selectin binding sites, completely blocked leukemic engraftment. Whereas L-selectin has no role in homing and engraftment of normal HSC, BCR-ABL-expressing L-selectin-deficient progenitors were profoundly defective for engraftment, with decreased disease clonality, increased disease latency, and frequent death of recipients from graft failure. Importantly, efficient engraftment and leukemogenesis of BCR-ABL-expressing L-selectin-deficient cells was restored by co-expression of a chimeric E/L-selectin molecule that is resistant to cell surface shedding. These results establish that BCR-ABL-expressing leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than normal HSC. Specific blocking of selectin-ligand interactions is a novel clinical strategy to exploit the differences in normal and Ph+ stem cells that may be beneficial in an autologous transplantation setting.

Hsp-90 Inhibition Decreases Survival of BCR-ABL T315I Leukemic Stem Cells in Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2184-2184
Author(s):  
Cong Peng ◽  
Julia Brain ◽  
Yiguo Hu ◽  
Linghong Kong ◽  
Ami Goodrich ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Inhibitory Effect ◽  
Hsp90 Inhibitor ◽  
Leukemia Stem Cells ◽  
Imatinib Treatment ◽  
Leukemic Stem Cells ◽  
Facs Analysis

Abstract Although advances have been made in the development of novel molecularly targeted drugs, a major therapeutic challenge in the treatment of patients with Philadelphia chromosome positive (Ph+) leukemia includes understanding how to target the leukemic stem cell. We used the bone marrow transplant (BMT) model of chronic myelogenous leukemia (CML) to study effects of imatinib mesylate and the novel, orally active heat shock protein 90 (Hsp90) inhibitor, IPI-504, on leukemic stem cells, based on our observation that unlike imatinib, IPI-504, prolongs survival in a murine model of drug-resistant T315I BCR-ABL-induced CML. We first identified BCR-ABL-expressing hematopoietic stem cells (HSCs) (Lin-c-Kit+Sca-1+) in mouse bone marrow as CML stem cells, as these cells sorted out by FACS from primary CML mice are sufficient to confer leukemia in recipient mice. We then investigated the effects of imatinib and IPI-504 on survival of leukemic stem cells from BCR-ABL T315I induced CML. Bone marrow cells from mice with T315I-induced CML were cultured under conditions that support survival and growth of stem cells, with or without IPI-504 or imatinib. FACS analysis of GFP+Lin-c-Kit+Sca-1+ cells showed that imatinib treatment did not lower the percentage and the number of leukemia stem cells, whereas IPI-504 treatment had a dramatic inhibitory effect on this population (p<0.001) at therapeutically achievable doses. To determine whether IPI-504 attenuates development of leukemia by specifically inhibiting stem cell survival, GFP+Lin-c-Kit+Sca-1+ cells were sorted from bone marrow of mice with BCR-ABL T315I-induced CML, and cultured with a placebo or IPI-504. When these cells were transferred into lethally-irradiated recipient mice, FACS analysis showed that myeloid leukemia cells were present in mice receiving the placebo-treated leukemic stem cells but not in mice receiving the IPI-504 treated leukemic stem cells. To examine whether IPI-504 inhibits leukemia stem cells in vivo, mice with BCR-ABL-T315I-induced CML were treated with a placebo, imatinib, or orally administered IPI-504 for six days. Bone marrow cells were analyzed by FACS for GFP+Lin-c-Kit+Sca-1+ cells. Consistent with the in vitro results, imatinib treatment did not lower the percentage and number of leukemia stem cells, as compared with the untreated group, whereas IPI-504 treatment had a dramatic inhibitory effect on the stem cells. Analysis of bone marrow from non-leukemic mice treated with IPI-504 for two weeks showed no change in levels of Lin-c-Kit+Sca-1+ cells, indicating that IPI-504 treatment did not inhibit survival of normal HSCs. These results provide a rationale for use of an Hsp90 inhibitor as a first-line treatment to inhibit leukemia stem cells and prevent emergence of imatininb-resistant clones in patients.

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