Inhibition of CML Stem Cell Growth By Targeting WNT Signaling Using a Porcupine Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3130-3130 ◽  
Author(s):  
Puneet Agarwal ◽  
Bin Zhang ◽  
YinWei Ho ◽  
Amy Cook ◽  
Ling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Biomedical Research ◽  
Fold Reduction ◽  
Cd34 Cells ◽  
Presence And Absence ◽  
Tki Treatment

Abstract BCR-ABL tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myeloid leukemia (CML). However, TKIs are unable to eradicate the leukemia stem cells (LSCs) from which CML arises, despite effective inhibition of BCR-ABL kinase activity in this population. There is evidence that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our previous studies have shown a role for Wnt signaling in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Palmitoylation of Wnts by the Porcupine acyltransferase (PORCN) is required for their secretion and activity. LGK974, a potent PORCN inhibitor, has recently been shown to inhibit Wnt signaling and shows in vivo efficacy against several Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). In this study, we investigated whether inhibition of Wnt secretion using LGK974 could sensitize CML stem/progenitor cells to TKI treatment. CML and normal CD34+ cells and purified CD34+CD38- stem cells and CD34+CD38+ committed progenitors were treated with LGK974 with or without addition of the BCR-ABL TKI (nilotinib), and in the presence and absence of human BM mesenchymal stromal cells (MSC). CML CD34+ cells showed enhanced Wnt signaling following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells as evidenced by increased phospho-LRP6 (ser1490) and β-catenin protein levels. Analysis of all 10 frizzled receptors expression by qRT-PCR revealed a significant upregulation of FZD4 (p=0.026, n=10), and FZD5 (p=0.003, n=10), but downregulation of FZD8 (p=0.009, n=10), in CML compared to normal CD34+ cells. Co-culture with MSC was associated with increased Wnt signaling in CML CD34+ cells as detected by increased expression and nuclear translocation of β-catenin protein, and increased expression of Wnt target genes including c-Myc, Cyclin-D1, PPARδ, and Axin2. Treatment with LGK974 reduced Wnt secretion from MSC measured in a TCF/LEF reporter assay (LGK 0.5μM, 0.74 fold reduction compared to control, p=0.016, n=5, LGK 1μM, 0.62 fold reduction, p=0.007, n=5) and potently inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with LGK974 (1μM) in combination with nilotinib (1μM) resulted in significantly increased inhibition of CML CFC growth compared to nilotinib alone, both in the presence and absence of MSC (34+38- cells LGK alone 82.75±6.38% inhibition of control, Nil alone 43.12±15.32%, combination 23.17±6.48%, p<0.05, n=4; 34+38+ cells LGK alone 87.65±6.13% inhibition of control, Nil alone 26.99±4.54%, combination 16.04±0.48%, p<0.05, n=4) Importantly, LGK974 inhibited CML CFC growth to a significantly greater extent than the normal CFC growth ( normal 34+38- cells LGK alone 88.09±6.85% inhibition of control, Nil alone 89.79±10.09%, combination 70.95±9.39%, p=ns, n=4; 34+38+ cells LGK alone 86.42±8.41% inhibition of control, Nil alone 87.43±4.66%, combination 73.39±6.91%, p=ns, n=4). However, LGK974 treatment did not induce apoptosis in CML or normal progenitors. These results suggest that the effects of LGK974 on clonogenic growth could be related to the long-term inhibition of LSC self-renewal. Therefore, studies designed to evaluate the effect of in vivo administration of LGK974 in a transgenic BCR-ABL mouse model of CML are in progress. Finally, our preliminary studies indicate that in vivo treatment of control healthy mice with LGK974 (5mg/kg bid p.o.), nilotinib (50mg/kg qd p.o.), or the combination for 4 weeks (n=7 per cohort) did not result in clinical toxicity or alter the short-term and long-term hematopoietic stem cell populations. In conclusion, our results indicate that treatment with the PORCN inhibitor LGK974 effectively inhibits both autocrine and paracrine WNT signaling in CML stem cells and inhibits their clonogenic capacity in vitro, and support further evaluation of this approach to selectively target CML stem cells. Disclosures Wang: Novartis Institute for Biomedical Research: Employment. McLaughlin:Novartis Institute for Biomedical Research: Employment.

Inhibition of CML Stem Cell Renewal By the Porcupine Inhibitor WNT974

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 54-54
Author(s):  
Puneet Agarwal ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Amy Cook ◽  
Ling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Cyclin D1 ◽  
Biomedical Research ◽  
Cell Renewal ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Tki Treatment

Abstract Treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs) fails to eradicate the leukemia stem cells (LSCs) from which the disease arises. Others and we have shown that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our studies indicate that microenvironmental Wnt signaling may play a role for in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Wnt secretion and activity requires their palmitoylation by the Porcupine acyltransferase (PORCN). WNT974 is a potent PORCN inhibitor that inhibits Wnt signaling and demonstrates in vivo efficacy against Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). We have investigated whether WNT974 could sensitize CML stem/progenitor cells to TKI treatment. We observed that CML CD34+ cells show enhanced increased phospho-LRP6 (ser1490) expression, β-catenin protein levels, and colony-forming cell (CFC) growth following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells (isolated from cord blood of healthy donors) indicating an enhanced Wnt signaling response. We observed significant upregulation of FZD4 and FZD5 expression in CML compared to normal CD34+ cells on qPCR analysis, which could potentially contribute to their enhanced Wnt sensitivity. CML CD34+ cells enhanced Wnt signaling following co-culture with human bone marrow mesenchymal stromal cells (MSC) as evidenced by increased β-catenin expression and nuclear translocation, and increased expression of the Wnt target genes c-Myc, Cyclin-D1, PPARδ, and Axin2. WNT974 exposure reduced Wnt secretion from MSC and inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with WNT974 (1μM) in combination with nilotinib (Nil, 1μM) resulted in significant inhibition of CML CFC growth, both in the presence and absence of MSC, and to a significantly greater extent than the normal CFC growth. Although, no difference in engraftment of normal CD34+ cells treated with individual drugs or combination in immunodeficient mice after 16 weeks was observed, treatment of CML CD34+ cells with WNT974 + Nil resulted in significantly decreased engraftment of BCR-ABL+ CML LSC. We further investigated the effects of WNT974 and Nil on CML hematopoiesis in vivo using a transgenic BCR-ABL mouse model of CML. BM cells from CML mice were transplanted into congenic wild-type FVB/N mice to generate CML-like disease in recipient mice. Three weeks after transplantation, mice were treated with Vehicle, WNT974 (5mg/kg bid p.o), Nil (50mg/kg qd p.o), or the combination for 2 weeks. Treatment with WNT974 + Nil resulted in significantly greater reduction in WBC levels, %neutrophils and myeloid cells (Gr-1+ CD11b+) in the peripheral blood of CML mice compared with Nil alone. Long-term hematopoietic stem cells (LTHSC) and committed progenitors (MPP, CMP, GMP) were significantly reduced in the BM and spleen of mice treated with combination compared to individual drugs. Next, we evaluated subsequent survival of mice after completion of 3 weeks of drug treatment. Whereas all control mice died by 30 days, mice treated with the combination of WNT974 + Nil showed significantly prolonged survival after discontinuation of treatment when compared to individual drug-treated mice (p=0.0146). To assess the effects on LSC self-renewal potential, BM cells from primary drug-treated mice were transplanted into normal secondary recipient mice. At 12 weeks post-transplantation, there was a significant reduction in LTHSC, MPP, and GMP populations in the BM of mice transplanted with cells from primary mice treated with the combination compared with individual drugs, indicating reduced LSC self-renewal. Finally, we evaluated the effect of in vivo treatment on Wnt/β-catenin signaling in leukemic cells. Significant downregulation of c-Myc, Cyclin D1, and Axin2 was seen on qPCR performed on c-Kit+ BM progenitors from mice treated with WNT974 and Nil+WNT974 compared to vehicle treated mice. Therefore, we conclude that inhibition of Wnt secretion and signaling by treatment with the PORCN inhibitor WNT974 effectively and selectively inhibits the self-renewal capacity of CML LSC. These results support the further evaluation of combinations of PORCN inhibitors with TKI treatment for selective targeting of CML LSC. Disclosures Wang: Novartis Institute of Biomedical Research: Employment. Mclaughlin:Novartis Institute of Biomedical Research: Employment.

scholarly journals Long-Term Cryopreservation Does Not Affect Quality of Peripheral Blood Stem Cell Grafts: A Comparative Study of Native, Short-Term and Long-Term Cryopreserved Haematopoietic Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972110360
Author(s):  
Daniel Lysak ◽  
Michaela Brychtová ◽  
Martin Leba ◽  
Miroslava Čedíková ◽  
Daniel Georgiev ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Statistical Significance ◽  
Extended Period ◽  
High Dose ◽  
Atp Production ◽  
Cd34 Cells ◽  
Negative Effect

Cryopreserved haematopoietic progenitor cells are used to restore autologous haematopoiesis after high dose chemotherapy. Although the cells are routinely stored for a long period, concerns remain about the maximum storage time and the possible negative effect of storage on their potency. We evaluated the effect of cryopreservation on the quality of peripheral stem cell grafts stored for a short (3 months) and a long (10 years) period and we compared it to native products.The viability of CD34+ cells remained unaffected during storage, the apoptotic cells were represented up to 10% and did not differ between groups. The clonogenic activity measured by ATP production has decreased with the length of storage (ATP/cell 1.28 nM in native vs. 0.63 in long term stored products, P < 0.05). Only borderline changes without statistical significance were detected when examining mitochondrial and aldehyde dehydrogenase metabolic activity and intracellular pH, showing their good preservation during cell storage. Our experience demonstrates that cryostorage has no major negative effect on stem cell quality and potency, and therefore autologous stem cells can be stored safely for an extended period of at least 10 years. On the other hand, long term storage for 10 years and longer may lead to mild reduction of clonogenic capacity. When a sufficient dose of stem cells is infused, these changes will not have a clinical impact. However, in products stored beyond 10 years, especially when a low number of CD34+ cells is available, the quality of stem cell graft should be verified before infusion using the appropriate potency assays.

Enhancing therapeutic effects and in vivo tracking of adipose tissue derived mesenchymal stem cells for liver injury using bioorthogonal click chemistry

Nanoscale ◽  
2020 ◽  
Author(s):  
Naishun Liao ◽  
Da Zhang ◽  
Ming Wu ◽  
Huang-Hao Yang ◽  
Xiaolong Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Stem Cell ◽  
Liver Injury ◽  
Mesenchymal Stem Cell ◽  
Liver Diseases ◽  
Therapeutic Effects

Adipose tissue derived mesenchymal stem cell (ADSC)-based therapy is attractive for liver diseases, but the long-term therapeutic outcome is still far from satisfaction due to low hepatic engraftment efficiency of...

scholarly journals A Novel Small Molecule CXCR4 Antagonist Potently Mobilizes Hematopoietic Stem Cells in Mice and Monkeys

2020 ◽  
Author(s):  
Xiao Fang ◽  
Xiong Fang ◽  
Yujia Mao ◽  
Aaron Ciechanover ◽  
Yan Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Small Molecule ◽  
Cxcr4 Antagonist ◽  
Hematopoietic Stem ◽  
Stromal Microenvironment ◽  
Stromal Cell Derived Factor ◽  
Hsc Transplantation

Abstract Background Hematopoietic stem cell (HSC) transplantation is an effective treatment strategy for many types of diseases. Peripheral blood (PB) is the most commonly used source of bone marrow (BM)-derived stem cells for current HSC transplantation. However, PB usually contains very few HSCs under normal conditions, as these cells are normally retained within the BM. This retention depends on the interaction between the CXC chemokine receptor 4 (CXCR4) expressed on the HSCs and its natural chemokine ligand, stromal cell-derived factor (SDF)-1α (also named CXCL12) present in the BM stromal microenvironment. In clinical practice, blocking this interaction with a CXCR4 antagonist can induce the rapid mobilization of HSCs from the BM into the PB.Methods C3H/HEJ, DBA/2, CD45.1+, CD45.2+ mice and monkeys were employed in colony-forming unit (CFU) assays, flow cytometry assays, and competitive/non-competitive transplantation assays, to assess the short-term mobilization efficacy of HF51116 and the long-term repopulating (LTR) ability of HSCs. Kinetics of different blood cells and the concentration of HF51116 in PB were also explored by blood routine examinations and pharmacokinetic assays. Results In this paper, we report that a novel small molecule CXCR4 antagonist, HF51116, which was designed and synthesized by our laboratory, can rapidly and potently mobilize HSCs from BM to PB in mice and monkeys. HF51116 not only mobilized HSCs when used alone but also synergized with the mobilizing effects of granulocyte-colony stimulating factor (G-CSF) after co-administration. Following mobilization by HF51116 and G-CSF, the long-term repopulating (LTR) and self-renewing HSCs were sufficiently engrafted in primary and secondary lethally irradiated mice and were able to rescue and support long-term mouse survival. In monkeys, HF51116 exhibited strong HSC mobilization activity and quickly reached the highest in vivo blood drug concentration. Conclusions These results demonstrate that HF51116 is a new promising stem cell mobilizer which specifically targets CXCR4 and merits further preclinical and clinical studies.

scholarly journals Silencing of long noncoding RNA HOXA11-AS inhibits the Wnt signaling pathway via the upregulation of HOXA11 and thereby inhibits the proliferation, invasion, and self-renewal of hepatocellular carcinoma stem cells

2019 ◽  
Vol 51 (11) ◽  
pp. 1-20 ◽  
Author(s):  
Jun-Cheng Guo ◽  
Yi-Jun Yang ◽  
Jin-Fang Zheng ◽  
Jian-Quan Zhang ◽  
Min Guo ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Stem Cells ◽  
Stem Cell ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Methylation Level ◽  
Wnt Signaling Pathway ◽  
The Self ◽  
Self Renewal

AbstractHepatocellular carcinoma (HCC) is a major cause of cancer-related deaths, but its molecular mechanisms are not yet well characterized. Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, including that of HCC. However, the role of homeobox A11 antisense (HOXA11-AS) in determining HCC stem cell characteristics remains to be explained; hence, this study aimed to investigate the effects of HOXA11-AS on HCC stem cell characteristics. Initially, the expression patterns of HOXA11-AS and HOXA11 in HCC tissues, cells, and stem cells were determined. HCC stem cells, successfully sorted from Hep3B and Huh7 cells, were transfected with short hairpin or overexpression plasmids for HOXA11-AS or HOXA11 overexpression and depletion, with an aim to study the influences of these mediators on the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo. Additionally, the potential relationship and the regulatory mechanisms that link HOXA11-AS, HOXA11, and the Wnt signaling pathway were explored through treatment with Dickkopf-1 (a Wnt signaling pathway inhibitor). HCC stem cells showed high expression of HOXA11-AS and low expression of HOXA11. Both HOXA11-AS silencing and HOXA11 overexpression suppressed the self-renewal, proliferation, migration, and tumorigenicity of HCC stem cells in vivo, as evidenced by the decreased expression of cancer stem cell surface markers (CD133 and CD44) and stemness-related transcription factors (Nanog, Sox2, and Oct4). Moreover, silencing HOXA11-AS inactivated the Wnt signaling pathway by decreasing the methylation level of the HOXA11 promoter, thereby inhibiting HCC stem cell characteristics. Collectively, this study suggested that HOXA11-AS silencing exerts an antitumor effect, suppressing HCC development via Wnt signaling pathway inactivation by decreasing the methylation level of the HOXA11 promoter.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

scholarly journals AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽  
2006 ◽  
Vol 107 (9) ◽  
pp. 3772-3778 ◽  
Author(s):  
André Larochelle ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Donald Orlic ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Genetic Manipulation ◽  
Retroviral Vectors ◽  
Lymphoid Cells ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Radiation Dose Determines Degree of Donor Chimerism after Nonmyeloablative Hematopoietic Cell Transplantation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1828-1828 ◽  
Author(s):  
Christoph Kahl ◽  
Marco Mielcarek ◽  
Mineo Iwata ◽  
Michael Harkey ◽  
Barry Storer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Radiation Dose ◽  
Low Dose ◽  
Donor Chimerism ◽  
Stem Cell Source ◽  
Cd34 Cells ◽  
Cell Source ◽  
Total Body

Abstract Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivo irradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.

Leukemic Stem Cells of Chronic Phase CML Patients Consistently Display Very High BCR-ABL Transcript Levels and Reduced Responsiveness to Imatinib Mesylate in Addition to Generating a Rare Subset That Produce Imatinib Mesylate-Resistant Differentiated Progeny.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 711-711 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Yun Zhao ◽  
Wing Yiu Chan ◽  
Emily Pang ◽  
Allen Eaves ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Imatinib Mesylate ◽  
Chronic Phase ◽  
Stem Cell Differentiation ◽  
Epigenetic Mechanism ◽  
Transcript Levels ◽  
Viable Cells ◽  
Cd34 Cells

Abstract Imatinib mesylate (IM) is an inhibitor of the BCR-ABL oncoprotein associated with human chronic myeloid leukemia (CML). IM therapy has shown remarkable effects in initial clinical trials, but both clinical and laboratory studies increasingly suggest that, on its own, IM may have limited curative potential, due to a reduced IM sensitivity of the more primitive, slowly proliferating CD34+ CML cells thought to be responsible for sustaining the disease in vivo. To investigate the basis of this unresponsiveness, we compared the IM sensitivity and BCR-ABL expression of FACS-purified subsets of lin−CD34+ cells from 4 CML chronic phase patients. None of these had been treated with IM and their cells at all stages of differentiation were exclusively leukemic; i.e., >95% of the lin−CD34+CD38−, lin−CD34+CD38+ and lin+CD34− cells were BCR-ABL+ (by direct FISH) and all longterm culture-initiating cell (LTC-IC) -derived CFCs were Ph+. In the absence of IM, suspension cultures initiated with these lin−CD34+CD38− CML cells (0.5–5% of the lin−CD34+ cells) showed a net expansion of viable cells after 3 weeks; 100x with and 10x without added growth factors (GFs). Addition of 0.1–10 μM/ml IM reduced the yield of viable cells in a dose-dependent fashion, particularly when GFs were not added (100-fold decrease with 10 μM/ml IM). Parallel cultures of the corresponding lin−CD34+CD38+ CML cells showed these did not expanded as much (~8x +GFs, 2x -GFs) and were more sensitive to IM (1000-fold decrease after 3 weeks in 10 μM/ml IM -GFs). Quantitative real-time RT-PCR analysis revealed BCR-ABL transcripts to be present in the most primitive, freshly isolated lin−CD34+CD38− cells (n=12) at >300-fold higher levels than in the terminally differentiating lin+CD34− CML cells (n=21), at >10-fold higher levels than the normal BCR transcripts in the same lin−CD34+CD38− cells, and at 40-fold higher levels than in the less primitive lin−CD34+CD38+ cells (n=12), indicating a correlation between decreasing BCR-ABL transcripts and increasing IM sensitivity during CML stem cell differentiation in vivo. Interestingly, maintenance of the lin−CD34+CD38− CML cells for 3 weeks in vitro with 10 μM/ml IM (±GFs) consistently selected for a subset of leukemic cells (80–100% BCR-ABL+ by FISH) that showed complete resistance to 5 μM/ml IM in CFC assays, in marked contrast to the CFCs in the starting lin−CD34+CD38− cells that were inhibited 5–10-fold by 5 μM/ml IM. Moreover, although the Ph was the sole abnormality present in all direct metaphases, initial CFCs and LTC-IC-derived CFCs from all samples, a 17p+ abnormality was seen in 4/4 metaphases obtained from one colony generated from the cells present in one of the 3-week IM-containing cultures, suggesting the selective survival of differentiating progeny of rare, pre-existing, IM-resistant stem cells. Consistent with this possibility was the finding that BCR-ABL transcript levels in the cells present in the 3 week cultures were reduced 50-fold relative to the input lin−CD34+CD38− cells. Taken together, these findings suggest a previously undescribed epigenetic mechanism of IM unresponsiveness characteristic of chronic phase CML stem cells, in addition to the silent accumulation of genetically-determined IM-resistant members as the CML stem cell population expands during the development of the chronic phase of the disease.

Increased Aldehyde Dehydrogenase Activity Indentifies a Subset of Human Leukemic Blasts with Stem Cell Characteristics and Correlates with Poor Prognosis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1345-1345
Author(s):  
Dan Ran ◽  
Mario Schubert ◽  
Larissa Pietsch ◽  
Isabel Taubert ◽  
Christian Wallenwein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Prognostic Factors ◽  
Aldehyde Dehydrogenase ◽  
Protein Level ◽  
Aldh Activity ◽  
Cd34 Cells ◽  
Dividing Cells

Abstract INTRODUCTION: Normal hematopoietic stem cells (HSC) are characterized by their ability to self-renew, to generate multiple cell-lineages, and show slow divisional kinetics. Leukemic stem cells (LSC) have been reported to show similar characteristics but their identification has been elusive. We have studied various methods and have identified aldehyde dehydrogenase (ALDH) staining as an optimal method for the enrichment of primary human LSC. MATERIAL&METHODS: Bone marrow samples were obtained from patients with newly diagnosed AML after informed consent. Mononuclear cells were stained with Aldefluor and sorted by flow cytometry according to their forward/side scatter characteristics and ALDH activity (ALDH+/ALDH−). Alternatively, primary AML samples were being enriched for CD34+ cells by magnetic column, then double-stained with CD34-antibodies and Aldefluor and sorted for the co-expression of CD34+ and ALDH+, respectively for CD34+ alone. Human Mesenchymal Stromal Cells (MSC), isolated from human bone marrow, were used as a surrogate model for the cellular microenvironment of the hematopoietic niche. Adhesion of various AML cell lines and subpopulations of primary leukemic cells (ALDH+, ALDH−, CD34+, CD34+/ALDH+, all blasts) to MSC was tested in the adhesion chamber assay. Semi-quantitative RT-PCR was used to analyze the gene expression of various adhesion molecules and Western- Blot analysis was performed to validate the PCR-results on protein level. The generation of secondary leukemic colonies was evaluated in a semi-solid methylcellulose medium, as well as in a long term co-culture system (LSC-IC assay; in analogy to the LTC-IC assay). RESULTS: The percentage of ALDH+ cells ranged from 0.01% to 13.2% with a median of 1.47% (n=55). Adhesion significantly differed in the ALDH+ and ALDH− subpopulations: 85±4% of ALDH+ cells but only 61±8% of ALDH− cells were adherent (n=11, p&lt;0.001). Adhesion molecules, such as CXCR4 and CD44, were highly expressed on the ALDH+ subpopulation both on mRNA level and protein level, in contrast to the ALDH− subpopulation. Analysis of the initial divisional kinetics on single cell base showed that the ALDH+ subpopulation contained more slow dividing cells whereas the majority of the ALDH− subpopulation consisted of fast-dividing cells (n=3; p&lt;0.01). The frequency of long term leukemic colony initiating cells (LSC-IC) was 3.82% in the ALDH+ but only 0.01% in the ALDH− (n=21; p&lt;0.01). In the CD34+ the LSC-IC frequency was 1.96% versus 0.01% in the CD34− (n=5, p&lt;0.01). The highest LSC-IC frequency could be monitored in ALDH+/CD34+ cells: 6.1% generated secondary leukemic colonies (n=5). These colonies, harvested after 7 weeks of cultivation, were examined for their immune phenotype and screened for cytogenetic aberrations by fluorescent in situ hybridization (FISH) and the chromosomal aberrations were consistent with the AML samples taken at diagnosis. Furthermore, the frequency of ALDH+ cells correlated significantly with adverse prognostic factors: patients with a high-risk karyotype had a mean of 2.9% ALDH+ cells (n=21); in contrast, patients with a normal karyotype had a mean of 0.4% ALDH+ cells in their bone marrow (n=34; p&lt;0.001). The ability of ALDH+ versus ALDH− subsets to generate secondary leukemia in the animal model is concurrently examined. DISCUSSION: In summary, measurement of the ALDH activity provides a useful tool for the isolation of a distinct AML-blast subpopulation with stem-cell like features (LSC). The ALDH+ subsets showed higher affinity to the surrogate niche (MSC), elevated expression of CD44, Cadherin-2, and CXCR4 and were associated with an increased frequency of secondary leukemic colonies in vitro (LSC-IC). Above all, the frequency of ALDH+ blasts correlated with clinical prognostic factors, which substanciates LSC as a relevant therapeutic target.

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