Two Distinct Mechanisms Contribute to Granulomonocytic Hyperplasia in Chronic Myelomonocytic Leukemias (CMML)

Abstract Abstract 309 Background: The granulomonocytic (GM) hyperplasia of CMML has been attributed to GM-CSF hypersensitivity triggered by mutations in the CBL/RAS pathway according to the prevailing model in juvenile myelomonocytic leukemias (Kotecha Cancer Cell 2008). Recurrent mutations affecting epigenetic (eg TET2 and ASXL1) and splicing (eg SRSF2) machineries, or cytokine signaling (N/KRAS, CBL, JAK2) are present in most CMML cases, but none is specific of CMML. In 224 CMML patients (pts), we found TET2 (58%), SRSF2 (47%) and ASXL1 (38%) to be the most frequently mutated genes; only 66 (35%) cases had mutations in cytokine signaling genes (CBL, N/KRAS, JAK2, FLT3, KIT) (abstract submitted). We analyzed the differentiation of CD34+populations from genetically annotated CMML pts to address the mechanisms of GM hyperplasia in CMML. Methods: CD34+ populations (hematopoietic stem cells [HSC]; multipotent [MPP]; common myeloid [CMP] and granulomonocytic progenitors [GMP] defined by the CD34/CD38/CD90/CD123/CD45RA panel; Majeti Cell Stem Cell 2007) from 28 genetically annotated CMML and TET2 mutated MPN (n=8) or MDS (n=5) cases were cloned and genotyped for each mutation identified in mature CD14+ cells, and differentiated in vitro. Results: Early clonal dominance, with at least one mutation in > 75% of HSC/MPP clones, was found in all cases. In 18/19 pts with ≥2 mutations, a linear succession of mutations was found, with signaling mutations often following TET2 or ASXL1 mutations. Contrasting with the dominance of first events in HSC/MPP, second events reached clonal dominance in GMP, suggesting that they provide a selective advantage during the early steps of myeloid differentiation. We next analyzed the clonogenicity of peripheral blood (PB) CD34+ cells in the presence of GM-CSF (10 ng/mL) in 20 CMML cases and 4 controls. GM-CSF hypersensitivity (clonogenicity > mean+2SD of controls) was found in 7 (35%) cases. A mutation in a signaling gene was found in 6/7 pts (1 homozygous JAK2, 1 homozygous CBL, 4 heterozygous N/KRAS mutations), compared to 3/13 in pts without GM-CSF hypersensitivity (2 JAK2, 1 CBL, all heterozygous; P=.02) Median WBC was 29.2 and 11.4 G/L in pts with and without GM-CSF hypersensitivity, respectively (P=.08). The proportion of GMP in bone marrow (BM) CD34+cells was not significantly different in 33 CMML pts compared to 15 age-matched controls. Clonogenicity of GMP was similar in CMML and controls, except for a trend toward increased clonogenicity in pts with mutations in signaling genes. In contrast, the proportion of MPP and CMP was higher in CMML than in controls (P<.01 and P <.05, resp.). In erythromyeloid conditions (SCF, IL-3, G-CSF & EPO), both CMP and to a lesser extent MPP had an increased ability to form GM colonies at the expense of erythroid colonies (P <.001 and P<.01, resp.). Compared to healthy CMP, CMML CMP had and increased ability to mature into GMP in short-term culture, and increased PU.1 mRNA expression (P<.05), without significant changes in the levels of GATA1, CEBPA and CEBPB. Finally, in 16 pts, the proportion of GM colonies differentiating from CMP at the expense of erythroid colonies was inversely correlated to patient hemoglobin level (P=.002). Thus, premature GM differentiation of CMP, and to a lesser extent MPP, appears as the dominant mechanism of GM hyperplasia in CMML, whereas GM-CSF hypersensitivity and GMP expansion contribute only in the minority of patients with mutations in signaling genes. We next explored a possible link between early clonal dominance of TET2 mutations and premature GM differentiation. In TET2 mutated MPN (n=8) or MDS (n=5), the PB monocyte count was significantly correlated to the size of the TET2-mutated clone in the CD34+/CD38− (P=.006) rather than in the CD34+/CD38+ population (P=.08). Finally, functional invalidation by shRNA of TET2 in CD34+/CD38− followed by culture in the presence of SCF, IL-3, G-CSF & EPO caused a GM expansion that was not observed in CD34+/CD38+ cells. Similar analyses are underway for ASXL1. Conclusion: Our results suggest that early clonal dominance of mutations affecting the epigenetic machinery leading to premature GM differentiation of multipotent progenitors, rather than GM-CSF hypersensitivity, is the main mechanism of GM hyperplasia in CMML. This suggests a model whereby a single mutation can lead to different phenotypes, depending on the stage of differentiation at which the mutation has gained clonal dominance. Disclosures: No relevant conflicts of interest to declare.

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Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽

10.1182/blood.v108.11.1652.1652 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1652-1652

Author(s):

Patrick Ziegler ◽

Steffen Boettcher ◽

Hildegard Keppeler ◽

Bettina Kirchner ◽

Markus G. Manz

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Human Mesenchymal Stem Cells ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

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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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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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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Innovative Hematopoietic Gene-Therapy Concepts for Hereditary Pulmonary Alveolar Proteinosis Utilizing Hematopoietic Stem Cell Derived Macrophages

Blood ◽

10.1182/blood.v126.23.4417.4417 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4417-4417

Author(s):

Nico Lachmann ◽

Christine Happle ◽

Takuji Suzuki ◽

Miriam Hetzel ◽

Kevin A. Link ◽

...

Keyword(s):

Gene Therapy ◽

Colony Formation ◽

Pulmonary Alveolar Proteinosis ◽

Hematopoietic Stem ◽

Gm Csf ◽

Protein Levels ◽

Donor Cells ◽

Cd34 Cells ◽

Alveolar Proteinosis

Abstract Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocyte/macrophage-colony-stimulating factor (GM-CSF) receptor genes (CSF2RA and CSF2RB), resulting in disturbed alveolar macrophage (AM) differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. We here introduce pulmonary transplantation of gene corrected hematopoietic stem cell (HSC)-derived macrophage progenitors (MP) as a novel, cause directed, and well-tolerated therapy for herPAP. In a Csf2rb-/- mouse-model, selective pulmonary engraftment of healthy donor cells upon pulmonary transplantation of MPs was demonstrated by flow- and chip cytometry. Profound reduction of alveolar-protein levels and significant improvement of clinical parameters such as lung function and lung densities on CT scans were observed for more than nine months. Subsequent in situ analysis of donor cells revealed in vivo differentiation towards an AM phenotype characterized by CD11chi, CD11blo, MHC-II+, CD14+, F4/80+ surface marker, poor antigen presentation capacity, high phagocytic activity and AM-typical morphology on electron microscopy. Similar results were obtained following pulmonary transplantation of MPs differentiated from lentivirally corrected Csf2rb -/- HSCs. In vitro these gene-corrected HSCs expanded up to 1045-fold while differentiating into typical alveolar macrophages with F4/80, CD11b, CD11c, CD68, as well as Csf2rb mRNA and protein (CD131) expression and reconstitution of GM-CSF receptor signaling. Transplanted herPAP mice displayed significant improvement of biomarkers in the bronchoalveolar fluid (cloudiness, turbidity, SP-D, MCP-1, M-CSF, and GM-CSF) and in AMs (mRNA for PU.1, PPARg and ABCG1). Moreover, administration of human CD34+ cell-derived MPs profoundly improved symptoms in a humanized herPAP mouse model. Here, transplantation of 1-2x106 human MPs led to long-term pulmonary engraftment and reduced alveolar protein levels by 50-70%. CT scans six months after transplantation revealed significant improvements in herPAP related signs, including marked reduction of expiratory lung densities and normalisation of inspiratory to expiratory lung volume ratio. Furthermore, to genetically correct human CSF2RA-patient derived CD34+ cells, we have generated SIN-lentiviral vectors expressing a codon-optimized human CSF2RA-cDNA in combination with EGFP (Lv.EFS.CSF2RA.EGFP) from an EFS1a-promoter. BaF3 cells transduced with this vector showed stable and longterm (>3 month) expression of CSF2RA (CD116) by flow cytometry and survived in hGM-CSF dependant assays even at low concentrations of GM-CSF (5 ng/ml) confirming the formation of functional hybrid receptors of the murine GM-CSF receptor ß-chain with the transgene. Characterization of GM-CSF receptor downstream signalling revealed 5-6-fold increased STAT5 phosphorylation by Western blot in response to hGM-CSF (over control cells). Conferring this vector to CD34+ cells of CSF2RA-deficient patients yielded efficient CD116 (CSF2RA) expression, and rescued hGM-CSF dependent colony formation as well as monocytoid differentiation. Of note, clonogenic growth by G-CSF control treatment revealed no differences in colony formation or differentiation capacity when compared to uncorrected patient samples. Furthermore, healthy Lv.EFS.CSF2RA.EGFP transduced CD34+ samples, while showing robust CD116 overexpression, exhibited no aberrations in biological functions such as colony formation or in vitro differentiation towards macrophages. Thus, we here describe an innovative, cause directed and highly effective hematopoietic gene therapy approach to herPAP. Given the longevity of the transplanted MP population in our model, the strategy also may serve as a proof-of-principle to incorporate long-lived differentiated, cell sources into current hematopoietic gene therapy concepts. Disclosures No relevant conflicts of interest to declare.

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Production of GM-CSF by CML Cells Can Modulate the Anti-Proliferative and Pro-Apoptotic Effects of Imatinib on CML CD34+ Cells.

Blood ◽

10.1182/blood.v106.11.2865.2865 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2865-2865 ◽

Cited By ~ 1

Author(s):

Alan B. Lyons ◽

Timothy P. Hughes ◽

Pongtep Viboonjuntra

Keyword(s):

Single Agent ◽

Chronic Phase ◽

Cell Number ◽

Culture Conditions ◽

Proliferation Index ◽

Cytokine Signaling ◽

Gm Csf ◽

Proliferative Effect ◽

Cd34 Cells

Abstract CML cells gain a survival advantage over normal cells due to dysregulated tyrosine kinase activity of BCR/ABL protein, which phosphorylates a number of proteins, potentially activating multiple signal transduction pathways. Imatinib specifically targets BCR/ABL protein and results in anti-proliferation and apoptosis. Even in the absence of mutation, the leukaemic clone may not be eradicated by imatinib, suggesting that leukaemic stem cells may not be absolutely reliant on BCR/ABL activity for survival. Reports on GM-CSF production by CML cells suggest a possible autocrine role, therefore we examined if GM-CSF could protect CML CD34+ cells from imatinib. CD34+ cells from peripheral blood of patients with CML in chronic phase (n=9) and from normal bone marrow donors (n=5), were labeled with CFSE (Carboxy-Fluorescein diacetate Succinimidyl Ester) to enable tracking of cell division. Normal and CML samples were cultured with and without GM-CSF (300pg/mL) to assess response to this cytokine as a single agent. CML CD34+ cells were cultured for 3 days in serum deprived medium with imatinib only, GM-CSF (300 pg/mL) + imatinib, GM-CSF (300pg/mL) + E21R (a GM-CSF analogue able to block cytokine binding) (10mg/mL), and GM-CSF (300pg/mL) + E21R (10mg/mL) + imatinib. In each condition, imatinib was titrated over the range of 0 to 10 microM. Cultures were analysed by flow cytometry to evaluate the proliferation index (PI), a ratio of final cultured cell to precursor cell number, where a PI of 1 represents no proliferation, and a PI of 2 corresponds to approximately three division cycles on average. Data are summarised in Table 1. Effect of GM-CSF and imatinib on proliferation Culture Proliferation Index Statistics using Student t test, symbols denote comparisons between culture conditions Normal Control 1.07+/−0.06 * NS Normal+GM-CSF 1.11+/−0.03 * # NS CML Control 1.43+/−0.18 ** p>0.01 #, & NS CML+GM-CSF 2.30+/−0.40 ** CML+10μM imatinib 1.18+/−0.09 *** NS CML+10μM imatinib+GM-CSF 1.54+/−0.19 @, & CML+10μM imatinib+GM-CSF+E21R 1.17+/−0.09 *** @ p>0.01 GM-CSF induced strong proliferation in all CML, but not normal samples. Imatinib reduced proliferation of CML CD34+ cells at 1 microM and above, and the addition of GM-CSF reduced this proliferative effect at concentrations of imatinib up to 10 microM. The protective effect of GM-CSF was clearly blocked using E21R. When the proliferation of CML CD34+ cells cultured within the total mononuclear fraction was compared to purified CD34+ cells, it was found that there was an approximate 40% enhancement of PI at each concentration of imatinib. This enhanced proliferation was inhibited by the addition of E21R, indicating GM-CSF may be produced by non-CD 34+ cells. ELISpot assay confirmed the production of GM-CSF by non-CD34+ CML cells (34±23%), but not CD34+ cells (0.06±0.02%). Fluorescent inhibitor of apoptosis (FLICA) assay showed that GM-CSF could reduce the proportion of cells with activated caspases induced by imatinib by over 40% during a 3 day in vitro culture, promoting increased cell survival. As the GM-CSF receptor and BCR/ABL may share common signaling pathways, during blockade of BCR/ABL activity by imatinib, GM-CSF can compensate to maintain cell viability and proliferation. These findings have implications for optimizing imatinib therapy by manipulating cytokine signaling.

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Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

Blood ◽

10.1182/blood.v122.21.2411.2411 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2411-2411

Author(s):

Hein Schepers ◽

Patrick M. Korthuis ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Fold Increase ◽

Negative Regulator ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells. We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells. To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice. Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures: No relevant conflicts of interest to declare.

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Cohesin Genes Are Negative Regulators of HSC Renewal

Blood ◽

10.1182/blood.v124.21.605.605 ◽

2014 ◽

Vol 124 (21) ◽

pp. 605-605

Author(s):

Roman Galeev ◽

Aurelie Baudet ◽

Anders Kvist ◽

Therese Törngren ◽

Shamit Soneji ◽

...

Keyword(s):

Bone Marrow ◽

Control Group ◽

Hematopoietic Stem ◽

Myeloid Malignancies ◽

Genome Wide ◽

Cd34 Cells ◽

Recurrent Mutations ◽

Major Player

Abstract The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. To gain deeper insights into the mechanisms underlying renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), we have developed global RNAi screens targeted to human cord blood derived CD34+ cells. In previous work such screens have allowed us to identify novel druggable targets to facilitate ex vivo expansion of HSPCs. Recently, we employed a near genome-wide screen (targeting 15 000 genes) to identify genes with an impact on renewal/differentiation of HSPCs, in a completely unbiased manner. Among the most prominent hits from this screen were many transcription factors and epigenetic modifiers and we found a strong enrichment of genes known to be recurrently mutated in hematopoietic neoplasms. A striking finding, was the identification of several members of the cohesin complex (STAG2, RAD21, STAG1 and SMC3) among our top hits (top 0.5%). Cohesin is a multimeric protein complex that mediates adhesion of sister chromatids as well as long-range interactions of chromosomal elements to regulate transcription. Recent large-scale sequencing studies have identified recurrent mutations in the cohesin genes in myeloid malignancies. Upon individual validation and targeting of the cohesin genes by lentiviral shRNA in human CD34+ cells, we found that their knockdown by independent shRNAs led to an immediate and profound expansion of primitive hematopoietic CD34+CD90+ cells in vitro. A similar expansion phenotype was observed in vivo following transplantation to primary and secondary immundeficient mice. Transplantation of CD34+CD38lowCD90+CD45RA- cells transduced with shRNA targeting STAG2 (the cohesin component with the strongest in vitro phenotype) into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in a significant increase in human reconstitution in the bone marrow 16 weeks post-transplantation compared to controls (31.3±4.4% vs 11.6±2.8% p=0.001). The engrafted mice showed a marked skewing towards the myeloid lineage as analyzed by CD33/CD15 expression in bone marrow (27.0±5.0% vs 13.0±2.6% p=0.013), as well as an increase in the more primitive CD34+CD38- population (2.8±0.6% vs 1.3±0.4% p=0.036). In secondary transplanted mice, 3/6 recipients in the STAG2 group maintained detectable levels of human chimerism while no engraftment was detected in the control group, indicating an increased expansion of HSPCs in vivo upon knockdown of STAG2. Global transcriptome analysis of cohesin deficient CD34+ cells 36 hours post shRNA transduction showed a distinct up-regulation of HSC specific genes coupled with down-regulation of genes specific for more downstream progenitors, demonstrating an immediate shift towards a more stem-like gene expression signature upon cohesin deficiency. This observation was consistent for all cohesin genes tested (STAG2, RAD21, STAG1 and SMC3). Our findings implicate cohesin as a novel major player in regulation of human HSPCs and, together with the recent discovery of recurrent mutations in myeloid malignancies, point toward a direct role of perturbed cohesin function as a true driver event in myeloid leukemogenesis. Our findings illustrate how global RNAi screens targeted to primary human HSPCs can identify novel modifiers of cell fate and may complement genome-wide sequencing approaches to guide the identification of functionally relevant disease-related genes in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

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RAB14 Regulates Human Erythropoiesis and Megakaryopoiesis

Blood ◽

10.1182/blood.v128.22.2661.2661 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2661-2661

Author(s):

Minjung Kim ◽

Tami J. Kingsbury ◽

Curt I. Civin

Keyword(s):

Conflicts Of Interest ◽

Culture Media ◽

Myeloid Cell ◽

Rab Gtpase ◽

Human Leukemia ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Gm Csf ◽

Human Cd34

We recently reported that RAB GTPase 14 (RAB14) knockdown (KD) increased the frequency and total numbers of erythroid cells generated in vitro in response to erythropoietin (EPO) from either the TF1 human leukemia erythropoietic model cell line or from primary human CD34+ hematopoietic stem-progenitor cells (HSPCs). RAB14 overexpression (OE) had the opposite effect. Thus, RAB14 functions as an endogenous inhibitor of human erythropoiesis (Kim et al., Br. J. Haematol., 2015). In contrast to the greater cell numbers generated in the presence of EPO, RAB14 KD TF1 cells grown in standard culture media containing granulocyte-macrophage colony-stimulating factor (GM-CSF; as the only cytokine) generated fewer total cells, compared to empty vector-transduced control TF1 cells. Furthermore, RAB14 KD TF1 cells cultured in GM-CSF media generated greater numbers of erythroid (CD34-/CD71+/CD235a+) cells, as compared to control TF1 cells, suggesting that RAB14 KD stimulated erythropoiesis even in the absence of EPO. Cells generated from RAB14 KD TF1 cells had higher GATA1 and lower GATA2 transcription factor expression, as compared to controls, demonstrating the cells had undergone the "GATA1/2 switch," a hallmark of erythropoiesis. Consistent with higher GATA1 levels, RAB14 KD TF1 cells generated cells with higher levels of b- and g-hemoglobins. Similarly, RAB14 KD in primary human CD34+ HSPCs generated greater numbers of erythroid cells, with or without exogenous EPO. RAB14 KD CD34+ HSPCs cultured in GM-CSF media generated fewer monocytic/granulocytic (CD13+/CD33+) cells, as compared to control CD34+ HSPCs. Interestingly, RAB14 OE CD34+ HSPCs cultured in thrombopoietin (TPO)-containing media generated higher numbers of megakaryocytic (CD34-/CD41a+/CD42b+) cells, as compared to control CD34+ HSPCs. In summary, (1) RAB14 KD in TF1 cells or primary human CD34+ HSPCs increased erythropoiesis in the presence or absence of EPO, but reduced myeloid cell differentiation, probably via the GATA1/2 switch; and (2) RAB14 OE in CD34+ HSPCs increased megakaryopoiesis in the presence of TPO. Thus, RAB14 normally serves as an endogenous hematopoietic decision-maker, physiologically inhibiting erythropoiesis and stimulating megakaryopoiesis (and possibly, to a lesser extent, mono-granulopoiesis). Disclosures No relevant conflicts of interest to declare.

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Direct Comparison of Wharton Jelly and Bone Marrow Derived Mesenchymal Stromal Cells to Enhance Engraftment of Cord Blood CD34+ Transplants

Blood ◽

10.1182/blood.v126.23.5410.5410 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5410-5410

Author(s):

Mark van der Garde ◽

Melissa Van Pel ◽

Jose Millan Rivero ◽

Alice de Graaf-Dijkstra ◽

Manon Slot ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Waste Product ◽

Human Platelets ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Co-transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) and mesenchymal stromal cells (MSC) enhances HSPC engraftment. For these applications, MSC are mostly obtained from bone marrow. However, MSC can also be sourced from Wharton's jelly (WJ) of the human umbilical cord, which as a 'waste product', is cheaper to acquire and without significant burden to the donor. Here, we evaluated the ability of WJ MSC to enhance HSPC engraftment. First, we compared cultured human WJ MSC with human bone marrow-derived MSC (BM MSC) for in vitro marker expression, immunomodulatory capacity and differentiation into three mesenchymal lineages. Although we confirmed that WJ MSC have a more restricted differentiation capacity, both WJ MSC and BM MSC expressed similar levels of surface markers and exhibited similar immune inhibitory capacities. Co-transplantation of either WJ MSC or BM MSC with CB CD34+ cells into NOD-SCID mice showed faster recovery of human platelets and CD45+ cells in the peripheral blood and a 3-fold higher engraftment in the BM, blood and spleen six weeks after transplantation when compared to transplantation of CD34+ cells alone. Upon co-incubation, both MSC sources increased the expression of adhesion molecules on CD34+ cells, although SDF-1-induced migration of CD34+ cells remained unaltered. Interestingly, there was an increase in CFU-GEMM when CB CD34+ cells were cultured on monolayers of WJ MSC in the presence of exogenous thrombopoietin, and an increase in BFU-E when BM MSC replaced WJ MSC in such cultures. Our results suggest that WJ MSC is likely to be a practical alternative for BM MSC to enhance CB CD34+ cell engraftment. Disclosures No relevant conflicts of interest to declare.

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