Nilotinib Inhibits Bcr-Abl Kinase Activity in CML Progenitor Cells More Effectively Than Imatinib but Is Equipotent in Inducing Growth Inhibition.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 744-744 ◽  
Author(s):  
Heiko Konig ◽  
Melissa Holtz ◽  
Hardik Modi ◽  
Paul Manley ◽  
Tessa L. Holyoake ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Inhibition ◽  
Progenitor Cells ◽  
Drug Exposure ◽  
Kinase Activity ◽  
Dose Range ◽  
Therapeutic Success ◽  
Abl Kinase ◽  
Signaling Mechanisms ◽  
Cd34 Cells

Abstract The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant stem cells. Mechanisms contributing to incomplete elimination of CML stem cells include resistance to proapoptotic effects of IM, persistent signaling through growth stimulating pathways and Abl kinase mutations resulting in IM resistance. We investigated whether nilotinib, a more potent Bcr-Abl tyrosine kinase (TK) inhibitor than IM, could more effectively target CML progenitors. CD34+ progenitors from CML patients and healthy donors were cultured with nilotinib (0–10μM) and IM (0–5μM) in growth factor (GF) containing medium. After 96h incubation, cells were harvested and assayed for colony-forming-(CFC) and long-term culture initiating cells (LTCIC). CD34+38- primitive progenitor cells (PPC) and CD34+38+ committed progenitor cells (CPC) labeled with CFSE were incubated under similar conditions and evaluated for proliferation and apoptosis assays by annexin-V staining and FACS analysis. The IC50 for CML LTCIC-suppression was 0.9μM for nilotinib and 0.8μM for IM. Normal LTCIC were not significantly suppressed at doses below 10μM nilotinib and 5μM IM. The IC50 for CML CFC suppression for nilotinib was 6.4μM and 1.0μM for IM. Normal CFC showed less growth inhibition and the IC50 was not reached at the dose range tested. CFSE assays indicated dose-dependent antiproliferative activity of both compounds with IC50-values of 3.0μM nilotinib and 1.8μM IM for CML PPC, and 8.2μM nilotinib and 4.0μM IM for CML CPC. In CML PPC, apoptosis significantly increased following nilotinib treatment (8±2.1% [control] to 22±2.7% [10μM], n=4, p=.015). IM treatment also increased apoptosis in CML PPC (to 35±3.8% [5μM], n=4, p=.0008). In CML CPC, apoptosis increased from 33±2.3% [control] to 50±1.7% (n=4, p=.002) with 10μM nilotinib, and to 53±2.1% (n=4, p=.001) with 5μM IM exposure. The effects on IM and nilotinib on Bcr-Abl-TK activity were investigated by Western blotting with anti-CrkL antibodies after overnight drug exposure of CML CD34+ cells in GF containing media. Importantly, 0.25μM nilotinib significantly reduced P-CrkL levels (from 85.7±4.1% [control] to 13.9±4.5%, n=4, p=.000002), whereas a higher concentration of IM (1–5μM) was needed to achieve similar inhibition. Nilotinib resulted in increased MAPK activity (to 13.3-fold [5μM]) in CML CD34+ cells. MAPK kinase activity was also increased following treatment with IM (to 22.7-fold [5μM]). P-AKT and P-STAT5 levels were not significantly changed in response to either drug. In contrast incubation with either drug in the absence of GF resulted in inhibition of MAPK, Akt and STAT5 activity in CML CD34+ cells. In conclusion, nilotinib is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors but does not induce greater suppression of progenitor growth. As with IM, inhibition of cell division represents the predominant mode of inhibition of progenitor growth. Nilotinib or IM treatment inhibits Bcr-Abl dependent MAPK, STAT5 and Akt activity, but does not inhibit these signaling mechanisms in CML progenitors in the presence of GF. Our results suggest that combined inhibition of Bcr-Abl TK and additional signaling mechanisms may be required to achieve elimination of CML progenitors by targeted therapies.

The Dual Src/Abl Kinase Inhibitor SKI-606 Effectively Inhibits Bcr-Abl Kinase Activity and Reduces Proliferation of CML Primitive Progenitor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1370-1370
Author(s):  
Heiko Konig ◽  
Simran K. Sindhu ◽  
Frank Boschelli ◽  
Tessa L. Holyoake ◽  
Stephen J. Forman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Drug Exposure ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Abl Kinase ◽  
Mapk Activity ◽  
Cd34 Cells ◽  
Ic50 Values ◽  
Dose Dependent

Abstract Imatinib mesylate (IM) is highly effective in the treatment of CML. However resistance to IM can develop in a subset of patients. In addition CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK or other growth stimulatory pathways, or Bcr-Abl kinase mutations resulting in IM resistance. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models, and is currently being investigated in phase1/2 clinical trials. Here, we investigated whether SKI-606 could effectively target CML primary progenitors. Flow cytometry selected CD34+CD38− primitive progenitor (PPC) and CD34+CD38+ committed progenitor cells (CPC) from untreated CML patients were cultured for 96h in growth factor (GF) supplemented medium in a range of concentrations of SKI-606 (0–0.5μM), and with 5μM IM for comparison. Cells were labelled with CFSE-prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells were similarly incubated with SKI-606 and subsequently plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. CFSE assays indicated significant dose dependent antiproliferative activity of SKI-606 with IC50-values of 0.2μM for CML PPC. SKI-606 resulted in moderate induction of apoptosis of CML PPC (from 22±7.2% [control] to 49±16.4% [0.5μM], n=3, ns). CFC-assays consistently revealed significant dose dependent growth inhibitory effects of SKI-606 with IC50-values of 0.1μM SKI-606. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1μM and 0.5μM SKI-606 significantly suppressed phospho-CrkL levels (from 94.7±3.5% [control] to 14.9±4.8%, n=4, p=.0001, and to 6.8±2.5%, n=4, p=.00003, respectively), whereas a higher concentration of IM (5μM) was needed to achieve a similar degree (13.1±5.0%, n=4, p=.0000007). Whereas treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity (n=3, p=.038), a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606 (n=3, ns). In conclusion, SKI-606 significantly inhibits CML progenitor proliferation and moderately induces apoptosis at lower concentrations than previously observed with IM, although the maximum growth suppression effects observed were not greater than those observed with high concentrations of IM. SKI-606 is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further studies investigating the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds are warranted.

Imatinib Mesylate Does Not Inhibit BCR-ABL Kinase Activity in CML Stem Cells In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1979-1979 ◽  
Author(s):  
Lucy J. Elrick ◽  
Ashley Hamilton ◽  
Michael W. Deininger ◽  
Tessa L. Holyoake
Keyword(s):  
Stem Cells ◽  
Imatinib Mesylate ◽  
Drug Exposure ◽  
Remission Rate ◽  
Specific Inhibitor ◽  
Chronic Phase ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Rare Cells

Abstract Imatinib mesylate (IM, Gleevec, Novartis), a specific inhibitor of the tyrosine kinase BCR-ABL, promotes an impressive complete cytogenetic remission rate in patients with chronic phase Chronic Myeloid Leukaemia (CML). In most cases however, molecular disease persists and may lead to eventual relapse. IM selectively interacts with BCR-ABL thereby inhibiting activation of downstream targets, in particular phosphorylation of CrkL. We have previously demonstrated the sensitivity of rare Ph+ CD34+ CML stem cells (<5% of total CD34+ cells) to the anti-proliferative but, not the pro-apoptotic effects of IM, hence their persistence in vitro. We hypothesised that these rare cells may also behave differently from total CD34+ cells in terms of their kinase response to IM. The effect of IM on the phosphorylation status of CrkL was therefore investigated, in total CD34+ and in primitive CD34+/38- cells from 6 untreated chronic phase CML patients. Ph+ CD34+ and CD34+/38- cells were cultured in serum free medium for up to 72h, in the presence and absence of IM (5mM). After 16 and 72h the phosphorylation status of CrkL was measured with an antibody specifically designed to recognise only the phosphorylated form of the protein. After 16h total CD34+ cells showed a significant dephosphorylation of CrkL in response to IM. However, after 72h the remaining viable BCR-ABL+ cells failed to show any de-phosphorylation of CrkL, consistent with an IM selected, resistant population of stem cells. Primitive CD34+/38- cells from the same patients exhibited no de-phosphorylation of CrkL after either 16 or 72h, confirming their innate/inherent resistance to the drug. These data show, for the first time, that very primitive CML cells exhibit BCR-ABL dependent resistance (i.e. BCR-ABL kinase remains active during drug exposure) and suggest that the drug is not reaching or binding its target at inhibitory concentrations. Novel approaches to eliminate these cells will be critical to the development of curative strategies for CML.

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.

scholarly journals Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Andrzej Eljaszewicz ◽  
Lukasz Bolkun ◽  
Kamil Grubczak ◽  
Malgorzata Rusak ◽  
Tomasz Wasiluk ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Progenitor Cells ◽  
Endothelial Progenitor Cells ◽  
Lymphoblastic Leukemia ◽  
Monocyte Subsets ◽  
Endothelial Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background. Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. Methods. We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. Results. We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. Conclusion. These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1397-1397
Author(s):  
Nadim Mahmud ◽  
Kazumi Yoshinaga ◽  
Craig Beam ◽  
Hiroto Araki
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (&gt;X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (&lt; X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 324-324
Author(s):  
Xiaoyan Jiang ◽  
Donna Forrest ◽  
Franck Nicolini ◽  
Karen Lambie ◽  
Kyi Min Saw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Clinical Responses ◽  
Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P&lt;0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and &gt;50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and &gt;40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P&lt; 0.001 with 5 μM and P&lt;0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

BMP4 and TGFbeta Differentially Regulate CD34+ Progenitor Development in Human Embryonic Stem Cells through SMAD-Dependent Pathway

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 889-889
Author(s):  
ZacK Z. Wang ◽  
Hao Bai ◽  
Melanie Arzigian ◽  
Yong-Xing Gao ◽  
Wen-Shu Wu
Keyword(s):  
Stem Cells ◽  
Smooth Muscle ◽  
Growth Factors ◽  
Progenitor Cells ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Ips Cells ◽  
Bmp Signaling ◽  
Cd34 Cells

Abstract Pluripotent stem cells derived from patients, including embryonic stem (ES) cells and “induced pluripotent stem” (iPS) cells, are a promising area of regenerative medical research. A major roadblock toward human clinical therapies using ES cells or iPS cells is to define the factors that direct ES cell differentiation into lineage specific cells. We previously established a simple and efficient human embryonic stem cell (hESC) differentiation system to generate CD34+/CD31+ progenitor cells that gave rise to hematopoietic and endothelial cells (Nat Biotech.25:317, 2007). To advance potential clinical application and to define the effects of growth factors on hematopoietic and vascular differentiation, we assessed hESC differentiation on human feeder cells in serum-free condition without intermediate embryoid body (EB) formation. We investigated the roles of BMPs, TGFbeta, VEGF, and FGF2 in directing hESC differentiation. Growth factors were added into culture at different time points to test their stage specific roles. Our study demonstrated that BMP proteins, including BMP2, BMP4, and BMP7, but not BMP9, had synergic effects to VEGF and FGF-2 on hESC differentiation to CD34+/CD31+ progenitor cells. BMP4 was essential to initial CD34+/CD31+ cell development, whereas VEGF and FGF2 promoted the differentiation in later stage, suggesting the sequential roles of BMP4, VEGF and FGF2 in directing hESC differentiation to CD34+/CD31+ progenitor cells. TGFbeta or activin promoted hESC differentiation into CD34+/CD31− cells that were unable to give rise to hematopoietic, endothelial, and smooth muscle cells. Furthermore, TGFbeta or activin activated Smad2/3 signaling, and suppressed BMP4-induced CD34+/CD31+ cells. Microarray analysis revealed that BMP4-induced CD34+ cells expressed hematopoietic, endothelial and smooth muscle genes, including GATA2, gamma globins, VE-Cad, KDR, CD31, Tie2, and aortic smooth muscle actin, whereas TGFbeta-induced CD34+ cells expressed pluripotent markers and endoderm markers, including Oct3/4, Sox2, and Nanog, HHEX, GATA6, and FoxA2. Both canonical BMP signaling (Smad1/5/8-dependent) and non-canonical BMP signaling (p38 MAPK and p42 ERK pathway) were activated by BMP4 in hESCs. Dorsomorphin specifically inhibited BMP4-mediated phosphorylation of Smad1/5/8, and blocked hESC differentiation into CD34+/CD31+ cells. In summary, BMPs and TGFbeta regulate distinct populations of CD34+ cells in hESCs. BMP-Smad1/5/8 pathway is critical for hematopoietic and vascular progenitor development.

Blocking of Cytokine Survival Signals along with Intense Bcr-Abl Kinase Inhibition May Eradicate CML Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3250-3250
Author(s):  
Devendra K Hiwase ◽  
Deborah L White ◽  
Jason A Powell ◽  
Verity A Saunders ◽  
Stephanie Zrim ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Activity ◽  
Short Term ◽  
Kinase Inhibition ◽  
Gm Csf ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Short Term Culture

Abstract Abstract 3250 Poster Board III-1 Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. Fig 1 In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Fig 1. In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1449-1449
Author(s):  
Naoya Uchida ◽  
Aylin Bonifacino ◽  
Allen E Krouse ◽  
Sandra D Price ◽  
Ross M Fasano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Transgene Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

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