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PDF file - 38K, This provides a supplemental table of gene expression data and background on the data set. The GEO accesssion number and hyperlink are included.

PDF file - 2870K, Figure 1 is a detailed characterization of the myeloid precursor cells used in the study.

PDF file - 23K

Purpose: An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage.Experimental Design: We developed an ex vivo primary human myeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O6-benzylguanine (6BG) and TMZ.Results: Exposure to 6BG/TMZ led to increases in p53, p21, γ-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6-methylguanine–DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ–treated versus control, 6BG-, or TMZ alone–treated cells, there were no changes in caspase-8 activity. In addition, there were no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative.Conclusions: In human myeloid precursor cells, 6BG/TMZ–initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human myeloid precursor cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity. Clin Cancer Res; 19(10); 2699–709. ©2013 AACR.

Purpose: An understanding of how hematopoietic cells respond to therapy that causes myelosuppression will help develop approaches to prevent this potentially life-threatening toxicity. The goal of this study was to determine how human myeloid precursor cells respond to temozolomide (TMZ)-induced DNA damage. Experimental Design: We developed an ex vivo primary human myeloid precursor cells model system to investigate the involvement of cell-death pathways using a known myelosuppressive regimen of O6-benzylguanine (6BG) and TMZ. Results: Exposure to 6BG/TMZ led to increases in p53, p21, γ-H2AX, and mitochondrial DNA damage. Increases in mitochondrial membrane depolarization correlated with increased caspase-9 and -3 activities following 6BG/TMZ treatment. These events correlated with decreases in activated AKT, downregulation of the DNA repair protein O6-methylguanine–DNA methyltransferase (MGMT), and increased cell death. During myeloid precursor cell expansion, FAS/CD95/APO1(FAS) expression increased over time and was present on approximately 100% of the cells following exposure to 6BG/TMZ. Although c-flipshort, an endogenous inhibitor of FAS-mediated signaling, was decreased in 6BG/TMZ–treated versus control, 6BG-, or TMZ alone–treated cells, there were no changes in caspase-8 activity. In addition, there were no changes in the extent of cell death in myeloid precursor cells exposed to 6BG/TMZ in the presence of neutralizing or agonistic anti-FAS antibodies, indicating that FAS-mediated signaling was not operative. Conclusions: In human myeloid precursor cells, 6BG/TMZ–initiated apoptosis occurred by intrinsic, mitochondrial-mediated and not extrinsic, FAS-mediated apoptosis. Human myeloid precursor cells represent a clinically relevant model system for gaining insight into how hematopoietic cells respond to chemotherapeutics and offer an approach for selecting effective chemotherapeutic regimens with limited hematopoietic toxicity. Clin Cancer Res; 19(10); 2699–709. ©2013 AACR.

In the production of lentiviral vector for clinical studies the purity of the final product is of vital importance. To remove plasmid and producer cell line DNA, investigators have incubated the vector product with Benzonase, a bacterially derived DNase. As an alternative we investigated the use of Pulmozyme, a U.S. Food and Drug Administration-approved human DNase for the treatment of cystic fibrosis, by comparing the efficiency of DNA removal from lentiviral vector preparations. A green fluorescent protein-expressing lentiviral vector was prepared by transient calcium phosphate transfection of HEK 293T cells and DNA removal was compared when treating vector after harvest or immediately after transfection. The effectiveness of DNase treatment was measured by quantitative PCR using primers for vesicular stomatitis virus glycoprotein G viral envelope plasmid. When treating the final product, 1-hr incubations (37°C) with Pulmozyme at 20 U/ml reduced plasmid DNA to undetectable levels. Longer incubations (up to 4 hr) did not improve DNA removal at lower concentrations and the effectiveness was equivalent to or better than Benzonase at 50 U/ml. Attempting to use Pulmozyme immediately after transfection, but before final medium change, as a means to decrease Pulmozyme concentration in the final product provided a 2-log reduction in DNA but was inferior to treatment at the end of production. Pulmozyme, at concentrations up to 100 U/ml, had no measurable effect on infectious titer of the final vector product. The use of Pulmozyme is likely to increase the cost of DNase treatment when preparing vector product and should be considered when generating clinical-grade vector products.

Purpose: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However, one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example, hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens, but in vivo models to assess potential toxicities have not been developed. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. Experimental Design: A proof-of-concept, temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-PrkdcscidIL2rgtm1Wjl/Sz (NOD/SCID/γchainnull), reconstituted with human hematopoietic cells, and the impact of treatment on human hematopoiesis was evaluated. Results: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow, flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor, lymphoid, and myeloid). Conclusions: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. Clin Cancer Res; 17(8); 2195–206. ©2011 AACR.

Objective Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin neg BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O 6 -methylguanine DNA methyltransferase (MGMT P140K ) and selected in vivo with submyeloablative or myeloablative alkylator therapy. Materials and Methods Lin neg BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT P140K and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K–transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals. Results Following submyeloablative therapy, 55% of the mice expressed MGMT P140K in the BM. Proviral integration was observed in ∼50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT P140K -positive cells for 5 to 6 months. Conclusions In vivo selection of MD9-P140K–transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.

Murine xenograft models are one of the primary tools used for screening of new therapeutic compounds and regimens. However, one major limitation of utilizing murine xenograft studies to assess therapeutic efficacy is that significant inter-species differences in drug sensitivity can exist between mice and humans. It is possible that the levels of a therapeutic compound reached in a mouse xenograft model may not be achievable in humans due to differential toxicity profiles. There is a growing need for in vivo models that can simulate efficacy of promising therapeutic regimens but at the same time can evaluate the potential for off-target human toxicity. Since bone-marrow toxicity can be a major life-threatening side effect of treatment, models to screen for the impact of treatment on human hematopoiesis would improve our ability to select compounds with decreased off-target toxicities. In this study, a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. As a proof-of-concept, the impact of a combination therapy consisting of O6-benzylguanine and temozolomide (O6-BG/TMZ) on human hematopoiesis in vivo was investigated since this regimen is currently being evaluated in clinical trials and the main dose-limiting toxicity in these patients is myelosuppression. A dose-intensive O6-BG/TMZ-dosing regimen that requires stem-cell rescue was developed that significantly inhibits the growth of human-glioblastoma xenografts in NOD/SCID/γchainnull mice. To monitor human hematotoxicity profiles, NOD/SCID/gammanull mice were next transplanted with human CD34+ cells and reconstitution confirmed one month post-transplantation by peripheral blood analysis. The dose-intensive regimen was then administered to NOD/SCID/gammanull mice reconstituted with human hematopoietic cells and the impact of treatment on human hematopoiesis evaluated. Flow cytometric analyses indicated that the human bone-marrow cells were significantly more sensitive to treatment than the murine bone-marrow cells in vivo, and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (CD34+ progenitor, CD45+CD19+ lymphoid, and CD33+ myeloid). This proof-of-concept study indicates that use of NOD/SCID/γchainnull mice with humanized bone marrow can be used as an in vivo toxicity measure of human hematopoiesis. This model holds promise as a new approach for monitoring the impact of anti-cancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2543. doi:10.1158/1538-7445.AM2011-2543

36 Chemoprotection Of Long-Term Repopulating Hematopoietic Stem Cells From Alkylator Therapy: in vivo Comparison of Gene-Transfer Vectors that Express a DNA Repair Protein.

Recombinant foamy virus vectors transduce noncycling and cycling cells, are stable episomally, and integrate into the host genome during cell division. Due to the cytoplasmic stability of this vector, a substantial lag period between transduction and cell division required for provirus integration is possible. Therefore, in transplantation studies that use minimally stimulated hematopoietic stem and progenitor cells (HSC), integration of the foamy virus vector in HSC may occur once HSC divide post-transplantation. We used a foamy virus vector, MD9-P140K-EGFP, that co-expresses a mutant form of O6-methylguanine DNA methyltransferase (MGMTP140K) and the enhanced green fluorescent protein (EGFP) to test the hypothesis that HSC could be transduced with a foamy virus vector and selected in vivo by alkylator-based chemotherapy. We also compared foamy virus transduction and selection to our previously optimized strategy using an oncoretrovirus vector to express MGMTP140K (SF1-P140K-IRES-EGFP). Lineage-depleted bone marrow (BM) from C57BL/6 mice was transduced for 10-16 hours with the foamy virus vector or transduced following a 2-day prestimulation with the oncoretrovirus vector. Data presented are from three primary transplant experiments analyzed over 6 months and one secondary transplantation experiment analyzed for 6 months. The bulk transduction efficiency using the foamy virus vector ranged from 12–25% and the CFU transduction efficiency was 55–57%. Transductions with the oncoretrovirus vectors resulted in similar bulk and CFU transduction efficiencies (55–60%). Similar numbers of progenitor colonies (oncoretrovirus vs. foamy virus) were observed. MGMT activity in pooled progenitor colonies was ~10-fold higher in EGFP+ versus EGFP− colonies. Although similar numbers of CFU were transduced using the two vector systems, significantly different levels of in vivo selection were obtained in primary recipient mice. Consistent with previous studies, selection of oncoretroviral vector-transduced cells resulted in high and sustained levels of EGFP+ cells in the PB and BM in primary and secondary recipient mice (80–99% EGFP+ with 2–3 cycles of 6BG/BCNU). For primary transplants using cells transduced with the foamy virus vector, EGFP expression in the PB peaked at 3 months post-treatment (26.2+/−4.0%) which represented a 4–6 fold increase compared to vehicle-treated mice. However, by 6 months EGFP expression dropped by 3-fold (9+/−1%). Western analysis of MGMT protein levels found in the BM at 6 months post-transplantation also showed a 3-fold decline in expression. In secondary reconstitution experiments, however, flow cytometry and Western analysis of MGMT expression indicated that EGFP expression in foamy virus-transduced HSC no longer correlated with MGMT expression. In fact MGMT expression levels following drug treatment were similar to those found in secondary recipient mice transplanted with oncoretroviral vector-transduced cells, suggesting that stem cells expressing MGMT were selected over time. These data demonstrate that although foamy virus transduction is not as efficient as the more commonly studied oncoretrovirus transduction strategy, a simple overnight protocol can be used to transduce minimally stimulated HSC with a foamy virus vector. These cells can be selected in vivo, can reconstitute mice for up to one year, and can maintain high levels of MGMT expression.

Top of pageAbstract The ability to deliver genetic sequences that encode therapeutic proteins to hematopoietic stem and progenitor cells (HSC) in the absence of cell cycle progression provides an opportunity to transduce polyclonal populations of stem cells without loss of long-term function. We constructed a foamy virus vector MD9-P140K-EGFP that co-expresses a mutant form of O6-methylguanine DNA methyltransferase (MGMTP140K) and the enhanced green fluorescent protein (EGFP) to study the outcome of transducing noncycling populations of HSC derived from human and mouse sources. We also compared in parallel transduction of HSC with an oncoretrovirus vector, SF1-P140K-EGFP, using standard transduction protocols. In the human studies, CD34+ cells derived from Granulocyte-Colony Stimulating Factor-mobilized peripheral blood (MPB) were used since MPB is difficult to transduce with oncoretrovirus vectors. We demonstrated previously that although MPB could be selected in vivo by expression of MGMTP140K using an oncoretrovirus vector and drug treatment, the number of selected HSC was significantly lower than that obtained when using UCB HSC presumably due to inefficient gene transfer. (Pollok et al, Hum. Gene Ther. 14:1703). Cell cycle analysis indicated that 98-99% of the MPB were in G0/G1 prior to transduction. MPB HSC were transduced on Retronectin-coated plates with the MD9-P140K-EGFP vector in the presence of G-CSF, Stem Cell Factor (SCF), and Thrombopoietin (TPO) or SCF, TPO, and Flt3-ligand at a multiplicity of infection (MOI) ranging from 10-20. Transduction on Retronectin-coated plates increased the transduction efficiency and also prevented detrimental cell fusion. In colony-forming unit (CFU) assays, similar numbers of CFU were present in transduced and nontransduced cultures, indicating that no gross toxicity occurred during the transduction. The MD9 vector preferentially transduced the committed progenitor pool regardless of the cytokine combination used. While the bulk transduction efficiencies ranged from 3%-5%, the CFU transduction efficiency ranged from 40%-70% in lineage-restricted progenitors (n=5). Consistent with our previous studies, transduction of HSC with the SF1 oncoretrovirus vector resulted in similar bulk and CFU transduction efficiencies (55%-60%). MGMT DNA repair activity in EGFP-positive CFU derived from foamy virus MD9-transduced HSC exhibited 50%-70% higher level of activity compared to EGFP-positive CFU derived from oncoretrovirus SF1-transduced HSC. In mouse studies, bone marrow from C57BL/6 mice was lineage depleted to enrich for noncycling HSC. In MD9 transductions, no significant toxicity was observed up to a MOI of 40. Bulk transduction efficiency ranged from 12%-25% and the CFU transduction efficiency was 55%-57% (n=3).Transduction with the oncoretrovirus vector resulted in similar bulk and CFU transduction efficiencies (55%-60%). Current studies are now using these conditions to evaluate in vivo the selection of minimally stimulated HSC transduced with the MD9-P140K-EFGP foamy virus vector.