SFK Inhibition with Dasatinib Results In Selective Targeting of Primitive Human Acute Myeloid Leukemia Stem and Progenitor Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1053-1053
Author(s):  
Cedric Emmanuel Dos Santos ◽  
Tinisha McDonald ◽  
Liang Li ◽  
Allen Lin ◽  
Ya-Huei Kuo ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Growth And Survival ◽  
Cd34 Cells ◽  
Dependent Inhibition ◽  
Stem And Progenitor Cells ◽  
Control Versus ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Abstract Abstract 1053 The Src family tyrosine kinases (SFKs) are abnormally activated in AML compared to normal CD34+ hematopoietic progenitors. Studies using pharmacological and siRNA approaches indicate an important role for the SFK Lyn in AML progenitor cell growth and survival (Dos Santos et al., 2008). However the role of SFKs in AML leukemic stem cell (LSC) growth and survival is not clear. SFK activity in Lin- CD34+ CD38- primitive stem/progenitor cells, Lin- CD34+ CD38+ committed progenitors, and Lin-CD34- cells from primary human AML (n=14) and cord blood (CB) (n=6) samples was measured by analyzing SFK phosphorylation using flow cytometry. We observed significant increase of SFK phosphorylation in AML compared to CB Lin- CD34+CD38- cells (3.8±1.9 versus 1.9±0.7, p=0.006), Lin- CD34+ CD38+ (3.9±1.7 versus 1.9±0.6, p=0.013) and Lin- CD34- cells (3.4±1.8 versus 1.1±0.5, p=0.0005). Dasatinib, a potent SFK and ABL kinase inhibitor, is approved for clinical use in chronic myeloid leukemia. We evaluated the effect of SFK inhibition using Dasatinib on the growth and viability of AML stem and progenitor cells. Dasatinib exposure resulted in dose-dependent inhibition of SFK phosphorylation in each subpopulation (in CD34+ CD38-, 3.0 for the control versus 1.9 with 100nM and 1.6 with 500nM, in CD34+ CD38+, 2.8 for the control versus 1.9 with 100nM and 1.6 with 500nM) after 30 minutes of drug treatment). The addition of Dasatinib (10-500nM) to methylcellulose progenitor assays resulted in dose-dependent inhibition of AML colony forming cell (CFC) growth (83.9±16.1% inhibition with 500nM, and 48.1±6.8% inhibition with 10nM Dasatinib, n=8), to a greater extent than CB CFC to (CFU-GM inhibition 62.6±1.5% with 500nM, and 1.1±16.3% with 10nM Dasatinib, n=4). Short-term exposure to Dasatinib (10-500nM) for 48 hours also resulted in significantly greater inhibition of AML CFC (73.6±13% with 500nM, and 41.7±10.8% with 10nM Dasatinib, n=8) compared to CB CFC (CFU-GM inhibition 23±9.1% with 500nM, and 1.3±11.3% with 10nM Dasatinib, n=4). Importantly Dasatinib treatment (200nM) also resulted in reduction of AML stem/primitive progenitor growth in long term culture-initiating cells (LTC-IC) assays (56±23, 8 % inhibition, p=0.003, n=4), suggesting that SFK inhibition may inhibit AML stem cell maintenance. The effect of Dasatinib on apoptosis was evaluated by labeling cells with Annexin V and DAPI. Treatment with Dasatinib resulted in significant increase in apoptosis of Lin- AML cells (41.5% ±10.7 of apoptosis with 200nM Dasatinib versus 25%±10.8 for the control, p=0,004, n=5) We studied the effects of Dasatinib on differentiation of Lin- CB (n=3) and AML cells (N=5) cultured with SCF, IL-3, GM-CSF, G-CSF and EPO. In normal CD34+, Dasatinib (100 nM) treatment resulted in increased CD33+ and CD14+ cells and reduced CD34+, CD11b+, CD15+, GPA+ and CD71+ cell numbers, indicating that SFK increased monocytic but reduced granulocytic and erythroid differentiation. Treatment of AML cells with Dasatinib resulted in markedly reduced numbers of CD34+, CD33+ and CD71+ cells, but increased numbers of CD11b cells, in 3 of 5 samples, indicating a trend towards increased granulocytic differentiation in contrast to normal progenitors. Our results indicate that SFK activity is increased in primary human AML stem and progenitor cells and suggest that SFK blockade with Dasatinib may reduce maintenance of AML LSC/ primitive progenitors, through inhibition of progenitor proliferation, induction of apoptosis and enhancement of differentiation. These results support further evaluation of SFK blockade with Dasatinib for targeting of AML stem and progenitor cells in preclinical and clinical studies. Disclosures: Bhatia: Novartis: Consultancy, Honoraria.

Persisting Chronic Myeloid Leukemia Stem and Progenitor Cells From Patients in Major Molecular Remission Under Imatinib Are Characterized by Low BCR/ABL Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2207-2207
Author(s):  
Ashu Kumari ◽  
Cornelia Brendel ◽  
Thorsten Volkmann ◽  
Sonja Tajstra ◽  
Andreas Neubauer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Soft Agar ◽  
Molecular Remission ◽  
Stem And Progenitor Cells ◽  
First Diagnosis

Abstract Abstract 2207 Poster Board II-184 Introduction: Treatment with the Abl-kinase specific inhibitor imatinib (IM) is very effective in chronic myeloid leukemia (CML). However, IM presumably fails to eradicate CML stem cells (HSC) leading to disease persistence and relapse after IM-discontinuation. Although causes of CML persistence under imatinib remain ill defined, quiescence and BCR/ABL-overexpression of CML stem and progenitor cells have been suggested as underlying mechanisms. We here set out to identify means to directly study persistence mechanisms in residual BCR/ABL-positive progenitor and stem cell clones from chronic phase CML patients in major molecular remission (mmR) under imatinib. Methods: Bone marrow specimens of twenty-one CML patients in at least major molecular remission (mmR) according to the international scale, first diagnosis (FD) patients (n=5) and healthy donors (n=4) were sorted into HSC, common myeloid progenitors (CMP), granulocyte/macrophage progenitors (GMP) and megakaryocate-erythrocyte progenitors (MEP) and BCR-ABL mRNA expression was directly assessed by quantitative real time (qPCR) and/or nested PCR (mRNA of 4.000 sorted cells). Alternatively, HSC, CMP, GMP and MEP were seeded into soft agar and mRNA was extracted from individual colony forming units (CFU) to assess BCR/ABL-mRNA expression by qPCR. Moreover, CFU of sub-fractions of first diagnosis CML patients were treated in vitro with IM at 3mM and BCR/ABL-expression of surviving CFU was compared with the BCR/ABL expression levels of mock-treated CML-CFU. In total, 595 soft agar colonies were analyzed. Results: By nested PCR, BCR/ABL-mRNA was readily detectable in the HSC compartments of 7 of 10 (7/10) CML patients in mmR. BCR/ABL was also detected in the CMP-, GMP-, and MEP-compartments in 6, 10 and 8 of the 10 patients, respectively. Real time qRT-PCR suggested only a trend toward stronger BCR/ABL positivity of the HSC compartment when compared to the other progenitor compartments (table 1). A detailed analysis of the BCR/ABL-expression of individual CFU from HSC-, CMP-, GMP-, and MEP-compartments of mmR patients revealed that persisting CML-CFU expressed significantly less BCR/ABL than first diagnosis CML-CFU obtained before imatinib therapy (table 1). This finding could be recapitulated in vitro: primary CML-CD34+ cells of first diagnosis CML patients (n=4) were seeded into soft agar in the presence or absence of 3 uM imatinib. After 14 days BCR/ABL expression only of BCR/ABL-positive CFU was compared. BCR/ABL-positive CML-CFU (n=30) that had survived imatinib exposure expressed significantly less BCR/ABL than mock-treated CML-CFU (n=175) (p<0.001). Work is in progress providing in vitro evidence that selection/induction of low BCR/ABL expression in immature progenitor and stem cells is a new mechanism of imatinib persistence in mmR patients via reducing oncogenic addiction from BCR/ABL. Conclusions: We showed that BCR/ABL-persistence is not confined to the quiescent CML-stem cell compartment, but seems to affect also the highly proliferative progenitor compartments. More intriguingly, persisting CML-HSC and -precursor cells express remarkably low levels of BCR/ABL when compared to first diagnosis HSC and progenitors, implying that low BCR/ABL expression reduces imatinib sensitivity in vivo. The simple model of selection / induction of low BCR/ABL expression as mechanism of imatinib persistence in CML would explain the low propensity of disease progression after achieving mmR, and the low genetic instability of CML clones from mmR patients. Disclosures: No relevant conflicts of interest to declare.

A Highly Selective Anti-ROR1 Monoclonal Antibody Inhibits Human Acute Myeloid Leukemia CD34+ Cell Survival and Self-Renewal.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2560-2560
Author(s):  
Larissa Balaian ◽  
Anil Sadarangani ◽  
George F. Widhopf ◽  
Rui-kun Zhong ◽  
Charles Prussak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
The Self ◽  
Self Renewal ◽  
Leukemia Stem Cell ◽  
Cd34 Cells ◽  
Selective Expression ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

A Novel 4-anilino-3-quinolinecarbonitrile Dual Src and Abl Kinase Inhibitor (SKI-606) Has In Vitro Activity on CML Ph+Blast Cells Resistant to Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1991-1991 ◽  
Author(s):  
Tiziana Grafone ◽  
Manuela Mancini ◽  
Emanuela Ottaviani ◽  
Matteo Renzulli ◽  
Frank Boschelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
G1 Phase ◽  
Inhibition Of Proliferation ◽  
Cd34 Cells ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Abstract The tyrosine kinase Bcr-Abl is the fusion product of a reciprocal translocation between chromosomes 9 and 22, known as Philadelphia chromosome and it is present in the leukemic cells of more than 95% of patients with chronic myeloid leukemia (CML). Overexpression of Bcr-Abl in myeloid cells activates various signaling pathways. Previous studies have demonstrated that certain Src family kinases, such as Hck and Lyn, are also targets of Bcr-Abl activity. Hck and Lyn are expressed and activated in CML blast-crisis patients and their increased expression correlates with disease progression or STI571 resistance in some CML patients. Resistance to STI571 seems to be mediated by amplification of or mutations in the Bcr-Abl gene, reducing sensitivity to this inhibitor; newer Abl inhibitors may be susceptible to the same mechanism of resistance. Alternative strategies for control of CML, including the biological relevance of the Bcr-Abl - Src family kinase pathway, are necessary. One such strategy is the use of a specific small molecule Src kinase inhibitor. Recently, a new class of compounds, 4-anilino-3-quinolinecarbonitrile Src kinase inhibitors, has been synthesized. One member of this class, SKI606, is a dual-specificity inhibitor of both Src family and Abl kinases. To investigate the effect in vitro of SKI-606, we analyzed human cell lines from CML patients in blast crisis (K562, MK2, LAMA) and CD34+ from 9 patients in CML blast crisis using a wide range of concentrations (0.01μM-10μM) of this novel agent. Cell cycle analysis, in particular for the cell lines, showed that a major effect of SKI606 is to alter cell cycle progression, producing G1/S arrest. SKI606 induced dose-dependent inhibition of proliferation with IC50 of 1μM at 24hr. Flow cytometric analysis with Annexin-V showed that SKI-606 induced apoptosis of 50% of cells at 48hr. Western blotting and immuno-blotting analyses showed reduced phosphorylation of Bcr-Abl and also of Lyn and Hck. We also demonstrated activation of Caspase-9, an effector cysteine-protease, after exposure to SKI606. These drug effects also reduced the oncogenic effects of the Bcr-Abl gene product in CD34+ cells from patients with CML blast crisis. SKI606 induced a dose-dependent inhibition of proliferation with an IC50 of 1μM at 48hr and induction of apoptosis at 72hr. Cytofluorimetric analysis after 72hr of exposure revealed marked accumulation of cells in the G1 phase of cell cycle, accompanied by a significant increase in the number of apoptotic cells. In some of these patient samples, we observed hypophosphorylation of Bcr-Abl, Hck and Lyn at low concentration of SKI606 (1uM at 24h, 10uM at 48h). Interestingly, CD34+ cells taken from two of our imatinib-resistant patients with Bcr-Abl point mutations (E255K and Y253H) in the P-loop region of the protein exhibited a significant increase of apoptosis (50%) and a block in G1 phase of the cell cycle after treatment with 1 μM SKI606 for 48h. Our study thus showed a potential therapeutic usefulness of the drug in treatment of CML, particularly in blast crisis phase. Ongoing gene expression profiles will contribute to further understanding of the drug mechanism.

Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3975-3975
Author(s):  
Szabolcs Fatrai ◽  
Djoke van Gosliga ◽  
Lina Han ◽  
Simon M. G. J. Daenen ◽  
Edo Vellenga ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Signal Transduction Pathways ◽  
Monocytic Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
P38 Pathway

Abstract Abstract 3975 Poster Board III-911 In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved. In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V. In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways. Disclosures: No relevant conflicts of interest to declare.

Nilotinib exerts equipotent antiproliferative effects to imatinib and does not induce apoptosis in CD34+ CML cells

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 4016-4019 ◽  
Author(s):  
Heather G. Jørgensen ◽  
Elaine K. Allan ◽  
Niove E. Jordanides ◽  
Joanne C. Mountford ◽  
Tessa L. Holyoake
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Imatinib Mesylate ◽  
Myeloid Leukemia ◽  
Caspase 3 ◽  
Inhibitory Concentration ◽  
Antiproliferative Effects ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Predominant Effect

Abstract Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34+ CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 μM. CML CD34+ cells were still able to expand over 72 hours with 5 μM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSEmax) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

scholarly journals STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2880-2888 ◽  
Author(s):  
Hein Schepers ◽  
Djoke van Gosliga ◽  
Albertus T. J. Wierenga ◽  
Bart J. L. Eggen ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Fold Reduction ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Precise Role ◽  
Term Maintenance

Abstract The transcription factor STAT5 fulfills a distinct role in the hematopoietic system, but its precise role in primitive human hematopoietic cells remains to be elucidated. Therefore, we performed STAT5 RNAi in sorted cord blood (CB) and acute myeloid leukemia (AML) CD34+ cells by lentiviral transduction and investigated effects of STAT5 downmodulation on the normal stem/progenitor cell compartment and the leukemic counterpart. STAT5 RNAi cells displayed growth impairment, without affecting their differentiation in CB and AML cultures on MS5 stroma. In CB, limiting-dilution assays demonstrated a 3.9-fold reduction in progenitor numbers. Stem cells were enumerated in long-term culture-initiating cell (LTC-IC) assays, and the average LTC-IC frequency was 3.25-fold reduced from 0.13% to 0.04% by STAT5 down-regulation. Single-cell sorting experiments of CB CD34+/CD38− cells demonstrated a 2-fold reduced cytokine-driven expansion, with a subsequent 2.3-fold reduction of progenitors. In sorted CD34+ AML cells with constitutive STAT5 phosphorylation (5/8), STAT5 RNAi demonstrated a reduction in cell number (72% ± 17%) and a decreased expansion (17 ± 15 vs 80 ± 58 in control cultures) at week 6 on MS5 stroma. Together, our data indicate that STAT5 expression is required for the maintenance and expansion of primitive hematopoietic stem and progenitor cells, both in normal as well as leukemic hematopoiesis.

Selective Targeting Of Inv(16)+ AML Stem Progenitor Cells By Inhibiting HDAC8

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 224-224
Author(s):  
Jing Qi ◽  
Sandeep Singh ◽  
Qi Cai ◽  
Ling Li ◽  
Hongjun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Control Group ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Primary Bone ◽  
Primary Bone Marrow ◽  
Acute Myeloid

Abstract Chromosomal inversion inv(16)(p13.1q22) which leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, occurs in over 8% of acute myeloid leukemia (AML) cases. The fusion product CBFβ-SMMHC (CM) inhibits differentiation of hematopoietic stem and progenitor cells (HSPCs) and creates pre-leukemic populations predisposed to acute myeloid leukemia (AML) transformation. The mutations of tumor suppressor p53 occur in approximately half of all cases of human cancer, but TP53 mutations are relatively rare in inv(16) AML. We have previously shown that CM expression leads to reduced acetylation of p53 and impaired p53 target gene activation through formation of aberrant protein complex with p53 and HDAC8 (Blood, 2012,120: A772.). Here, we showed that CM interacts with p53 both in CM transformed mouse primary bone marrow cells as well as in AML stem and progenitor cells from inv(16) patients. When HDAC8 selective pharmacological inhibitor 22d directed against its catalytic sites (ChemMedChem 2012, 7:10, 1815-24;) were used to treat inv(16) mouse primary bone marrow progenitor cells and inv(16)+ CD34+ stem progenitor cells from patients, Ac-p53 levels were remarkably increased as shown by western blot. We further assessed the p53 target genes expression after HDAC8 inhibitor 22d treatment by qRT-PCR assay in inv(16)+ CD34+ stem progenitor cells (n=8), and observed variable levels of activation in p53 targets (Fold activation: p21:2.25-fold, hdm2:1.17-fold, 14-3-3σ: 3.12-fold, puma: 2.39-fold), indicating p53 was re-activated. Similar results were also shown in CM transformed mouse bone marrow progenitor cells. Importantly, we found that 22d treatment significantly inhibit the growth of inv(16)+ AML CD34+ cells (n=9) rather than normal CD34+ cells (n=7) , (AML IC50= 6.509 μM, vs Normal cells IC50=13.83 μM, p=0.0003). Meanwhile, 22d selectively induces apoptosis of inv(16)+ AML stem and progenitor cells while sparing normal HSPCs (AML LD50= 10.24 μM, vs NL LD50= 46.36 μM, p=0.001). To evaluate whether the effect of HDAC8i is mediated by p53, we knocked down p53 with a lentiviral vector expressing shRNA against p53 (or non-silencing shRNA) in AML CD34+ cells, and treated the cells with HDAC8 inhibitor 22d (5-20 µM). We showed that despite the inter-sample variability, knocking down p53 expression in all AML samples tested (n=3) led to reduced HDAC8i-induced apoptosis, suggesting that p53 contributes to the apoptosis effect induced by HDAC8i (22d) in inv(16)+ AML cells. Importantly, by taking advantage of our conditional knock-in mouse model (Cbfb56M/+/Mx1-Cre), which develops AML under induced expression of CBFß-SMMHC (Cancer Cell, 2006, 9:1, 57-68), we were able to perform the ex vivo treatment assay by treating primary leukemic cells (marked with dTomato) with either DMSO (as vehicle control) or with HDAC8 inhibitor 22d (10μM) for 48h, followed by transplantion into congenic mice (control group n=8, treatment group n=7). We observed reduced short-term engraftment of leukemic cells that are treated with 22d (10 μM) at 4 weeks post-transplantation in the peripheral blood (Donor cell%: control group=5.99%, treatment group=0.178%, P=0.0093). Interestingly, engraftment of cord blood CD34+ cells at 16 weeks post-bone marrow transplantation was not reduced after treatment with 22d (10 μM) (human CD45+ %: control=66.2% versus treatment=63.4%, p=0.9), indicating the effect by HDAC8 inhibition is selective for leukemic cells. In conclusion, we have identified a novel mechanism whereby CBFβ-SMMHC inhibits p53 fucntion, and may further implicate inhibition of HDAC8 as a promising approach to selectively target inv(16)+ AML stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoetic Stem and Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4619-4619
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Liquid Culture ◽  
Erythroid Differentiation ◽  
Apoptotic Response ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been demonstrated that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). Mutation of ASXL1 leads to truncation of the protein and thereby to a loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in hematopoietic system in vitro mimics the changes observed in MDS-patients. We down regulated ASXL1 in CD34+ cord blood (CB) cells using a lentiviral approach and obtained a 40-50% reduction of ASXL1 expression. Colony forming (CFC) assays revealed that erythroid colony formation was significantly impaired (p=0.01) and, to some extent, granulocytic and macrophage colony formation (p=0.09, p=0.05 respectively). As MDS can affect all hematopoietic lineages, we first stimulated cell differentiation along the myeloid or erythroid lineage in liquid culture. Upon culturing shASXL1 CB CD34+ cells in suspension, we observed a modest reduction in expansion (two-fold at week1) under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p=0.02). The increase of cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. This phenotype is similar to what has been observed in patients, where increased cell death of progenitors occurs, and suggests that ASXL1 loss may reflect an MDS-like phenotype in this culture setting. Furthermore, as MDS is considered a hematopoietic stem cell (HSC)-driven disorder, we tested whether HSCs were affected by ASXL1 loss. Long-term culture initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, we performed cultures on stromal layers with or without cytokines. shASXL1 CB CD34+ cells cultured on MS5 stromal layer showed a modest, two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. In patients, mutations in ASXL1 are frequently accompanied by a loss of p53. Possibly, loss of p53 is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our first data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies compared to shASXL1/shSCR cells, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, when using erythroid liquid conditions, a decrease in apoptosis compared to the ASXL1 single mutation could be observed. Nevertheless, no transformation occurred and ASXL1 mutated cells were eventually lost in the double hit model despite reduced apoptosis, suggesting that the p53 axis might not be sufficient as the second hit for full transformation. In conclusion, our data indicate that mutations in ASXL1 may lead to an increase in cell death and reduced progenitor output in vitro, which may reflect disease development and progression as seen in patients. Unexpectedly, MS5 stromal did not alter the negative phenotype caused by ASXL1 knock down. Therefore, studies are ongoing to investigate whether an already established MDS microenvironment will influence ASXL1 mutation positively. To this end, we are using healthy human mesenchymal stem cells (MSC) and patient derived MDS MSCs. Disclosures No relevant conflicts of interest to declare.

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Blood ◽  
2009 ◽  
Vol 114 (8) ◽  
pp. 1498-1505 ◽  
Author(s):  
Aleksandra Rizo ◽  
Sandra Olthof ◽  
Lina Han ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Oxygen Species ◽  
Self Renewal ◽  
Human Cd34 ◽  
Bmi1 Expression ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Cord Blood Cd34

Abstract High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34+ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that down-modulation of BMI1 in cord blood CD34+ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34+ cells, down-modulation of BMI1 impaired long-term expansion, whereby self-renewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and self-renewal of normal and leukemic stem and progenitor cells, and that expression of BMI1 protects cells against oxidative stress.

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