Selective Targeting of CML Progenitor/Stem Cells by the Class 1 Histone Deacetylase (HDAC) Inhibitor MS275 in Combination with Imatinib.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2791-2791
Author(s):  
Su Chu ◽  
YinWei Ho ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Hdac Inhibitor ◽  
Hdac Inhibitors ◽  
Normal Cells ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Selective Targeting ◽  
Class 1

Abstract Abstract 2791 BCR-ABL tyrosine kinase inhibitors (TKI) effectively inhibit CML stem/progenitor cell proliferation and induce remission in CML patients, but do not completely eliminate primitive leukemia stem cells, which persist as potential sources of relapse. There is considerable interest in identifying additional therapeutic strategies to selectively induce apoptosis in CML stem and progenitor cells. We have shown that the pan-HDAC inhibitor Pabinostat, that targets Class 1, Class 2 and Class 4 HDACs, can eliminate CML stem cells in vitro and in vivo when combined with Imatinib (Cancer Cell 17:427, 2010). However pan-HDAC inhibitors can have significant toxicity to normal stem cells, which limits their clinical utility. Here we used siRNA knockdown to determine the role of individual HDACs in proliferation and apoptosis of CML compared with normal CD34+ cells. CD34+ cells were transfected with siRNAs to 11 individual HDAC enzymes and control non-specific siRNA, using an Amaxa nucleofector system. 50–80% knockdown of target gene expression was achieved. Knockdown of individual Class 1, 2 and 4 HDACs resulted in increased apoptosis of CML CD34+ cells, which was increased in combination with IM. On the other hand, normal CD34+ cells demonstrated increased apoptosis in response to knockdown of Class 2 and 4 HDACs, but not Class 1 HDACs. These results suggested that inhibition of Class I HDACs may allow selective targeting of CML stem/progenitor cells while sparing normal cells, and led us to evaluate the effects of MS275, an orally available HDAC Class I specific inhibitor currently being evaluated in clinical trials in solid tumors, against CML and normal stem/progenitor cells. MS275 (1μM) exposure increased acetylated Histone H3 and histone H4 levels in CML CD34+ cells, as analyzed by Western blots and flow cytometry. Treatment of CML CD34+CD38- cells and CD34+CD38+ cells with MS-275 resulted in significantly increased apoptosis compared with their normal counterparts (17.93±1.1% for CML vs. 7.9±2.3% for normal CD34+CD38- cells, p=0.01; 20.1±4.6% for CML vs. 9±1.9% for normal CD34+CD38+ cells, p=0.05). Combination of MS275 with IM resulted in significant enhancement of apoptosis of CML but not normal cells (72±11.1% for CML vs. 6.3±1.87% for normal CD34+CD38- cells, p=0.02; 41.3±9.8% for CML vs. 8.45±1.75% for normal CD34+CD38+ cells, p=0.04). Quiescent CML stem cells are especially resistant to TKI-induced apoptosis. The combination of MS275 and IM resulted in significantly increased apoptosis of non-dividing CML but not normal CD34+38- cells (72.1±14.3% for CML vs. 10.6±2.0% for normal cells, n=3). Cell cycle analysis by Ki67 and DAPI labeling demonstrated significant increase in G0, and decrease in S/G2/M phase, in CML but not normal CD34+ cells treated with MS275, alone or combination with IM. Consistent with these findings, we observed downregulation of the anti-apoptotic protein Mcl1 and up-regulation of the cell cycle inhibitor p21 in MS-275 treated CML CD34+ cells. MS275 treatment also significantly reduced colony forming cell growth from CML but nor normal CD34+CD38+ and CD34+CD38- cells in methylcellulose progenitor culture. To further evaluate the effects of MS275 on normal stem cells, normal CD34+ cells were treated with MS275, IM or the combination of MS275 and IM for 72 hours in vitro, followed by injection into sub-lethally irradiated (300 cGy) NSG mice and evaluation of human cells engraftment after 12 weeks. These studies showed that treatment with MS275 did not significantly affect engraftment of normal stem cells in NSG mice. In conclusion, the selective Class1 HDAC inhibitor MS-275 significantly increases apoptosis in CML primitive and committed progenitors, including non-proliferating cells, which is further significantly enhanced in combination with IM. In contrast to pan-HDAC inhibitors, MS275 treatment does not affect survival or growth of normal stem cells. These results indicate that use of inhibitors specific to Class 1 HDAC enzymes could avoid toxicity to normal HSC associated with pan-HDAC inhibitors, allowing more selective and effective targeting of CML LSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Treatment in Vitro with a Combination of Bcl-Xl Inhibitor-ABT-737 and a JAK2 Inhibitor Selectively Eliminates JAK2V617F MPN Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 752-752 ◽  
Author(s):  
Min Lu ◽  
Wei Zhang ◽  
Jiapeng Wang ◽  
Yan Li ◽  
Xiaoli Wang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Lower Percentage ◽  
Normal Cells ◽  
Jak2 Inhibitor ◽  
Cd34 Cells ◽  
Hel Cells ◽  
Jak2 Inhibitors

Abstract Abstract 752 The Ph- myeloproliferative neoplasm (MPN) are associated with excessive production of red cells, platelets and granulocytes which largely determines their clinical manifestations. A mutation in the JAK2 tyrosine kinase (JAK2V617F) was identified in the majority of patients with MPNs. The JAK2V617F mutation has been shown to play a pivotal role in the pathogenesis of MPNs. We have reported that erlotinib (Tarceva), a kinase inhibitor which inhibits the epidermal growth factor induced kinase activity, is also a potent inhibitor of JAK2V617F activity. It has been shown that erythroblasts from patients with polycythemia vera (PV) express elevated levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL. In addition, we have recently documented that megakaryocytes derived from patients with primary myelofibrosis (PMF) undergo a delayed pattern of apoptosis in vitro which might be attributed to the over-expression of Bcl-xL. We hypothesize that a combination of a JAK2V617F inhibitor and a Bcl-xL inhibitor might be capable of selectively eliminating MPN cells while sparing normal cells, and therefore, providing an optimal treatment strategy for Ph− MPNs. We then evaluated the combinations of a JAK2V617 inhibitor (erlotinib or INCB018424) and a Bcl-xL inhibitor (ABT-737) for their ability to selectively eliminate MPN (JAK2V617F positive) cells while sparing normal cells in vitro using a variety of screening systems. We first tested the ability of each of the JAK2 inhibitors alone or in combination with ABT-737 to induce death of HEL cells, which harbors JAK2V617F. Among these treatments, ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1 nM) were shown to have similar capability of inducing HEL cell apoptosis (50-70%) which was significantly greater than that by each of the single agents (<5%). However, the combination of ABT-737 (0.25 uM) plus erlotinib (1.0 uM) was the least potent of inducing normal CD34+ cells to undergo apoptosis (∼5%) as compared to ABT-737 plus INCB018424 (∼20%). We next examined the effects of each of the JAK2 inhibitors alone or in combination with ABT-737 on CD34+ cells isolated from patients with PV or PMF. PV or PMF CD34+ cells were incubated in the absence or presence of agent(s) for 4 days; the percentage of apoptotic cells was then determined using Annexin V/PI staining by flow cytometry. A fraction of both untreated and treated CD34+ cells were assayed for hematopoietic colonies in the presence of SCF, IL-6, IL-3, G-CSF and EPO; individual colonies were then randomly plucked for JAK2V617F genotyping using nested allele-specific PCR. Similar to HEL cells, the percentage of MPD CD34+ cells undergoing apoptosis were similar when cells were treated with ABT-737 (0.25 uM) plus either erlotinib (1.0 uM), or INCB018424 (1.0 nM) (∼40%), but were significantly higher than that of MPN CD34+ cells treated with each of the agents alone (∼5%). Interestingly, when the percentages of JAK2V617F positive colonies within each of the treated and untreated cell populations were analyzed, MPN CD34+ cells treated with ABT-737 plus each of the JAK2 inhibitors contained a significantly lower percentage of JAK2V617F positive colonies than those untreated or treated with each of the single agents. These data indicate that the combination of ABT-737 plus a JAK2 inhibitor specifically targets on the JAK2V617F positive progenitor cells. Collectively, we identified an agent combination of ABT-737 (0.25 uM) plus a JAK2 inhibitor that is capable of selectively eliminating JAK2V617F positive MPN progenitor cells while sparing normal progenitor cells in vitro. Disclosures: No relevant conflicts of interest to declare.

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.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Different Histone Deacetylase Inhibitors Affect Distinct Cellular Targets within the Hierarchy of Hematopoietic Stem / Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1721-1721
Author(s):  
Hiroto Araki ◽  
Ronald Hoffman ◽  
Nadim Mahmud
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Recently several laboratories have examined the in vitro effects of chromatin modifying agents on hematopoiesis. We have previously reported that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) to cultures of human cord blood (CB) CD34+ cells containing SCF, thrombopoietin and FLT-3 ligand (cytokines) resulted in a 10-fold expansion of SCID repopulating cells (SRC) (Araki et al. Blood2004: 104:881a). Recently others have shown that another HDAC inhibitor, valproic acid (VA) resulted in an expansion of CB CD34+ cells and murine hematopoietic stem / progenitor cells (HSPC) (DeFelice et al. Cancer Res.2005:65:1505, Beg et al. Cancer Res.2005:65:2537). In our current studies we have compared the efficacy of VA, TSA or 5azaD as single agents or in combination to promote the expansion of CB HSPC in vitro. The frequency and fold expansion of colony forming cells (CFC), cobblestone area-forming cells (CAFC) as well as SRC generated from CB CD34+ cells after 9 days of culture were examined. The addition of cytokines alone result in a 1.5-fold expansion of CD34+CD90+ cells. By contrast the addition of cytokines with VA led to a 65-fold expansion of the numbers of CD34+CD90+ cells as compared to a 1.3-fold, 5.6-fold, 4.2-fold or 12.5-fold expansion of CD34+CD90+ cells in cultures receiving cytokines with 5azaD, TSA or 5azaD/VA or 5azaD/TSA respectively. In vitro biological assays (CFC, CAFC) were performed to determine the correlation between CD34+CD90+ cell expansion and function. Cultures receiving cytokines alone or cytokines with VA had the greatest degree of expansion of CFC (14.4 and 18.6-fold respectively). By contrast cultures receiving cytokines alone contained only 70% of the numbers of CAFC as did the primary CB CD34+ cells. Cultures receiving VA or 5azaD/TSA had the greatest degree of expansion of CAFC numbers (9.6-fold and 11.5-fold respectively). The marrow repopulating potential of these various expanded cell populations were then assayed by transplanting them into NOD/SCID mice. CD34+ cells from cultures receiving cytokines alone or cytokines with 5azaD/VA were devoid of human hematopoietic cell chimerism. By contrast, all NOD/SCID mice receiving grafts from cultures treated with cytokines with 5azaD/TSA had evidence of human multilineage hematopoietic engraftment (7.5% ± 3.7%). Cells from cultures treated with cytokines with VA are capable of engraftment in 2 out of 6 mice with a barely detectable level of human cell chimerism (0.11%, 0.14%). We then assessed using western blot analysis whether the chromatin modifying agents might alter HSC function by upregulating HOXB4 protein levels. HOXB4 protein was detectable in cells cultured in the presence of cytokines with VA, cytokines with 5azaD/VA, cytokines with 5azaD/TSA but only cells treated with cytokines with 5azaD/TSA contained readily assayable SRC. These studies suggest that treatment with different chromatin modifying agents are capable of altering the differentiation program of distinct populations of HSPC. Some treatments (VA, 5azaD/VA) primarily affect CFC and CAFC but not SRC. While 5azaD/TSA targets CAFC and SRC but not CFC. In addition, although HOXB4 may participate in HSC self-renewal, additional genes are likely altered following 5azaD/TSA treatment which are required for the maintenance of SRC potential.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

Hypoxia Potentiates Notch-Induced Expansion of Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2640-2640
Author(s):  
Jianfei Fu ◽  
Heather D. Huntsman ◽  
Ayla Cash ◽  
Patali S. Cheruku ◽  
Richard H. Smith ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Number Of Cells

Abstract Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O2 tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholow) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. Disclosures Cheruku: Novartis: Research Funding. Larochelle:Novartis: Research Funding.

scholarly journals Thrombopoietin Affects the Behavior of Myelofibrosis Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2145-2145
Author(s):  
Xiaoli Wang ◽  
Cing Siang Hu ◽  
Yan Li ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Fold Increase ◽  
Absolute Number ◽  
Human Platelets ◽  
Similar Degree ◽  
Free Expansion ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Expansion Media

Abstract Recently the presence of myelofibrosis (MF) stem cells (MF-SC) in the spleens of MF patients which when transplanted into NOD/SCID/IL2R null (NSG) mice were capable of generating multiple hematopoietic lineages that belonged to the malignant clone has been observed. Although MF is characterized by marrow megakaryocyte (Mk) hyperplasia, limited numbers of human marrow Mks were, however, observed in these transplanted mice and evidence of both marrow and splenic fibrosis 9 months after the transplantation was lacking (Wang X, et al. J Clin Invest. 2012; 122:3888). Splenic MF CD34+ cells did retain the capacity to differentiate in vitro into CD41a+ and CD61+ Mks in the presence of thrombopoietin (TPO). We therefore investigated whether the administration of romiplostim, a novel human TPO peptide mimetic which lacks homology to endogenous TPO in MF humanized mice, might create appropriate environmental cues which affect the behavior of MF-SCs. Elevated levels of TPO (345±114ng/ml) were detected in MF plasmas (n=13) as compared to levels detected in normal plasma (10±4ng/ml, n=6, P=0.049), indicating the possibility that TPO affects MF-SCs and MF hematopoietic progenitor cells (HPC). Following the culture of CB (n=3) or PB MF CD34+ cells (n=4) for 1 and 2 wks in serum free expansion media (SFEM) supplemented with SCF (50ng/ml) + romiplostim (100ng/ml), the numbers of total cells, CD34+Lin- cells and assayable HPCs, including CFU-Mk, CFU-GM and BFU-E, CD41a+CD34-CD15- cells and CD15+CD34-CD41a- cells generated were greater (CB) or similar (MF) to the number generated in the cultures containing SCF + TPO (100ng/ml). Moreover, similar proportions of colonies (CFU-GM+BFU-E) generated in cultures supplemented with SCF+TPO or SCF+ romiplostim (100ng/ml, 1000ng/ml) were JAK2V617F-positive. These findings suggest that both romiplostim and TPO are capable of promoting MF-SC and HPC proliferation in vitro. To assess the effects of romiplostim on human (h) platelet production, CB CD34+ cells (5×105) were transplanted via the tail vein into eight- to nine-week-old sublethally irradiated (240 cGy) NSG mice. One week after transplantation, mice were treated with water or 10, 100 or 1000 µg/kg of romiplostim. Both the percentage and number of hCD41a+ platelets in the PB increased 4 days following the treatment with 10ug/kg romiplostim, and peaked at day 8 with a 2.58±0.29 fold increase in the percentage (P<0.05) and 3.51±1.32 fold increase in the absolute number (P<0.05) of hCD41a+ platelets being observed. The number of human platelets in the PB, were reduced to the levels detected in mice not receiving romiplostim by day 15. MF splenic CD34+ cells (5-10×105) were next transplanted into romiplostim (10ug/kg or 30ug/kg) treated or control NSG mice. Two months after the transplantation, a 1.83±0.62 fold increase in the number of hCD41a+ cells was observed in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not treated with romiplostim. Moreover, a 7.13±2.13 fold elevation in the percentage of hCD45+ cells was detected in the PB of mice receiving splenic MF CD34+ cells and romiplostim (10ug/kg) as compared with mice not receiving romiplostim. Moreover, enhanced hCD45+ cell chimerism was achieved in both the marrow (55.6%, 7.2%) and spleen (47.7%, 1.5%) of mice receiving splenic CD34+ cells from 2 MF patients and romiplostim as compared with mice not receiving romiplostim (marrow: 30.4%, 5.6%; spleen: 23.6%, 0.9%), respectively. The splenic MF CD34+ cells receiving or not receiving romiplostim had a similar differentiation patterns in the marrow and spleen, however, the numbers of hCD33+, hCD19+, hCD3+, hCD41a+ and hCD15+ cells detected in mice treated with romiplostim were dramatically greater than those detected in mice not receiving romiplostim. Greater numbers of hCD34+ cells were detected in the BM (7.1%; 3.5%) of recipient mice receiving romiplostim as that detected in mice not receiving romiplostim (6.6%; 0.6%). Furthermore, a similar degree of hCD45+ cell and hCD34+ cell chimerism was observed in both the marrow and spleen of mice receiving splenic MF CD34+ cells and romiplostim 4 months after the transplantation. These findings suggest that the administration of thrombopietin agonist has a profound effect on the behavior of MF-SCs and that the elevated levels of TPO documented in MF patients likely has important effects on the biology of MF-SCs. Disclosures Wang: The MPN Research Foundation (MPNRF) and the Leukemia & Lymphoma Society (LLS) : Research Funding.

153 DIFFERENT MOLECULAR MECHANISMS FOR HISTONE DEACETYLASE INHIBITOR-INDUCED APOPTOSIS IN DOG FIBROBLASTS AND MESENCHYMAL STEM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 206
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
G. A. Kim ◽  
Y. K. Jo ◽  
Y. B. Choi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Molecular Mechanisms ◽  
Hdac Inhibitors ◽  
Normal Cells ◽  
Apoptotic Genes ◽  
Induced Apoptosis

Histone deacetylase (HDAC) inhibitors have been applied to cancer research for a therapeutic purpose or somatic cell nuclear transfer for improvement of embryonic reprogramming. Considering the ubiquitous expression of HDAC in normal cells, effects of HDAC inhibitors on normal cells need to be evaluated in detail. Therefore, we aimed to investigate molecular mechanisms of HDAC inhibitor-induced apoptosis in mesenchymal stem cells and compare with the results from fibroblasts. Beagle skin fibroblasts (BF) and adipose-derived mesenchymal stem cell (MSC) lines were established from a 7-year-old beagle. Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and RCMEp (K-stem Cell Ltd, Seoul, Korea) were used as culture media for BF and MSC, respectively. A Food and Drug Administration-approved HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), was dissolved in dimethyl sulfoxide (DMSO). On passage 4, cells were subcultured to reach 50% of confluency, and cultured with 5 μM of SAHA. Culture medium containing the same volume of DMSO used in the SAHA group was used for the control. After 24 h, all cells were harvested, RNAs were extracted, and cDNA were synthesised. Transcript expression of anti-apoptotic genes (BFL, MCL, BCLxl, BCL2) and pro-apoptotic genes (BAX, BID, BIM) were analysed using RT-PCR. Two-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) with Bonferroni post-tests was performed for statistical analysis. Expression of MCL and BCLxl was significantly decreased in both groups. However, although BFL expression was remarkably increased only in BF (16.7-fold), BCL2 was significantly increased in MSC (7.2-fold) after SAHA treatment. Also, transcript of BAX was significantly increased in MSC (1.5-fold), and BID was significantly decreased in BF (0.3-fold). These results would be helpful to understand different molecular mechanisms of apoptosis induced by HDAC inhibitor on fibroblasts and mesenchymal stem cells. This study was supported by RDA (#PJ010928032015), IPET (#311062-04-3SB010), NRF (#2014R1A1A2059928), Research Institute for Veterinary Science, Nestle Purina PetCare, and the BK21 plus program.

Drug Screening of Primary Human Pediatric Pre-B Cell Acute Lymphoblastic Leukemia (pre-B ALL) Cells in Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4295-4295
Author(s):  
Jae-Hung Shieh ◽  
Tsann-Long Su ◽  
Jason Shieh ◽  
Malcolm A.S. Moore
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 4295 Pre-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common leukemia in children and is treatable. However, no in vitro nor in vivo models are available to investigate their pathophysiology other than a number of established cell lines that grow in the absence of any cytokine dependence or stromal interaction. We developed a serum-free MS-5 cell (a murine bone marrow stromal cell line) co-culture system that is capable of expanding human primary pre-B ALL CD34+CD19+ cells in vitro. To define a population of pre-B ALL initiating cells, our study reveals that a sorted CD34bright population displays a slow proliferation and maintains a high % of CD34+ cells. In contrast, CD34dim cells/CD34− cells fraction shows a higher proliferation but expanded cells lost CD34 antigens. A group of alkylating molecules (BO-1055, -1090, 1099, -1393 and -1509) was evaluated for proliferation of the pre-B ALL CD34+ cells, the pre-B ALL CD34− cells, human mesenchymal stem cells (hMSC), murine MSC (MS-5 cells and Op9 cells), human bone marrow derived endothelial cells (BMEC), and human cord blood (CB) CD34+ cells, as well as for a week 5 cobblestones area forming (CAFC) assay with CB CD34+ cells. BO-1055 molecule efficiently suppressed the growth of pre-B ALL CD34+ cells (IC50 = 0.29 μM) and CD34− cells (IC50 = 0.31 μM). In contrast, IC50 of BMEC, MSC, CB CD34+ cells and CAFC are >10, >25, 8, and >5 μM, respectively. Pre-B ALL cells expressing green fluorescent protein (GFP) and luciferase (GFP-Lu-pre-B ALL) were created, and a xenograft of the GFP-Lu-pre-B ALL cells to NOD/SCID IL2R gamma null (NSG) mice was established. The in vivo effect of BO-1055 to the GFP-Lu-pre-B ALL cells in NSG mice is under investigation. Our stromal culture system supports primary pre-B ALL cells and closely recapitulates the growth of primary human pre-B ALL cells in their niche in vivo. Based on this co-culture system, we identified BO-1055 as a potential therapeutic agent with an excellent toxicity window between pre-B ALL cells and normal tissues including BMEC, MSC and hematopoietic progenitor/stem cells. The in vitro stromal co-culture system combined with the xenograft model of GFP-Lu-pre-B ALL cells provides an efficient and powerful method to screen new drugs for pre-B ALL therapy. Disclosures: No relevant conflicts of interest to declare.

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