RNAi-Mediated Inhibition of Mcl-1 Expression Enhances Apoptosis in Imatinib-Treated CML Progenitors

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1669-1669
Author(s):  
Su Chu ◽  
Ravi Bhatia
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Cell Expansion ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Control Shrna ◽  
Cd34 Cells

Abstract Abstract 1669 Tyrosine kinase inhibitor (TKI) treatment inhibits proliferation in CML stem/progenitor cells, but only modestly increases apoptosis. Residual leukemia stem cells remain a potential source of disease relapse in IM-treated patients. The Bcl-2 family of anti-apoptotic proteins plays a central role in the regulation of apoptosis. Several Bcl-2 inhibitors are being evaluated in preclinical and clinical studies and there is considerable interest in evaluating their ability to induce apoptosis in CML stem and progenitor cells. However these agents have considerable toxicity possibly related to lack of selectivity for individual family members. We performed a functional siRNA screen to determine the role of individual Bcl-2 family members in maintaining survival of in CML and normal CD34+ cells. CML and normal CD34+ cells were transfected with siRNAs targeting Bcl-2, Bcl-2L1, Bcl-2L2, Bcl-2L10, Mcl-1 and Bcl2A1. In this screen Mcl-1 knockdown resulted in significant reduction in viability of CML CD34+ cells, with or without co-treatment with IM (1uM). Significant reduction in normal CD34+ viability was not seen. These results were validated using different siRNA sequences to knockdown Mcl1 expression. Increased apoptosis of CML but not normal CD34+ cells was seen (23±8% for CML vs. 4.2±1.5% for normal CD34+ cells, n=3, p<0.5). CML CD34+ cell apoptosis was further enhanced by combination of Mcl-1 inhibition with IM treatment (48±15% for CML vs. 7.2±3% for CB progenitors, p<0.1). To further evaluate the role of Mcl-1 in regulating CML CD34+ cell growth, an anti-Mcl-1 shRNA construct was cloned into the pHIV7-SF-RFP lentivirus vector. Cord blood and CML CD34+ cells were transduced with Mcl-1 specific or control, non-specific shRNA expressing vectors. Western blotting demonstrated effective knockdown of Mcl-1 protein levels in Mcl-1 shRNA transduced CD34+cells (82% reduction in CML and 78% in normal CD34+ cells). CD34+ RFP+ cells were selected by flow cytometry and cultured in presence and absence of IM. A significant increase in apoptosis was seen in Mcl-1 knockdown CML CD34+ cells compared with control shRNA-transduced cells, and further increase in apoptosis was seen following IM treatment (4.7±0.5 for control shRNA-transduced cells VS 25.7±2.1 for Mcl-1 knockdown cells). Mcl-1 knockdown CML CD34+ cells generated fewer colonies in methylcellulose progenitor culture (93 colonies for control siRNA transduced cells vs. 31 colonies for Mcl-1 knockdown cells) and demonstrated reduced cell expansion following culture with growth factor (SCF; IL3; GM-CSF and G-CSF) compared with control shRNA transduced cells (383,750± 172,476 for control shRNA-transduced cells 224,250± 87,044 for Mcl-1 knockdown cells). Cell expansion was further reduced with IM treatment. Mcl-1 knockdown resulted in complete loss of erythroid colony formation. Analysis of cell differentiation by flow cytometry after culture for 4 or 7 days revealed that Mcl-1 knockdown resulted in reduced generation of both erythroid (GPA+) and myeloid (CD33+ and CD14+) cells. In contrast to the results of the initial siRNA studies, shRNA-mediated Mcl-1 knockdown also resulted in significantly increased apoptosis of normal CD34+ cells (12.6± 1.6% for control shRNA-transduced cells and 24.5± 0.9% for Mcl-1 knockdown cells) associated with reduced colony formation and reduced growth in culture (1.265e+006± 273,892 for control shRNA-transduced cells 589,000 ± 188,082 for Mcl-1 knockdown cells). We conclude that RNAi-mediated Mcl-1 knockdown inhibits CML CD34+ cell survival and proliferation and enhances apoptosis after IM treatment, but also reduces viability of normal CD34+ cells. Since Mcl-1 protein expression is subject to multiple levels of regulation, our results suggest that strategies to selectively target Mcl-1 regulatory mechanisms active in CML but not normal progenitors may be less toxic and have greater clinical utility than direct targeting of the protein. Disclosures: No relevant conflicts of interest to declare.

ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4316-4316
Author(s):  
Hendrik Folkerts ◽  
Maria Catalina Gomez Puerto ◽  
Albertus T.J. Wierenga ◽  
Koen Schepers ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

Aldehyde Dehydrogenase (ALDH) Vs CD34 for Predicting Engraftment After Autologous Hematopoietic Progenitor Cell (autoHPC) Transplant

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4441-4441
Author(s):  
Muthu Veeraputhiran ◽  
Lakshmikanth Katragadda ◽  
Bart Barlogie ◽  
Michele H. Cottler-Fox
Keyword(s):  
Flow Cytometry ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Correlation Coefficients ◽  
Threshold Dose ◽  
Hematopoietic Progenitor Cell ◽  
Aldh Activity ◽  
Cd34 Cells ◽  
Fresh Products

Abstract Abstract 4441 Introduction: When the cell surface marker CD34 became available for enumerating HPC there was controversy as to whether it was useful for predicting time to engraftment after autologous peripheral blood HPC transplant (autoHPCT). It was demonstrated that a threshold dose of 2.0–2.5 × 106 CD34+ cells/kg in the product, measured before cryopreservation, predicted reliable engraftment within a reasonable period of time. CD34 is expressed on the surface of HPC ranging from primitive to committed progenitor cells. ALDH is highly expressed in primitive cells such as HPC, endothelial progenitor cells and mesenchymal stromal cells. ALDHbr cells are by definition viable, while measurement of viable CD34+ cells requires an additional assay. We tested the correlation of CD34 and ALDH activity in fresh HPC products and then examined the usefulness of these two assays for predicting time to WBC and platelet engraftment after autoHPCT. Materials and Methods: We identified 42 consecutive HPC apheresis products used for autoHPCT for which data on CD34+ numbers and viability as well as ALDHbr cell numbers were available. Data from 5 of these product were not used for predicting engraftment due to lack of engraftment data. Viability was assesed by flow cytometry on pre-cryopreservation (fresh) and post-thaw samples by looking at propidium iodide or 7-AAD uptake within the CD34+ population identified using the ISHAGE method. ALDH activity was measured by flow cytometry on pre-freeze samples using the manufacturers prescribed method. HPC were cryopreserved using a controlled rate freezer and 10% DMSO, then stored in liquid nitrogen. Data on time to WBC and platelet engraftment were determined by chart review. The patents received a median of 3.78 × 106 CD34+ cells/kg (range 2.43–8.59 × 106 CD34+ cells/kg) for autoHPCT. Regression analyses were performed to see if viable CD34 in the fresh product, and in the thawed product, correlated with ALDH and whether any of these measures correlate with time to WBC (ANC >500/mm3) or platelet engraftment (>20,000/mm3). Results: The correlation coefficients for ALDHbr vs viable CD34+ cells pre-cryopreservation (r=0.97; n=42) and for ALDHbr cells vs post-thaw CD34+ cells (r=0.96; n=42) were both significant. The percent of variation for ALDHbr cells to WBC engraftment (r2 = 2.2%; n=37) and for CD34+ cells both pre-freezing and post-thaw to WBC engraftment (r2 =1.9%; n=37) were not significant. The r2 for ALDHbr cells and platelet engraftment was 0.9%, the r2 for CD34+ cells and platelet engraftment was 1.1% pre-freeze (n=37) and was 0.9% post-thaw (n=37) were also not significant. Conclusion: Enumeration of HPC by ALDH correlates well with CD34, suggesting that the two assays are equivalent when analyzing autologous peripheral blood. Neither the number of ALDHbr cells or CD34+ cells in fresh products, nor the number of viable CD34+ cells in the thawed products, correlates with time to WBC and platelet engraftment. Since our patients received at least 2.4 × 106 CD34+ cells/kg, our observations support previous data showing no correlation between cell dose and time to engraftment if more than the HPC threshold dose for engraftment is given for transplant. Disclosures: No relevant conflicts of interest to declare.

Treatment with Pegylated Interferon Alpha 2a in Combination with the Bcl-Xl Inhibitor ABT-737 Specifically Targets JAK2V617F Positive Hematopoietic Progenitor Cells From Patients with Polycythemia Vera.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3916-3916 ◽  
Author(s):  
Min Lu ◽  
Wei Zhang ◽  
Daniel Yoo ◽  
Dmitriy Berenzon ◽  
Yan Li ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Interferon Alpha ◽  
Colony Formation ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Hematopoietic Progenitor Cells ◽  
Low Doses ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Abstract 3916 Poster Board III-852 Polycythemia vera (PV) is a Philadelphia chromosome negative chronic myeloproliferative neoplasm (MPN) which is characterized by acquisition of a mutation in JAK2 (JAK2V617F). The administration of a pegylated form of interferon-alpha-2a (Peg IFNa-2a) to patients with PV has recently been reported to lead to hematological remissions and a reduction of the JAK2V617F allele burden in most patients receiving this modality of therapy. The mechanism underlying this profound clinical response of PV patients to Peg IFNa-2a has been the subject of a great deal of speculation. In order to evaluate the mechanism by which Peg IFNa-2a affects hematopoiesis in PV patients, CD34+ cells isolated from cord blood and the peripheral blood of patients with PV were cultured in semisolid media in the presence and absence of 200 and 500 U of Peg IFNa-2a. These relatively low doses of Peg IFNa-2a did not alter hematopoietic colony formation by CB CD34+ cells but inhibited PV CFU-GM colony formation by 35% and 50%, and BFU-E colony formation by 60% and 80%, respectively. Furthermore, the hematopietic colonies that formed in the presence of Peg IFNa-2a were composed of far fewer cells than those cultured in the presence of cytokines alone. In addition, individual hematopoietic colonies were plucked and the JAK2 genotype was assessed by nested allele-specific PCR assay. Exposure of PV CD34+ cells to Peg-IFNa-2a (500U) resulted in a reduction in the proportion of JAK2V617F-positive hematopoietic progenitor cells from 81.7±16.3% to 50.3±27.6% (p=0.004). Samples from 81.9% of the PV patients (9 of 11 samples) responded in this fashions to Peg IFNa 2a treatment. We then showed that incubation of PV CD34+ cells but not CB CD 34+ cells with 200 and 500U of Peg IFNa-2a resulted in increased rates of apoptosis by 4.3% and 15.3%, respectively. Erythroblasts and megakayoctes from patients with PV have been previously shown to be characterized by over-expression of the anti-apoptotic proteins Bcl-xL. We then examined if the effects of IFNa-2a could be enhanced by addition of the Bcl-xL inhibitor-ABT-737. After 2 days of treatment, Peg IFNa 2a plus ABT-737 induced significantly greater degree of apoptosis (∼50%) of a JAK2V617F positive erythroleukemia cell line (HEL cells) as compared to treatment with each agent alone, (Peg-IFNa-2a, <5%; ABT-737, 20%). PV CD34+ cells were incubated with Peg IFNa 2a (500 U) alone, ABT-737 (0.25 uM) alone or ABT-737 plus Peg IFNa 2a for 4 days and the numbers of cells were decreased by 35%, 40% and 65 %, respectively; and the corresponding percentage of apoptotic cells was 20%, 15% and 60%, respectively. Western blot analysis showed that the Bcl-xL protein level in PV but not CB mononuclear cells was reduced by treatments with ABT-737 alone or in combination with Peg IFNa 2a. Furthermore, treatment of PV CD34+ cells with ABT-737 plus Peg IFNa 2a (200U) lead to the appearance of a smaller proportion of JAK2 V617F-positive (46.7±26%) hematopoietic progenitor cells as compared to cells incubated with cytokines alone (81.7±17%) or cytokines plus Peg IFNa 2a (69±20%). These data suggest that low doses of Peg IFNa 2a selectively and directly eliminate Jak2V617F hematopietic progenitor cells which likely accounts for the therapeutic responses that have been observed with the use of this agent in the clinic. The enhanced elimination of JAK2V617F hematopoietic progenitor cells observed with the combination of ABT-737 and Peg-IFNa-2a suggests that this strategy might be an even more optimal approach for the treatment of JAK2V617F positive MPN which merits further testing in the clinic. Disclosures: No relevant conflicts of interest to declare.

CXCL9 and IL32 Regulate Progenitor Expansion and Protect Hematopoietic Progenitor Cells From Chemotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1316-1316
Author(s):  
Huili Lu ◽  
Wei Han ◽  
Abdulgabar Salama ◽  
Anja Moldenhauer
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Group Versus ◽  
Control Group ◽  
Stem Cell Expansion ◽  
Apoptosis Rate ◽  
Cd34 Cells

Abstract Abstract 1316 Background: We have reported that the cytokines CXCL9 and IL32 regulate murine bone marrow regeneration post chemotherapy, but the reasons for this effect and whether they work directly on progenitor cells remain unclear. Methods: Human CD34+ cells from cord blood were incubated with CXCL9 or IL32. Cell numbers were determined on a weekly basis, and one-week expanded cells were seeded on top of a confluent MS-5 stroma cell layer to determine the number of cobblestone-area forming and long-term culture initiating cells (LTC-IC). Apoptosis rates after incubation with CXCL9/IL32 and SCF/G-CSF/IL3 prior to Ara-C treatment (300mM, 1 h) were assessed by Annexin V detection. Subsequently, signaling pathways after stimulation with IL32 or CXCL9 were examined using the luminex map technology. Results: CXCL9 did not influence CD34+ cell expansion, while IL32 enhanced the expansion rates significantly [6.69±1.38 versus to 3.57±0.70 fold in the control group, p<0.05]. However, more LTC-ICs after CXCL9 treatment (1357±123 of CXCL9 group versus 1081±119 of control group, p<0.05) were found, while IL32 reduced the number of LTC-ICs (78±8 of IL32 group, p<0.005). That suggests that CXCL9 kept more primitive LTC-ICs quiescent instead of entering expansion during the one-week incubation, while IL32 rather stimulated the differentiation of LTC-ICs. Since SCF, G-CSF and IL-3 are the most widely used hematopoietic growing factors (HGF) in stem cell expansion, we detected their roles during chemotherapeutical treatment in vitro. We observed enhanced apoptosis during Ara-C treatment when the cells were incubated with SCF or IL3. But when CXCL9 or IL32 were added, results were different. Both CXCL9 and IL-32 reduced the apoptosis rate resulting from Ara-C treatment, when SCF is present (26.37±1.12% of CXCL9+SCF group, 29.97±0.72% of IL32+SCF group, versus 35.52±1.21% of SCF alone, p <0.005 and p<0.05), but none of them affected IL-3 related apoptosis. Especially the effect of CXCL9 was inhibited using antibodies to its receptor CXCR3 (37.97±1.50% with anti-CXCR3 versus 35.52±1.21% of SCF alone, p= 0.09, versus 26.37±1.12% of CXCL9+SCF group p<0.05). G-CSF alone did not influence Ara-C induced apoptosis, but in combination with IL32 the apoptosis rate increased (23.37±0.09% of IL32+G-CSF versus 19.59±0.79% of G-CSF alone, p<0.005). That suggests that IL32 could regulate stem cell expansion differently through various pathways in collaboration with SCF and G-CSF. In fact, IL32 reduced STAT5 and p38 activity, while CXCL9 activated p38 and JNK pathways of CD34+ cells in combination with SCF. Conclusions: Our results demonstrate that both CXCL9 and IL32 can regulate stem cell expansion in vitro; CXCL9 could protect HPCs from chemotherapy and therefore support the following recovery, IL32 could help progenitor cells to expand, differentiate rapidly and thereby enhance the regeneration of the hematologic system. Disclosures: No relevant conflicts of interest to declare.

Suppression of CML Progenitor but Not Stem Cells Requires Simultaneous Inhibition of KIT and BCR-ABL1.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2778-2778
Author(s):  
Zhimin Gu ◽  
Amie S. Corbin ◽  
Thomas O'Hare ◽  
Anna M. Eiring ◽  
Tian yi Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Colony Formation ◽  
Kinase Activity ◽  
Colony Growth ◽  
Newly Diagnosed ◽  
Cd34 Cells ◽  
Simultaneous Inhibition

Abstract Abstract 2778 In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition. Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p<0.003). When PPY-A was combined with KIT activation by SCF, proliferation was restored, indicating KIT signaling must be inactivated to induce cell death by BCR-ABL1 inhibition. Immunoblot analysis of Mo7eBCR-ABL1 cells revealed that culture in SCF rapidly activated AKT and ERK1/2 in the presence but not absence of PPY-A. Simultaneous inhibition of AKT with LY294002 abolished SCF-mediated rescue of cell proliferation, whereas ERK1/2 inhibition with PD98059 only partially abrogated SCF rescue. These data indicate that SCF rescue of Mo7eBCR-ABL1 cells upon BCR-ABL1 inhibition critically depends on AKT. To assess BCR-ABL1 vs. KIT inhibition in primary cells, CD34+ cells from newly diagnosed CML patients (n=4) and normal controls (n=3) were cultured in semisolid medium supplied with IL-3 and GM-CSF (no SCF), in the presence of 1 μM PPY-A combined with shKIT or 1 μM BAW667. KIT inhibition by shKIT or 1 μM BAW667 reduced CFU-GM formation by 40% compared to controls (p<0.04) even in the absence of SCF, with no effects were seen in normal CD34+ cells, indicating that BCR-ABL1-dependent KIT activation occurs in the absence of SCF stimulation. PPY-A reduced colony formation by 54.7%, while PPY-A plus shKIT and PPY-A plus BAW667 suppressed CFU-GM colony formation by 79.7% and 72.1%, comparable to the effects of imatinib (71.9%). Addition of SCF partially rescued colony growth from the effects of PPY-A, consistent with results on Mo7eBCR-ABL1 cells. In a separate set of experiments lineage-negative (Lin−) cells from newly diagnosed patients (n=4) were cultured on HS-5 stromal cells containing K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, followed by clonogenic assays. Only the PPY-A / K44.2 combination suppressed CFU-GM; isolated BCR-ABL1 or KIT block did not. These data demonstrate that both BCR-ABL1 and KIT contribute to CML progenitor cell survival under physiologically relevant conditions, and that inhibition of both pathways is required for imatinib-mediated suppression of CML progenitor cells. To assess the role of KIT vs. BCR-ABL1 inhibition on primitive CML cells we performed long-term culture-initiating cell (LTC-IC) assays on M2–10B4 murine stromal cells, using Lin− cells from newly diagnosed patients (n=3). Cultures were performed with K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, with colonies plated at 1, 3, and 6 weeks. At 1 week colonies were reduced by 30% with K44.2 and 70% with PPY-A, but by 90% with the PPY-A / K44.2 combination or with imatinib. In contrast, at 6 weeks colony formation was unaffected by K44.2 but reduced by >95% with PPY-A, the PPY-A / K44.2 combination or imatinib. Week 3 colony growth was intermediate. Consistent with the LTC-IC assay, KIT inhibition with BAW667 enhanced PPY-A suppression of colony formation in Lin−CD34+CD38+ progenitor cells from newly diagnosed patients (n=3) by 18.7% (p<0.05), with no significant effect on primitive Lin−CD34+CD38− cells (7.7%, p=ns). Our findings suggest KIT inhibition is much more critical for suppression of mature progenitors compared to primitive CML cells. Since AKT is active in CML progenitors but suppressed by TGFβ in stem cells (Nature, 2010;463(7281):676; JCI, 2011;121(1):396), we speculate that upon BCR-ABL1 inhibition CML progenitors but not stem cells switch to an SCF-dependent mode of AKT activation, which renders these cells uniquely sensitive to dual inhibition of BCR-ABL1 and KIT signaling. Disclosures: No relevant conflicts of interest to declare.

Oncogene Dosage Is a Major Determinant of Leukemogenic Transformation by AE9a

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1358-1358
Author(s):  
Kevin A. Link ◽  
Shan Lin ◽  
Kakajan Komurov ◽  
Mark Wunderlich ◽  
James C. Mulloy
Keyword(s):  
Fusion Protein ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Human Cell ◽  
Colony Formation ◽  
Cell Expansion ◽  
Conflicts Of Interest ◽  
Full Length ◽  
Hematopoietic Stem ◽  
Fusion Protein Expression

Abstract Abstract 1358 Chromosomal translocation (8;21) results in the formation of the AML1-ETO (AE) leukemia-associated fusion protein. Roughly 70% of AE+ patient samples also harbor an alternatively spliced variant of AE, termed AE9a. AE9a promotes the leukemic transformation of mouse hematopoietic stem/progenitor cells without the need for secondary external cooperating oncogenes. By comparison, leukemia development in response to expression of full length AE requires one or more additional events. We extended these findings into a human cell system to determine whether AE9a is able to promote the transformation of human CD34+ cord blood (CB) cells to acute myeloid leukemia (AML). Transduction of CD34+ CB cells with AE9a promotes long-term culture potential, enhanced replating capacity, and expansion of CD34+ progenitor cells similar to full length AE. While we have not yet been able to generate AE9a leukemia in xenograft assays (despite several attempts), subcutaneous injection of AE9a cells promotes formation of a robust granulocytic sarcoma which is not seen using cells expressing full length AE. Several of our analyses have highlighted the importance of increased AE9a fusion protein expression in its enhanced function relative to full length AE, a concept that was also observed using the MLL-AF9 leukemia oncogene (Chen et. al, Cancer Cell, 2008). Indeed, flow cytometric sorting of freshly transduced AE or AE9a cultures based on high and low fusion protein expression revealed the importance of expression level for both AE and AE9a. High AE expression completely blocked colony formation and decreased cell expansion. These effects correlated with a dramatic upregulation of the cell cycle inhibitor p21. Conversely, high levels of AE9a did not upregulate p21 to the same degree, and colony formation and cell expansion were not as dramatically affected. In contrast, flow sort for cells expressing low levels of AE or AE9a had significantly less remarkable effects on cell function and p21 expression. Unexpectedly, AE9a-low cells eventually came to resemble the AE9a-high in terms of AE9a protein levels. These effects were not seen for full length AE sorted cultures. To determine whether similar effects were occurring in the murine AML model, we examined the effects of low and high expression levels of AE9a in mouse fetal liver cells. Corroborating our findings with the human cell model, AE9a-low samples promoted significantly less AML as compared to AE9a-high cells. This was not due to lack of engraftment of these cells or to loss of AE9a expression. Similar effects were found in vitro, where AE9a-low cells were unable to replate in methylcellulose assays while AE9a-high cells as well as AE-high and AE-low cells showed enhanced replating ability. Strikingly, the AML that did develop in mice transplanted with AE9a-low cells reverted to a very high level of AE9a protein expression, implying that increased expression of AE9a is essential for function. To better understand the mechanistic link between increased AE9a expression and AML development, we performed global gene expression analysis of murine progenitor cells expressing AE or AE9a (after three weeks of in vivo expansion). Analysis revealed a subset of genes that were highly repressed in AE9a expressing cells as compared to full length AE. Given that AE9a lacks the carboxy-terminal NHR3 and NHR4 domains of AE that interact with the NCoR and SMRT corepressors, the finding of significantly more repressed genes in AE9a cells is quite surprising. Network analysis has uncovered potential signaling pathways that may be involved in AE9a mediated transformation. Uncovering the mechanism of AE9a function could lead to a better understanding of t(8;21)-associated function in human AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Revisiting the IGF-1R Signaling Pathway in Myeloproliferative Neoplasms: Quantification of IGF-1 Receptor May be Useful in the Differential Diagnosis of Polycythemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3226-3226 ◽  
Author(s):  
Guanfang Shi ◽  
Stacey Baptiste ◽  
Maryna Yarotska ◽  
Hemant Sindhu ◽  
Ching Wong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Differential Diagnosis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Signal Pathway ◽  
Interferon Α ◽  
Cd34 Cells ◽  
Igfbp 3

Abstract Endogenous erythroid colony formation in vitro is one of the important criteria for the diagnosis of polycythemia vera (PV). Extensive studies performed in the last decade suggest that IGF-1 plays a significant role in the erythropoiesis and endogenous colony formation in PV. Therefore, IGF-1R signal pathway is important in the pathogenesis of PV. We elected to revisit IGF-1R pathway by flow cytometry technique in PV and other myeolproliferative neoplasms (MPNs). 32 MPN patients (including 11 PV, 19 myelofibrosis (MF), 12 essential thrombocytopenia (ET), 8 secondary PV , and 17 age-matched normal controls were studied. Methods: 1) plasma IGF-1, IGFBP-1, IGFBP-3 were assayed by ELISA(R&D Systems, Minneapolis, MN) ; 2) quantification of IGF-1R . Peripheral blood mononuclear cells (MNC) harvested from Ficoll-Paque separation were stained with phycoerythrin conjugated antibody against IGF-1 receptor (R&D Systems), and assayed by flow cytometry using FACS Calibur system (BD Biosciences, San Jose, CA; 3) quantification of IGF-1R phosphoration (pIGF-1R) after IGF-1 stimulation. MNC cells were suspended in serum-free α-MEM medium for 2 hours, then starved cells were treated with or without 10 nM IGF-1 for 3 min and assayed for pIGF-1R by flow cytometry using Alexa Fluor 647 conjugated antibody against phosphorylated IGF-1 receptor (BD Biosciences); 4) Interferon alpha (IFNα) Treatment. IFN-α (Schering Corporation, Kenilworth, NJ) was added to the cell culture at 100 unit per ml. Cells were incubated with IFN-α for either 2 hours or 3 days and then collected for further pIGF-1R analysis. The results showed 1) No difference in plasma IGF-1 levels in MPN and controls; IGFBP-1 and IGFBP-3 were significantly elevated in MPN than controls but the change of IGFBP-3 in MF patients was not significant. 2) the PV group had significantly elevated IGF-1R expression than secondary PV (Fig 1). Mean ± SE (MFI expression) were 529 ± 84 and 90 ± 30 in PV versus secondary PV, respectively (p=0.004). 3) The untreated PV group had significantly higher IGF-1R than MF, ET, and controls. MF and ET were not different from controls, but 8/19 (43%) in MF and 6/12 (50%) in ET have elevated IGF-1R than controls. 4) No difference in IGF-1R expression in Jak2 (+) versus Jak2 (-) in MPN patients. Mean ± SE (MFI) were 293.2 ± 62.1 and 164.8 ± 47.7 in JAK2(+) versus JAK2 (-) respectively (p=NS). 5) Preliminary studies showed PV has a significant elevated pIGF-1R than controls in unstimulated PB CD34+ cells, but not in MNC cells. 6) IFN-α decreased IGF-1R expression and pIGF-IR about 10% in the PV group. Conclusions: 1) IGF1R signal pathway is more active in PV than MF, ET and controls. 2) Quantification of IGF-1R expression by flow cytometry may be helpful in differential diagnosis in primary versus secondary PV. 3) IGF-1R expression is not correlated with JAK2 status. 4) IGF-1R phosphorylation is more elevated in unstimulated CD34+ cells of PV patients than in controls suggesting autonomous erythroid colony formation in PV may be associated with IGF-1R activation. 5) interferon-α has some inhibitory effects in IGF-1R expression and phosphorylation but is unlikely to be the main mechanism of its induced remission of JAK2 –mutant allele-burden effects. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 319-327 ◽  
Author(s):  
K Sawada ◽  
M Ieko ◽  
A Notoya ◽  
T Tarumi ◽  
K Koizumi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Clonal Growth ◽  
Growth Patterns ◽  
Leukemic Cells ◽  
Human Macrophage ◽  
Cluster Number ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Action of interleukin-3, G-CSF, and GM-CSF on highly enriched human hematopoietic progenitor cells: synergistic interaction of GM-CSF plus G-CSF

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 110-114 ◽  
Author(s):  
I McNiece ◽  
R Andrews ◽  
M Stewart ◽  
S Clark ◽  
T Boone ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Synergistic Interaction ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Normal Human ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony

Abstract Purified preparations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and interleukin 3 (IL-3 or multi-CSF) alone and in combination, have been compared for their stimulatory effects on human granulocyte-macrophage colony forming cells (GM-CFC). In cultures of unseparated normal human bone marrow, the combinations of G-CSF plus IL-3 and GM-CSF plus IL-3 stimulated additive numbers of GM colonies, while GM-CSF plus G-CSF stimulated greater than additive numbers of GM colonies, compared with the sum of the colony formation obtained with each factor alone. Cultures of unseparated bone marrow, harvested from patients four to six days after administration of 5-fluorouracil (5-FU), resulted in additive GM colony formation with GM-CSF plus G-CSF, GM-CSF plus IL-3, and G-CSF plus IL-3. In order to address the possibility of secondary factor involvement in the synergistic interaction of GM-CSF and G-CSF, CD33+/CD34+ colony forming cells were separated from normal and post FU marrow by two color fluorescence activated cell sorting. In cultures of CD33+/CD34+ cells the combination of GM-CSF plus G-CSF stimulated a synergistic increase in GM colonies while GM-CSF plus IL-3 stimulated additive numbers of colonies. These results suggest that GM-CSF, G-CSF, and IL-3 stimulate distinct populations of GM-CFC. Furthermore GM-CSF and G-CSF interact synergistically and this action is a direct effect on progenitor cells not stimulated by GM-CSF or G-CSF alone.

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