Bone Marrow CFU-S Research Articles

FSL-1: A Synthetic Peptide Increases Survival in a Murine Model of Hematopoietic Acute Radiation Syndrome.

In the current geopolitical climate there is an unmet need to identify and develop prophylactic radiation countermeasures, particularly to ensure the well-being of warfighters and first responders that may be required to perform on radiation-contaminated fields for operational or rescue missions. Currently, no countermeasures have been approved by the U.S. FDA for prophylactic administration. Here we report on the efficacious nature of FSL-1 (toll-like receptor 2/6 agonist) and the protection from acute radiation syndrome (ARS) in a murine total-body irradiation (TBI) model. A single dose of FSL-1 was administered subcutaneously in mice. The safety of the compound was assessed in non-irradiated animals, the efficacy of the compound was assessed in animals exposed to TBI in the AFRRI Co-60 facility, the dose of FSL-1 was optimized, and common hematological parameters [complete blood cell (CBC), cytokines, and bone marrow progenitor cells] were assessed. Animals were monitored up to 60 days after exposure and radiation-induced damage was evaluated. FSL-1 was shown to be non-toxic when administered to non-irradiated mice at doses up to 3 mg/kg. The window of efficacy was determined to be 24 h prior to 24 h after TBI. FSL-1 administration resulted in significantly increased survival when administered either 24 h prior to or 24 h after exposure to supralethal doses of TBI. The optimal dose of FSL-1 administration was determined to be 1.5 mg/kg when administered prior to irradiation. Finally, FSL-1 protected the hematopoietic system (recovery of CBC and bone marrow CFU). Taken together, the effects of increased survival and accelerated recovery of hematological parameters suggests that FSL-1 should be developed as a novel radiation countermeasure for soldiers and civilians, which can be used either before or after irradiation in the aftermath of a radiological or nuclear event.

Ribonuclease Inhibitor 1 (RNH1) Regulates Myeloid Lineage Choice through Cyclin-Dependent Kinase 1

Background: Myelopoiesis is a complex process, it begins with myeloid lineage choice determination in haemopoietic stem cells (HSCs). Still we do not have complete molecular understanding of how myeloid lineage choices were determined. Although transcription factors expression majorly determine the lineage choices at HSCs level, recent study suggest that ratios of total PU.1:GATA1 protein levels are not the central mechanism that initiates HSPC lineage decisions . Further, cell fate commitment is linked to the cell cycle regulation, however whether the cell cycle itself regulates lineage choices in HSCs is not known. Previously, we have published that Ribonuclease inhibitor (RNH1) deletion in mice leads to defect inembryonic erythropoiesis. Here, we report that RNH1 regulates myeloid lineage choice through cyclin-dependent kinase 1 (CDK1) mediated cell cycle regulation. Methods: We have generated hematopoietic-specific conditional knockout mice by crossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Phenotypic analysis, Bone marrow transplantation (BMT) experiments, cell cycle and CFU assays were performed. To understand molecular mechanism RNA seq, mass spectrometry, confocal microscopy and biochemical studies were performed. Histopathology performed in AML bone marrow biopsies. RNH1 was knocked out in several AML cell lines and analyzed myeloid differentiation and performed CFU assays. Results: Rnh1-deficiency in both Vav1-iCre and Mx1-Cre models resulted in elevated levels of myeloid cells but decreased lymphoid cells and haemoglobin (HGB) levels in peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type (WT) and Rnh1-/- mice. However, the number of erythroid cells and lymphoid cells was significantly decreased, whereas myeloid cells were significantly increased. We analysed the progenitor populations in the BM. In line with increased myelopoiesis phenotype, granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-/- mouse BM, suggesting that RNH1 could affect HSC function. Supporting this, Rnh1-/- HSCs failed to engraft lethally irradiated mice in competitive and non- competitive BMT experiments. Intriguingly, despite increased myelopoiesis in Rnh1-/- mice, conventional myeloid genes that support myelopoiesis were not increased in Rnh1-/- HSCs by RNA-Seq studies. Rather we found increased expression of genes related to cell cycle, kinetochore and DNA damage in Rnh1-/- HSCs. Corroborating this, Rnh1-/- HSCs showed significant increase in G1 cycle phase compared to WT. At the molecular level, we found that RNH1 directly binds to CDK1 and also inhibits CDK1 kinase function. Supporting this, molecular docking studies suggest that RNH1 potentially binds with CDK1. In agreement with this, loss of RNH1 in HSCs increased CDK1 protein levels as well as increased CDK1 phosphorylation at Thr161. Further, CDK1 inhibitor treatment decreased myelopoiesis and rescued erythropoiesis and lymphopoiesis in Rnh1-/- mice. We checked whether RNH1 mediated myelopoiesis is relevant in any disease models and can be targeted. Interestingly, we found increased RNH1 expression in AML patients bone marrow biopsies independent of their genetic mutation. AML blasts have myeloid differentiation arrest and therapies that resolve differentiation arrest represent a powerful treatment strategy. Interestingly, RNH1 deletion in AML cell lines leads to increased expression of the myeloid markers, increased cell death and formed less colonies compared to wild type cells. Summary: Collectively, our studies suggest that loss of RNH1 in HSCs leads to dysregulation in cell cycle through increased CDK1 activity. Dysregulation in cell cycle decreases HSCs stemness and repopulating potential, and influences cell fate decisions by increasing myelopoiesis. Since loss of RNH1 increased myelopoiesis without increase of myeloid transcription factors expression and dependent on CDK1, we call this pathway 'alternative myelopoiesis'. Targeting alternative myelopoiesis could be a potential therapy for AML.

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Impaired in vitro erythropoiesis following deletion of the Scl (Tal1) +40 enhancer is largely compensated for in vivo despite a significant reduction in expression.

The Scl (Tal1) gene encodes a helix-loop-helix transcription factor essential for hematopoietic stem cell and erythroid development. The Scl +40 enhancer is situated downstream of Map17, the 3' flanking gene of Scl, and is active in transgenic mice during primitive and definitive erythropoiesis. To analyze the in vivo function of the Scl +40 enhancer within the Scl/Map17 transcriptional domain, we deleted this element in the germ line. Scl(Δ40/Δ40) mice were viable with reduced numbers of erythroid CFU in both bone marrow and spleen yet displayed a normal response to stress hematopoiesis. Analysis of Scl(Δ40/Δ40) embryonic stem (ES) cells revealed impaired erythroid differentiation, which was accompanied by a failure to upregulate Scl when erythropoiesis was initiated. Map17 expression was also reduced in hematopoietic tissues and differentiating ES cells, and the Scl +40 element was able to enhance activity of the Map17 promoter. However, only Scl but not Map17 could rescue the Scl(Δ40/Δ40) ES phenotype. Together, these data demonstrate that the Scl +40 enhancer is an erythroid cell-specific enhancer that regulates the expression of both Scl and Map17. Moreover, deletion of the +40 enhancer causes a novel erythroid phenotype, which can be rescued by ectopic expression of Scl but not Map17.

Pentoxifylline Enhances the Radioprotective Properties of γ-Tocotrienol: Differential Effects on the Hematopoietic, Gastrointestinal and Vascular Systems

The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5-13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

AbstractAbstract 404Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures:No relevant conflicts of interest to declare.

Alterations in Bone Marrow Stroma with Aging (36.5)

Abstract B lymphopoiesis arrests in rabbits by 16 weeks of age, and data suggest that the bone marrow (BM) environment is responsible for this. We hypothesize that this arrest is due to differences in the BM stroma of young and adult rabbits. Using limiting dilution analysis to quantify the number of CFU in BM of 2 to 6 wk-old rabbits, we found an 8-10 fold decrease in the number of stromal progenitors by 6 wk-of-age. This decrease in stromal progenitors is reflective of the age-related decline in progenitor B cells in BM. Histology of BM sections reveals trabecular bone formation in young rabbits but not in adults; in contrast, BM from adults shows large amounts of adipose tissue. We tested the capability of stromal progenitors to differentiate into adipocytes or osteoblasts in vitro , and found that stromal progenitors from BM of young rabbits displayed a higher propensity to form oseoblastic colonies than do stromal progenitors from BM of adults, which more readily formed adipocytic colonies. We conclude that with aging, the number of BM stromal progenitors decreases dramatically, and further, that these progenitors from adults more readily differentiate into adipocytes than into osteoblasts. We suggest that these changes in BM are responsible for the arrest of B lymphopoiesis.

High Levels of Multilineage Hematopoietic Cell Gene Marking Following Lentiviral Vector Transduction and Transplantation of Steady-State Bone Marrow CD34+ cells in a Non-Human Primate Model.

Abstract 2464Poster Board II-441Non-human primate autologous hematopoietic stem cell (HSC) transplantation models have been invaluable in developing the ex vivo transduction protocols used in human gene transfer clinical trials. Previous studies of HSC gene transfer using gamma-retroviral and lentiviral vectors in these models have almost exclusively utilized cytokine-mobilized peripheral blood (PB) or bone marrow (BM) CD34+ cells as target cells. The administration of the combination of granulocyte colony stimulating factor (G-CSF) plus stem cell factor (SCF) became standard as a priming method prior to HSC collection after it was shown that SCF coupled with G-CSF resulted in significantly improved long-term gene transfer levels, compared to mobilization with G-CSF alone (Hematti et al., Blood 2003 101:2199-2205). However, SCF is not available in the US for human gene transfer trials due to its history of causing severe anaphylactic reactions. For this reason and to define a clinically relevant gene transfer protocol for sickle cell disease, in which G-CSF administration is also contraindicated, we evaluated lentiviral stem cell gene transfer using steady-state BM CD34+ cells in the pigtail macaque transplantation model. Bone marrow was harvested from the long bones and CD34+ cells were enriched using an anti-CD34 antibody and immunomagetic beads (92% purity). Cells were prestimulated overnight in serum-free medium containing 100 ng/ml of FLT-3 ligand, thrombopoietin, and stem cell factor. Cells were then exposed for 14 hours to a VSV-G pseudotyped, HIV-based lentiviral vector encoding GFP at an MOI of 40. Cells were collected, washed, and placed back into culture in serum-free medium containing the above cytokines for an additional 24 hours. Subsequently, cells were collected (15 × 106/kg) and transplanted into an animal which had been conditioned with 950 cGy. Marking of the graft, as assessed by flow cytometry for GFP expression, was 74% and all hematopoietic colonies (CFU) obtained in methylcellulose cultures were GFP positive. Neutrophil recovery (ANC > 500) was observed on day 20. One month following transplant, 36% of peripheral blood granulocytes (GRANS) were GFP positive. At two months post-transplant, marking levels were: GRANS 27%, RBC 43%, and platelets (PLT) 22%. These levels have remained steady out to 6 months post-transplantation: GRANS 21%, lymphocytes 20%, RBC 17%, and PLTS 16%. A BM aspirate was obtained and analysis of BM CFU showed that 186 of 508 (37%) were GFP positive. Southern blot analysis and q-PCR on DNA from peripheral blood leukocytes and BM showed a vector copy number ranging from 0.6 to 1.0 over time, suggesting that the positive cells contained several vector copies. No evidence of clonal dominance has been observed by Southern blot analysis using restriction enzymes which cut only once in the vector. Insertion site analysis using deep sequencing technology is in progress. A second animal has recently been transplanted (11 × 106 cells/kg) with graft marking of greater than 50%. These data are encouraging as they suggest that therapeutic levels of lentiviral vector gene transfer can be obtained using CD34+ cells from steady-state bone marrow as the HSC cell source. This is important since SCF cannot be used in humans and for patients with sickle cell disease, administration of G-CSF is also contraindicated. Disclosures:No relevant conflicts of interest to declare.

Overlapping but Distinct Role of p21WAF1/CDKN1 and Survivin in Hematopoietic Progenitor Cell Proliferation.

Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in cell cycle control, anti-apoptosis and cell division. Our previous studies and others have shown that Survivin and the cyclin dependent kinase inhibitor p21WAF1/CDKN1 (p21) are functionally associated and are involved in cell cycle, anti-apoptosis and cytokinesis in cancer cells and in normal hematopoietic progenitor cells (HPC). P21 is highly expressed in quiescent hematopoietic stem cells (HSC) in steady state, but the proportion of quiescent HSCs in G0 phase is reduced in p21−/− mice. In contrast, p21 has been shown as positive regulator on cell cycle of normal HPC since p21 deficiency results in fewer total CFU in mouse bone marrow (BM) cells with fewer CFU in S-phase and retrovirus transduction of p21 in p21 deficient bone marrow cells restores total and cycling CFU. We have previously reported that Survivin increases the proliferation of mouse primary HPC and that this enhancing effect is on HPC proliferation is absent when p21 is functionally deleted, suggesting that p21 is required for Survivin to enhance HPC proliferation. In addition, ITD-Flt3 mutations that are normally expressed in patients with acute myeloid leukemia and associate poor prognosis increase expression of both Survivin and p21, implicating their involvement in aberrant proliferation of HPC expressing ITD-Flt3. Herein we have characterized the functional association between p21 and Survivin in normal and transformed cell proliferation. Antagonizing wild-type Survivin in mouse BaF3 cells by retrovirus transduction of a T34A dominant negative mutant Survivin or anti-sense increased p21 expression, even though Survivin requires p21 to enhance HPC proliferation. Ectopic p21 in Survivin+/+ primary mouse bone marrow cells increased the number of immunophenotypically defined c-kit+, lin− (KL) cells, which is consistent with a positive role of p21 in HPC proliferation, however; ectopic expression of p21 failed to increase HPC proliferation in Survivin deficient primary bone marrow cells, suggesting that p21 alone is not sufficient to substitute for Survivin's enhancing function on normal HPC proliferation. Over-expression of ITD-Flt3 enhanced growth factor independent proliferation of primary mouse marrow c-kit+, Sca-1+, lin− (KSL) cell number; however, co-expression of p21 with ITD-Flt3 dramatically decreased the number of growth factor independent KSL cells (80±6% reduction: P<0.01). Furthermore, the inhibitory effect of p21 on KLS proliferation was further enhanced by Survivin knockout bone marrow cells (64±5% reduction compared with presence of Survivin: P<0.05). These findings indicate that Survivin and p21 have a overlapping but distinct roles in regulating normal HPC proliferation and that manipulating p21 and Survivin may represent a potential therapeutic target for acute leukemia cells expressing ITD-Flt3.

The effects of the EW dipeptide optical and chemical isomers on the CFU-S population in intact and irradiated mice

The influence of Glu–Trp (EW) synthetic dipeptide isomers on hemopoietic progenitor cells and certain immune response reactions is determined by their optical and chemical properties. Thus, the all l-amino acid containing dipeptides l-Glu–l-Trp and l-γGlu–l-Trp have no effect on proliferation of committed and pluripotent CFU-S in intact bone marrow. The optical isomers of the Glu residue are an essential determinant of the EW dipeptide biological activity. The inversion of the amino acid optical form imparts suppressor properties: d-Glu–d-Trp, d-γGlu–d-Trp, d-Glu–l-Trp and d-γGlu–l-Trp inhibit proliferation of hemopoietic progenitors in intact bone marrow. The type of the peptide bond between l-Glu and Trp is another important factor for the biological activity of the l-Glu-containing peptides. Unlike l-Glu–d-Trp with α-peptide bond, the dipeptide l-γGlu–d-Trp with γ-peptide bond stimulates CFU-S-8 proliferation in intact bone marrow. The diverse effects of the EW optical isomers on hemopoietic progenitors underlie the radioprotective properties of the d-Glu-containing dipeptides and the radiotherapeutic ones of the l-Glu dipeptides. In animals, pre-irradiation injection of d-Glu–d-Trp, d-γGlu–d-Trp, d-Glu–l-Trp, d-γGlu–l-Trp, or post-irradiation injection of l-Glu–l-Trp, l-γGlu–l-Trp promoted regeneration of the hemopoietic progenitor population.

Predictors of Organ Allograft Tolerance Following Hematopoietic Cell Transplantation

Using the miniature swine large animal model we have attempted to determine the relationship between tolerance and the presence of donor cells in the bone marrow, thymus and lineages of peripheral blood in a series of hematopoietic cell transplant recipients receiving delayed donor allografts without immunosuppression. Twenty-two animals receiving hematopoietic cell transplantation and a delayed organ allograft were analyzed. Assays for presence of donor CFUs in bone marrow (by PCR), thymic chimerism (by FACS and PCR/Southern Blot), peripheral blood chimerism (by FACS), and in vitro responsiveness to donor MHC were performed. Presence of donor BM CFUs, thymic chimerism and multilineage peripheral blood chimerism at the time of organ transplantation all correlated precisely with subsequent allograft tolerance (p < 0.001, p < 0.001, p < 0.005 respectively). These parameters were therefore accurate predictors (Positive Predictive Value (PPV) = 100% in all) of tolerance. In vitro assays of responsiveness were also highly associated (p < 0.002, p < 0.002 respectively), but were not as accurate predictors of subsequent organ tolerance (CML PPV = 80%). Engraftment, as indicated by the presence of donor derived CFU in the bone marrow, detectable thymic chimerism and multilineage peripheral blood chimerism are reliable predictors of subsequent donor allograft acceptance in hematopoietic cell transplant recipients.

Effects of structural and “mixed” isomers of Glu-Trp dipeptide on normal hemopoietic stem cells

We studied the effects of optical (dd-, ll-, dl-, and ld-dipeptides with alpha-bond, EW) structural isomers and cyclic (dd-, ll-, dl-, and ld-dipeptides with gamma-bond, iEW) analogs of Glu-Trp synthetic dipeptide on the population of normal hemopoietic stem cells. Dipeptides containing lGlu (lGlu-lTrp, lGlu-dTrp) injected to mice were inert towards committed bone marrow CFU-S; dGlu-containing dipeptides (dGlu-dTrp, dGlu-lTrp) inhibited the growth of CFU-S-8; and LiGlu-dTrp stimulated these cells. Inhibitory or stimulatory effects of optical and chemical isomers of Glu-Trp dipeptide are determined by optical orientation and nature of peptide bond of Glu residue. The effects of cyclic and mixed peptides towards colony formation are similar to those of the corresponding linear dipeptides.

Studies of the Bcl Inhibitor GX15-070 in Multiple Myeloma Demonstrate Substantial Pre-Clinical Activity.

GX15-070 is a small-molecule antagonist of the BH3-binding groove of the Bcl-2 family of proteins and can thus induce apoptosis by inhibiting the interaction between pro- and anti-apoptotic proteins. In addition, GX15-070 activates a cellular pathway, which leads to the activation of the pro-apoptotic BH3-only protein Bim, a natural cellular inhibitor of anti-apoptotic members. Together, the direct inhibition of anti-apoptotic Bcl-2 family proteins and the activation of pro-apoptotic Bim by GX15-070 can sensitize tumor cells to apoptosis. Bcl-2 family members, particularly Bcl-XL and Mcl-1 have been implicated in the survival of myeloma cells and may be responsible, in part, for drug resistance. Consequently, GX15-070 was tested against the MMRC 12 validation cell line panel, a panel of 12 standardized and annotated human myeloma cell lines (HMCLs). Western blots preformed on 8 HMCLs confirmed expression of Bcl-2, Mcl-1 and/or Bcl-XL. Basal levels of Bcl-2 and MCl-1 were relatively similar across all HMCLs studied whereas levels of Bcl-XL were variable GX15-070 inhibited the viability of all 12 myeloma cell lines as determined by MTT assay. The mean IC50 was 215 nM; range (6.4-678nM) and sensitivity did not correlate with level of Bcl-2, MCL-1 or Bcl-XL expression. On the other hand cytotoxicity to normal human blood lymphocytes, stroma and bone marrow CFUs was not observed at concentrations which were effectively cytotoxic to HMCL. Time course experiments demonstrate that apoptosis and cleavage of caspase 3 began by 12 hours and continued to increase over a 96 hour period. Co-culture with human bone marrow stroma cells (BMSCs) failed to protect HMCL from GX15-070 induced cytotoxicity. Similarly, potent MM growth factors, Interleukin 6 (IL-6) and Insulin like growth factor (IGF)-1 did not confer resistance to GX15-070. Of interest, overexpression of Bcl-2 has been shown to attenuate PS-341-induced cell death (Mitisades); however, the combination of PS-341 and GX15-070 did not reveal synergistic interaction in preliminary results. Studies looking at other drug combinations are planned. Finally, GX15-070 produced cytotoxic responses in the first primary myeloma sample tested with further testing ongoing. Mouse xenograft studies are also ongoing and will be presented. These studies indicate extensive pre-clinical activity of GX15-070 in multiple myeloma and set the stage for planned Phase II clinical trials.

1006. Preliminary Results of a Phase I Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG and BCNU Therapy of Advanced Malignancies

We have extensively studied the G156A MGMT mutation which confers O6alkylguanine DNA alkyltransferase (AGT) that is resistant to O6benzylguanine (BG). In a Phase I clinical trial at our institution, BG was shown to inhibit tumor AGT at 120mg/m2 and the maximum tolerated dose of BCNU was 40 mg/m2. In the laboratory, we have shown that the retrovirus MFG-G156A-MGMT confers BG and BCNU resistance ex vivo into human CD34+ cells. We have also reported that G156A-MGMT transduced murine hematopoietic progenitors repopulate the bone marrow and can be enriched up to 1000-fold after 2 cycles of BG and BCNU. These studies demonstrate the strong capacity of this model to confer BG and BCNU resistance and a selection advantage in vivo. Based on these data, we initiated a clinical trial using gene transfer into autologous CD34+ cells in patients with advanced malignancies. Patients undergo CD34+ mobilization with G-CSF and GM-CSF. CD34+ enriched cells (CliniMACS, Miltenyi) are transduced with MFG-G156A MGMT (produced in PG13 cells by the NGVL, K. Cornetta, Director) in the presence of the fibronectin fragment CH-296 (provided by Takara Bio Inc) and the cytokine combination SCF, Tpo, and Flt-3 ligand for 86 hours. The cell culture is harvested on day 4 and the entire culture is re-infused into the patient who received BG and BCNU 48 hours prior. Patients receive subsequent cycles of BG and BCNU every 6 weeks with analysis of marrow and blood cells for evidence of gene transfer prior to each cycle. In the first three patients, the frequency of gene transfer measured by proviral containing CFU in the post transduction culture was 20 ± 7% and 6.3 ± 3 % of the cells expressed G156A-AGT by flow cytometry. An average of 4.4 ± 2 x 107viable mononuclear cells with a CD34 purity of 93 ± 5% were infused. In one patient, 10% of bone marrow CFU showed presence of the provirus 5 weeks after infusion but prior to the second cycle of BG and BCNU. In this same patient, insertion site analysis by LAM-PCR showed a similar banding pattern in both the lymphocyte-monocyte and granulocyte fractions, indicating multilineage reconsitituion by a single transduced stem cell. Our preliminary results demonstrate efficient gene transfer into cytokine stimulated CD34+ cells using the MFG-G156A-MGMT vector and these cells can be infused without toxicity. Given the strong degree of selection observed in pre-clinical models, we anticipate enrichment for transduced cells following each cycle of chemotherapy.

The Tel-PDGFRbeta fusion gene produces a chronic myeloproliferative syndrome in transgenic mice.

Chronic myelomonocytic leukemia (CMML) is a pre-leukemic syndrome that displays both myelodysplastic and myeloproliferative features. The t(5;12) chromosomal translocation, present in a subset of CMML patients with myeloproliferation fuses the amino terminal portion of the ets family member, Tel, with the transmembrane and tyrosine kinase domains of platelet-derived growth factor receptor beta (PDGFRbeta) gene. To investigate the role of this fusion protein in the pathogenesis of CMML, we expressed the Tel-PDGFRbeta fusion cDNA in hematopoietic cells of transgenic mice under the control of the human CD11a promoter. Transgenic founders and their offspring express the transgene specifically in hematopoietic tissues and develop a myeloproliferative syndrome characterized by: overproduction of mature neutrophils and megakaryocytes in the bone marrow; splenomegaly with effacement of splenic architecture by extramedullary hematopoiesis; an abnormal population of leukocytes co-expressing lymphoid and myeloid markers; and increased numbers of colonies in in vitro bone marrow CFU assays. All mice expressing the transgene exhibited at least one of these features of dysregulated myelopoiesis, and 20% progressed to a myeloid or lymphoid malignancy. This murine model of CMML parallels a myeloproliferative syndrome in humans and implicates the Tel-PDGFRbeta fusion protein in its pathogenesis.

IL-17 Stimulates Granulopoiesis in Mice: Use of an Alternate, Novel Gene Therapy-Derived Method for In Vivo Evaluation of Cytokines

IL-17 is a novel cytokine secreted principally by CD4+ T cells. It has been shown to support the growth of hemopoietic progenitors in vitro; however, its in vivo effects are presently unknown. Adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to the liver (5 x 10(9) plaque-forming units (PFU) intravenous) resulted in a transiently transgenic phenotype, with dramatic effects on in vivo granulopoiesis. Initially, there was a significant increase (fivefold) in the peripheral white blood count (WBC), including a 10-fold rise in the absolute neutrophil count. This was associated with a doubling in the spleen size over 7-14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated. There was a profound stimulation of splenic hemopoiesis as demonstrated by an increase in total cellularity by 50% 7 days after gene transfer and an increase in hemopoietic colony formation. A maximal increase in frequency of high proliferative potential colonies (HPPC) (11-fold) and CFU-granulocyte-macrophage (GM) and CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM) (CFU) (6-fold) was seen on day 3 after IL-17 gene transfer. Both CFU and HPPC remained significantly elevated in the spleen throughout day 21, but at reduced levels compared with day 3. Bone marrow CFU and HPPC were elevated on day 3 only by 75% and 25%, respectively, without changes in total cellularity. Thus, murine IL-17 is a cytokine that can stimulate granulopoiesis in vivo. Since IL-17 is principally produced by CD4+ T cells, this cytokine could have therapeutic implications in AIDS-related bone marrow failure and opportunistic infections.

Enhancement of bone marrow radioprotection and reduction of WR-2721 toxicity by Ocimum sanctum

The radioprotective effect of the leaf extract of Ocimum sanctum (OE) in combination with WR-2721 (WR) was investigated on mouse bone marrow. Adult Swiss mice were injected intraperitoneally (i.p.) with OE (10 mg/kg on 5 consecutive days), or 100–400 mg/kg WR (single dose) or combination of the two or double-distilled water (DDW) and whole-body exposed to 4.5 Gy γ-irradiation (RT). Metaphase plates were prepared from femur bone marrow on days 1, 2, 7 and 14 post-treatment and chromosomal aberrations were scored. The maximum number of aberrant cells was observed at 24 h after irradiation in all the groups. However, pretreatment with OE or WR individually resulted in a significant decrease in aberrant cells as well as different types of aberrations. The combination of the two further enhanced this effect; resulting in a 2-fold increase in the protection factor (PF=6.68) compared to 400 mg/kg WR alone. The percent aberrant cells decreased linear-quadratically with WR dose when given individually, while in the OE+WR pretreatment animals the values showed a linear dose response. Combination of OE with WR doses above 200 mg/kg completely eliminated rings, polyploidy and pulverization of chromosomes. Percent aberrant cells decreased with time in all groups, though the values remained higher than normal even on day 14 in the RT alone as well as those treated with single agent+RT. WR doses above 200 mg/kg before RT resulted in significantly higher frequency of aberrant cells compared to RT and OE+RT groups on day 14, suggesting delayed WR toxicity; but combination of OE with WR brought down these values to normal level, indicating that OE combination, in addition to enhancing WR protection, may also act as a detoxifier. The protective effect of OE and WR is also reflected in the enhancement of bone marrow CFU survival. Both OE and WR possessed significant free radical scavenging activity in vitro. The combination of the two further enhanced this effect, suggesting that the enhanced free radical scavenging activity by combining the two protectors results in the higher bone marrow cell protection. The significant elevation in chromosome protection obtained by combining OE with WR, with reduction in the latter's toxicity at higher doses, suggests that the combination may have promise for radioprotection in humans.

Flt3 Ligand Synergizes With Granulocyte-Macrophage Colony-Stimulating Factor or Granulocyte Colony-Stimulating Factor to Mobilize Hematopoietic Progenitor Cells Into the Peripheral Blood of Mice

AbstractPeripheral blood progenitor cells (PBPC) are increasingly being used in the clinic as a replacement for bone marrow (BM) in the transplantation setting. We investigated the capacity of several different growth factors, including human flt3 ligand (FL), alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF ) or granulocyte colony-stimulating factor (G-CSF ), to mobilize colony forming cells (CFU) into the peripheral blood (PB) of mice. Mice were injected subcutaneously (SC) with growth factors daily for up to 10 days. Comparing the single agents, we found that FL alone was superior to GM-CSF or G-CSF in mobilizing CFU into the PB. FL synergized with both GM-CSF or G-CSF to mobilize more CFU, and in a shorter period of time, than did any single agent. Administration of FL plus G-CSF for 6 days resulted in a 1,423-fold and 2,717-fold increase of colony-forming unit–granulocyte-macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) in PB, respectively, when compared with control mice. We also followed the kinetics of CFU numerical changes in the BM of mice treated with growth factors. While GM-CSF and G-CSF alone had little effect on BM CFU over time, FL alone increased CFU-GM and CFU-GEMM threefold and fivefold, respectively. Addition of GM-CSF or G-CSF to FL did not increase CFU in BM over levels seen with FL alone. However, after the initial increase in BM CFU after FL plus G-CSF treatment for 3 days, BM CFU returned to control levels after 5 days treatment, and CFU-GM were significantly reduced (65%) after 7 days treatment, when compared with control mice. Finally, we found that transplantation of FL or FL plus G-CSF–mobilized PB cells protected lethally irradiated mice and resulted in long-term multilineage hematopoietic reconstitution.

Receptor induction regulates the synergistic effects of substance P with IL-1 and platelet-derived growth factor on the proliferation of bone marrow fibroblasts.

The neuropeptide substance P (SP) stimulates CFU in bone marrow (BM) cultures. Although the methylcellulose matrix used in these assays does not provide an appropriate substratum to support adherent-dependent cells, we have observed that cultures containing optimal SP (10(-8)-10(-10) M) develop confluent areas of reticular/fibroblastoid-like cells with CFUs predominantly localized within their vicinity. Characterization (cytochemical and immunofluorescence) of the reticular/fibroblastoid-like cells indicated that they were fibroblasts, the major constituent of the BM stroma. Hemopoietic effects by SP are mediated by the stroma that expresses SP receptors. We studied the effects of SP (10(-7)-10(-11) M) with suboptimal platelet-derived growth factor-BB (PDGF-BB; 5 ng/ml) and IL-1alpha (2 ng/ml), two fibrogenic cytokines, and also hemopoietic regulators. SP by itself and in synergy with either cytokine induced fibroblast proliferation. At optimum SP, IL-1alpha induced 1.6 times the proliferation of PDGF-BB (87 +/- 7 vs 55 +/- 5; n = 12; p < 0.05). The effects of SP were blunted by a specific neurokinin-1 antagonist. Scatchard analysis indicated that SP binds to BM fibroblasts with an approximate Kd of 5 nM. SP induced steady state mRNA for IL-1 receptor IL-1RI and PDGF-BB (PDGF-AR, PDGF-BR) receptors by 7.5-, 6.2-, and 10.5-fold, respectively. Their up-regulation may be partly responsible for the synergistic effects of SP and their ligands. Induction (3-fold) of neurokinin-1 mRNA by IL-1alpha compared with no induction by PDGF-BB may explain the preferred synergism between SP and IL-1alpha. This study indicates that induction of SP, IL-1alpha, and PDGF-BB receptors is important to their synergistic effects on BM fibroblast proliferation. These results bring new insights into stroma-mediated hemopoietic regulation.