Effect of the Neddylation Inhibitor Pevonedistat on Normal Hematopoietic Stem Cell Subsets and Immune Cell Composition

Abstract Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications. Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of 'stemness'. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc. Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM. Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells. Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve 'stemness' of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. Figure 1 Figure 1. Disclosures Majidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.

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Lower Hematopoietic Progenitor Cell Counts and Yields at Subsequent Donations Is Influenced By a Shorter Inter-Donation Interval between the First and Subsequent Mobilizations

Blood ◽

10.1182/blood-2019-123492 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1962-1962

Author(s):

Sandhya R. Panch ◽

Brent R. Logan ◽

Jennifer A. Sees ◽

Bipin N. Savani ◽

Nirali N. Shah ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Time Interval ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Peripheral Blood Cd34 ◽

Cell Counts ◽

Cd34 Cells

Introduction: Approximately 7% of unrelated hematopoietic stem cell (HSC) donors are asked to donate a subsequent time to the same or different recipient. In a recent large CIBMTR study of second time donors, Stroncek et al. incidentally found that second peripheral blood stem cell (PBSC) collections had lower total CD34+ cells, CD34+ cells per liter of whole blood processed, and CD34+ cells per kg donor weight. Based on smaller studies, the time between the two independent PBSC donations (inter-donation interval) as well as donor sex, race and baseline lymphocyte counts appear to influence CD34+ cell yields at subsequent donations. Our objective was to retrospectively evaluate factors contributory to CD34+ cell yields at subsequent PBSC donation amongst NMDP donors. Methods. The study population consisted of filgrastim (G-CSF) mobilized PBSC donors through the NMDP/CIBMTR between 2006 and 2017, with a subsequent donation of the same product. evaluated the impact of inter-donation interval, donor demographics (age, BMI, race, sex, G-CSF dose, year of procedure, need for central line) and changes in complete blood counts (CBC), on the CD34+ cell yields/liter (x106/L) of blood processed at second donation and pre-apheresis (Day 5) peripheral blood CD34+ cell counts/liter (x106/L) at second donation. Linear regression was used to model log cell yields as a function of donor and collection related variables, time between donations, and changes in baseline values from first to second donation. Stepwise model building, along with interactions among significant variables were assessed. The Pearson chi-square test or the Kruskal-Wallis test compared discrete variables or continuous variables, respectively. For multivariate analysis, a significance level of 0.01 was used due to the large number of variables considered. Results: Among 513 PBSC donors who subsequently donated a second PBSC product, clinically relevant decreases in values at the second donation were observed in pre-apheresis CD34+ cells (73.9 vs. 68.6; p=0.03), CD34+cells/L blood processed (32.2 vs. 30.1; p=0.06), and total final CD34+ cell count (x106) (608 vs. 556; p=0.02). Median time interval between first and second PBSC donations was 11.7 months (range: 0.3-128.1). Using the median pre-apheresis peripheral blood CD34+ cell counts from donation 1 as the cut-off for high versus low mobilizers, we found that individuals who were likely to be high or low mobilizers at first donation were also likely to be high or low mobilizers at second donation, respectively (Table 1). This was independent of the inter-donation interval. In multivariate analyses, those with an inter-donation interval of >12 months, demonstrated higher CD34+cells/L blood processed compared to donors donating within a year (mean ratio 1.15, p<0.0001). Change in donor BMI was also a predictor for PBSC yields. If donor BMI decreased at second donation, so did the CD34+cells/L blood processed (0.74, p <0.0001). An average G-CSF dose above 960mcg was also associated with an increase in CD34+cells/L blood processed compared to donors who received less than 960mcg (1.04, p=0.005). (Table 2A). Pre-apheresis peripheral blood CD34+ cells on Day 5 of second donation were also affected by the inter-donation interval, with higher cell counts associated with a longer time interval (>12 months) between donations (1.23, p<0.0001). Further, independent of the inter-donation interval, GCSF doses greater than 960mcg per day associated with higher pre-apheresis CD34+ cells at second donation (1.26, p<0.0001); as was a higher baseline WBC count (>6.9) (1.3, p<0.0001) (Table 2B). Conclusions: In this large retrospective study of second time unrelated PBSC donors, a longer inter-donation interval was confirmed to be associated with better PBSC mobilization and collection. Given hematopoietic stem cell cycling times of 9-12 months in humans, where possible, repeat donors may be chosen based on these intervals to optimize PBSC yields. Changes in BMI are also to be considered while recruiting repeat donors. Some of these parameters may be improved marginally by increasing G-CSF dose within permissible limits. In most instances, however, sub-optimal mobilizers at first donation appear to donate suboptimal numbers of HSC at their subsequent donation. Disclosures Pulsipher: CSL Behring: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; Bellicum: Consultancy; Amgen: Other: Lecture; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Honoraria. Shaw:Therakos: Other: Speaker Engagement.

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Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽

10.1182/blood.v124.21.4363.4363 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4363-4363

Author(s):

Alexandre Janel ◽

Nathalie Boiret-Dupré ◽

Juliette Berger ◽

Céline Bourgne ◽

Richard Lemal ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Confocal Microscopy ◽

Hematopoietic Stem Cell ◽

Biological Samples ◽

Mesenchymal Cells ◽

Hematopoietic Stem ◽

Bm Niche ◽

Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v95.9.2813.009k20_2813_2820 ◽

2000 ◽

Vol 95 (9) ◽

pp. 2813-2820 ◽

Cited By ~ 215

Author(s):

Lisa Gallacher ◽

Barbara Murdoch ◽

Dongmei M. Wu ◽

Francis N. Karanu ◽

Mike Keeney ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Surface Markers ◽

Cell Surface Markers ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

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

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Multicolor Flow Cytometry Allows Quantitative Analysis of Signal Transduction in Murine and Human Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v112.11.5393.5393 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5393-5393

Author(s):

Tamara Riedt ◽

Claudia Lengerke ◽

Lothar Kanz ◽

Viktor Janzen

Keyword(s):

Signal Transduction ◽

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Intracellular Signaling ◽

Hematopoietic Stem ◽

Specific Antibodies ◽

Human Cd34 ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract The regulation of cell cycle activity, differentiation and self-renewal of stem cells are dependent on accurate processing of intrinsic and extrinsic signals. Traditionally, signaling pathway activation has been detected by immunobloting using phospho-specific antibodies. However, detection of signal transduction in rare cells within heterogeneous populations, such as hematopoietic stem and progenitor cells (HSC) has been difficult to achieve. In a recently reported approach to visualize signaling in selected single c-Kit+ Sca-1+ Lin− (KSL) bone marrow cells, cells were sorted onto glas slides by flow cytometry and signaling was detected by confocal fluorescence microscopy, a very time consuming method that thus restricts the number of cells that can be analysed simultaneously. Moreover it permits only qualitative, but not quantitative signaling evaluation (Yamazaki et al., EMBO J. 2006). Here, we report a new protocol allowing quantitative measurement of signaling activity in large numbers of defined murine and human hematopoietic cells. The cells are stained with established surface markers and then phospho-specific antibodies are used to detect the levels of active intracellular signaling molecules. Signals are quantified by flow cytometry fluorescence measurement. Importantly, the protocol developed in our laboratory enables preservation of surface marker staining identifying the cells of interest inspite the fixation and permeabilization procedures necessary for intracellular signaling detection. This applies also for antigens previously reported to be particularly vulnerable to standard fixation and permeabilization approaches (e.g. the murine stem cell markers c-Kit and Sca1). Thus, our protocol provides an easy and reliable method for quantifying the activation degree of several intracellular signaling pathways on single cell level in defined hematopoietic (stem) cells within the heterogeous bone marrow (BM) compartment. Using cytokines known to exert a biological effect on HSCs, we have examined the susceptibility of KSL murine BM cells and human BM CD34+ cells to cytokine-induced signaling. We have performed extensive dosage titration and time course analysis for multiple cytokines (SCF, TPO, Flt-3, IL-3, IL-6, Ang-1, SDF-1α, TGF-β, and BMP-4) and signaling pathways (ERK, Akt, p38MAPK, Jak-Stat, TGF-β/BMP-Smad) in murine KSL BM cells. The activation intensity and the duration of signal activity as measured by the expression of corresponding phosphorylated proteins were cytokine specific. The obtained results can be used as a platform to explore signaling alterations in distinct compartments of the hematopoietic system, and may provide mechanistical insights for observed bone marrow defects (e.g impaired ERK signaling pathway has been detected as a possible cause of hematopoietic defects in Caspase-3 mutant murine HSCs, Janzen et al, Cell Stem Cell 2008). Furthermore, we could show that the technique is also applicable to human BM cells and that the human hematopoietic stem cell marker CD34 is also preserved by our fixation and permeabilization protocol. Preliminary results suggest that cytokines induce similar signaling activation in human CD34+BM cells collected from healthy donors. As observed in mouse KSL BM cells, stimulation of human CD34+cells with human stem cell factor (hSCF) induced activation of the ERK but not the Akt pathway. Ongoing experiments analyse the stimulatory effects of other cytokines such as thrombopoietin (TPO) and fms-related tyrosine kinase 3 (Flt-3) and their corresponding pathways. Moreover, comparative studies are underway analyzing cross-reactivity between mouse and human cytokines, aiming to provide insights into cytokine-induced biases in commonly used xenotransplantation models.

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Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3229.3229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3229-3229 ◽

Cited By ~ 1

Author(s):

Ivana N Micallef ◽

Eric Jacobsen ◽

Paul Shaughnessy ◽

Sachin Marulkar ◽

Purvi Mody ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cd34 Cells ◽

Low Platelet Count ◽

Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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

Blood ◽

10.1182/blood.v116.21.1449.1449 ◽

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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Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro

Blood ◽

10.1182/blood.v118.21.4803.4803 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4803-4803

Author(s):

Manja Wobus ◽

Jing Duohui ◽

David M Poitz ◽

Katrin Müller ◽

Rainer Ordemann ◽

...

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Osteogenic Differentiation ◽

Oxygen Tension ◽

Cell Phenotype ◽

Low Oxygen ◽

Hematopoietic Stem ◽

Hypoxic Conditions ◽

Lower Oxygen

Abstract Abstract 4803 Background: Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions. Design and Methods: Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions. Results: In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC. Conclusions: Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro. Disclosures: No relevant conflicts of interest to declare.

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Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Blood ◽

10.1182/blood.v122.21.143.143 ◽

2013 ◽

Vol 122 (21) ◽

pp. 143-143 ◽

Cited By ~ 4

Author(s):

Saar Gill ◽

Sarah K Tasian ◽

Marco Ruella ◽

Olga Shestova ◽

Yong Li ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Research Funding ◽

Conditioning Regimen ◽

Hematopoietic Stem ◽

Gm Csf ◽

Nsg Mice ◽

Cd34 Cells ◽

Society Research

Abstract Engineering of T cells with chimeric antigen receptors (CARs) can impart novel T cell specificity for an antigen of choice, and anti-CD19 CAR T cells have been shown to effectively eradicate CD19+ malignancies. Most patients with acute myeloid leukemia (AML) are incurable with standard therapies and may benefit from a CAR-based approach, but the optimal antigen to target remains unknown. CD123, the IL3Rα chain, is expressed on the majority of primary AML specimens, but is also expressed on normal bone marrow (BM) myeloid progenitors at lower levels. We describe here in vitro and in vivostudies to evaluate the feasibility and safety of CAR-based targeting of CD123 using engineered T cells (CART123 cells) as a therapeutic approach for AML. Our CAR consisted of a ScFv derived from hybridoma clone 32716 and signaling domains from 4-1-BB (CD137) and TCR-ζ. Among 47 primary AML specimens we found high expression of CD123 (median 85%, range 6-100%). Quantitative PCR analysis of FACS-sorted CD123dim populations showed measurable IL3RA transcripts in this population, demonstrating that blasts that are apparently CD123dim/neg by flow cytometry may in fact express CD123. Furthermore, FACS-sorted CD123dimblasts cultured in methylcellulose up-regulated CD123, suggesting that anti-CD123 immunotherapy may be a relevant strategy for all AML regardless of baseline myeloblast CD123 expression. CART123 cells incubated in vitro with primary AML cells showed specific proliferation, killing, and robust production of inflammatory cytokines (IFN-α, IFN-γ, RANTES, GM-CSF, MIP-1β, and IL-2 (all p<0.05). In NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with the human AML cell line MOLM14, CART123 treatment eradicated leukemia and resulted in prolonged survival in comparison to negative controls of saline or CART19-treated mice (see figure). Upon MOLM14 re-challenge of CART123-treated animals, we further demonstrated robust expansion of previously infused CART123 cells, consistent with establishment of a memory response in animals. A crucial deficiency of tumor cell line models is their inability to represent the true clonal heterogeneity of primary disease. We therefore engrafted NSG mice that are transgenic for human stem cell factor, IL3, and GM-CSF (NSGS mice) with primary AML blasts and treated them with CART123 or control T cells. Circulating myeloblasts were significantly reduced in CART123 animals, resulting in improved survival (p = 0.02, n=34 CART123 and n=18 control animals). This observation was made regardless of the initial level of CD123 expression in the primary AML sample, again confirming that apparently CD123dimAML may be successfully targeted with CART123 cells. Given the potential for hematologic toxicity of CART123 immunotherapy, we treated mice that had been reconstituted with human CD34+ cells with CART123 cells over a 28 day period. We observed near-complete eradication of human bone marrow cells. This finding confirmed our finding of a significant reduction in methylcellulose colonies derived from normal cord blood CD34+ cells after only a 4 hour in vitro incubation with CART123 cells (p = 0.01), and was explained by: (i) low level but definite expression of CD123 in hematopoietic stem and progenitor cells, and (ii) up-regulation of CD123 upon myeloid differentiation. In summary, we show for the first time that human CD123-redirected T cells eradicate both primary human AML and normal bone marrow in xenograft models. As human AML is likely preceded by clonal evolution in normal or “pre-leukemic” hematopoietic stem cells (Hong et al. Science 2008, Welch et al. Cell 2012), we postulate that the likelihood of successful eradication of AML will be enhanced by myeloablation. Hence, our observations support CART-123 as a viable therapeutic strategy for AML and as a novel cellular conditioning regimen prior to hematopoietic cell transplantation. Figure 1. Figure 1. Disclosures: Gill: Novartis: Research Funding; American Society of Hematology: Research Funding. Carroll:Leukemia and Lymphoma Society: Research Funding. Grupp:Novartis: Research Funding. June:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding. Kalos:Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding.

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A Randomized Comparison of Low-Dose Cyclophosphamide + G-CSF Versus G-CSF Alone Mobilization after Lenalidomide-Bortezomib-Dexamethasone (RVD) Induction in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.847.847 ◽

2014 ◽

Vol 124 (21) ◽

pp. 847-847

Author(s):

Raija Silvennoinen ◽

Tuija Lundan ◽

Pekka Anttila ◽

Jouni Heiskanen ◽

Marjaana Säily ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Cell Count ◽

Primary Endpoint ◽

Research Funding ◽

Low Dose ◽

Median Number ◽

Cell Harvesting ◽

Cd34 Cells ◽

Neutrophil Engraftment

Abstract Introduction Autologous stem cell transplantation (ASCT) is the standard treatment in multiple myeloma (MM) for eligible patients below 70 years of age. The main mobilization treatment in Europe consists of combination of cyclophosphamide (CY) and G-CSF. It is questionable if CY is useful in mobilization. CY as an alkylating agent might also have some negative long-term effects. Bortezomib seems not to have negative impact on autologous stem cell harvesting, but prolonged use of lenalidomide might hamper mobilization. There are only a few studies regarding autologous stem cell mobilization after RVD induction. We designed this randomized study as a substudy of the Finnish Myeloma Group Study (NCT01790737) to compare the results of CY+G-CSF versus G-CSF mobilization in autologous stem cell harvesting. The primary endpoint is the percentage of patients reaching ≥ 3 x 106/kg CD34+ cells (or ≥ 6 x 106/kg for two transplants), with ≤ 2 apheresis after low-dose CY+G-CSF vs. G-CSF mobilization. Secondary endpoints are need for plerixafor, graft cellular composition and engraftment after ASCT. Patients and methods This phase 2 study will include 80 patients below 70 years of age with symptomatic MM and eligible for ASCT. At registration the patients are randomized equally to arm A) CY 2 g/m2 + G-CSF 5 μg/kg or arm B) G-CSF 10 μg/kg. Before mobilization three RVD induction cycles are given. Each 3-week RVD cycle includes lenalidomide 25 mg daily on days 1−14, bortezomib 1.3 mg/m2 subcutaneously on days 1, 4, 8, 11, and dexamethasone 160 mg/cycle. By this schedule the first harvest is estimated to be on day +10 in CY + G-CSF and on day +5 in G-CSF group. Apheresis will start with blood CD34+ level >10 x 106/l. The target cell yields for one and two grafts are ≥ 3 and ≥ 6 x 106 CD34+ cells/kg, respectively, and the minimum for one graft ≥ 2 x 106/kg. Plerixafor is given if B-CD34+ cell count is less than 10 x 106/l on days +10 or +5, respectively, or if the first apheresis product contains < 1 x 106/kg CD34+ cells. G-CSF support is used after ASCT if the number of CD34+ cells in the graft is less than 3 x 106/kg. Engraftment will be assessed by blood neutrophil count > 0.5 x 109/l, and unsupported platelets > 20 x 109/l. Results Fifty-six patients have been included, and the mobilization data for the first 37 patients are available for analysis. The primary endpoint was reached in 90% of the patients (18/20) in arm A and in 82% (14/17) in arm B (p=NS). The median number of apheresis to reach the goal in arms A and B is one (1-3) and two (1−3), respectively (p=0.03). The median number of harvested cells in the two arms is 6.2 (2.2−12.1) and 4.8 (2.9−7.6) x 106/kg, respectively (p<0.01). All patients achieved the minimum collection target of ≥ 2 x 106/kg CD34+ cells in both arms, and the target yield of ≥ 3 x 106/kg was reached in 95% (19/20) in arm A and in 94% (16/17) in arm B. Plerixafor was used in two (12%) patients in arm B. The median blood CD34+ cell count on the first apheresis day was 55.9 (13.0−118.6) and 35 (16.0−148.5) x 106/l for arms A and B, respectively (p=0.44). The median time from mobilization day 1 to the first apheresis day was 10 (10−13) days in arm A and 5 (5−6) in arm B. The median number of CD34+ cells transplanted, was 4.1 (2.2−7.3) and 3.2 (2.3−4.7) x 106/kg in arms A and B, respectively (p=0.01). In arm A the median neutrophil engraftment was on day +14 (9−28) (16 patients) and in arm B on day +15 (11−25) (15 patients) (p=0.68). The median platelet engraftment days were +13 (8−22) and +11 (8−21) in arms A and B, respectively (p=0.67). At ASCT the response rates in arm A were ≥ VGPR 65% (13/20), PR 25% (5/20), and 10% (2/20) were progressing, and the respective rates for arm B were 70% (12/17), 18% (3/17),and 12 % (2/17). Conclusions Preliminary results of this randomized mobilization substudy with a limited number of patients show no clinically significant differences between the number of harvested CD34+ autologous stem cells. However, in CY+G-CSF group the CD34+ cell target was reached by less aphereses. After short induction course of RVD it seems possible to harvest also for two autografts with G-CSF only mobilization. However, when compared to historical data the CD34+ cell counts in blood and grafts were lower than after bortezomib + dexamethasone induction, and also neutrophil engraftment seemed to be slower after lenalidomide-based induction therapy. We conclude that CY can be omitted in the mobilization regimen for MM patients who have responded to short course of RVD. Disclosures Silvennoinen: Janssen-Cilag: Research Funding; Celgene: Research Funding; Janssen-Cilag: Honoraria; Sanofi: Honoraria; Celgene: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding.

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The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽

10.1182/blood.v126.23.1169.1169 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1169-1169

Author(s):

Kam Tong Leung ◽

Karen Li ◽

Yorky Tsin Sik Wong ◽

Kathy Yuen Yee Chan ◽

Xiao-Bing Zhang ◽

...

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Peripheral Blood ◽

Scid Mouse ◽

Conflicts Of Interest ◽

Chimeric Mice ◽

Hematopoietic Stem ◽

Cell Engraftment ◽

Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ cells. Disclosures No relevant conflicts of interest to declare.

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