Supplementary Data 1-5 from Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

1Division of Experimental Hematology, Department of Pediatrics, Cincinnati Children’s Research Foundation and Cincinnati Children’s Hospital Medical Center, OH; 2Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis; 3Division of Pathology, Department of Pediatrics, Cincinnati Children’s Research Foundation and Cincinnati Children’s Hospital Medical Center, OH; and 4Hoxworth Blood Center, University of Cincinnati College of Medicine, OH

Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

Retroviral-mediated delivery of the P140K mutant O-methylguanine-DNA methyltransferase (MGMT) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O-benzylguanine) of endogenous MGMT. Because detoxification of O-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT activity, we show that MGMT expression by weaker cellular promoter/ enhancers is sufficient for in vivo protection/selection following treatment with O-benzylguanine/temozolomide. Conversely, the highest level of MGMT activity did not promote efficient in vivo protection despite mediating detoxification of O -alkylguanine adducts. Moreover, very high expression of MGMT was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT to the nucleus/chromatin. These data show that very high expression of MGMT has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT. [Cancer Res 2008;68(15):6171–80] Introduction Retroviral-mediated transfer of drug resistance genes into hematopoietic stem cells (HSC) has been investigated for its potential to achieve the dual aim of preventing severe dose-limiting toxicity of cancer chemotherapy and enrichment of gene-modified cells in vivo (reviewed in ref. 1). Of several drug resistance genes proposed for this application, one of the best characterized is O-methylguanine-DNA methyltransferase (MGMT ; ref. 2). MGMT confers protection against clinically relevant O-alkylating agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; Carmustine) and temozolomide (Temodar), which are used in the treatment of a variety of tumors, predominantly brain tumors (3, 4). Hematotoxicity is dose limiting in many treatment protocols using O-alkylating agents (5, 6), likely due to low expression of MGMT in the HSC and progenitor (P) compartment (7). In addition, elevated expression of MGMT has been found to be a mechanism of drug resistance in tumor cells, particularly glial tumors, which have poor prognosis (8, 9). To circumvent tumor resistance, pseudosubstrate inhibitors of endogenous MGMT, such as O-benzylguanine and O-(4-bromothenyl)guanine (lomeguatrib), have been developed. These agents ablate MGMT activity and sensitize tumors to the cytotoxic effects of O-alkylating agents (10, 11). However, in human clinical trials the combination of O-benzylguanine or lomeguatrib with temozolomide or BCNU has been shown to exacerbate myelosuppression (12–14). This increased toxicity likely relates to the depletion of MGMT activity in bone marrow HSC/P. Several mutant versions of MGMT are resistant to such pseudosubstrate inhibitors, yet retain alkyltransferase activity. Of these, the P140K mutant (MGMT) seems to be optimal, combining resistance to pseudosubstrate inhibitors with efficient repair of cytotoxic O-alkylguanine adducts (15–18). Thus, one goal of current studies is to achieve transgenic expression of MGMT in bone marrow HSC/P. Coupled with O-alkylating agent treatment and O-benzylguanine depletion, tumor cells will be sensitized while simultaneously protecting the bone marrow compartment to effect a widened therapeutic window. Clinical studies are currently under way using gamma-retrovirus vectors expressing MGMT in patients with poor-prognosis brain tumors (19). In addition, we and others have shown that treatment of mice and dogs that have been transplanted with HSC transduced with O-benzylguanine–resistant MGMT vectors allows significant in vivo selection of transduced cells (16, 20–23). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: David A. Williams, Children’s Hospital Boston, 300 Longwood Avenue, Karp 07212, Boston, MA 02115. Phone: 617-919-2697; Fax: 617730-0934; E-mail: DAWilliams@childrens.harvard.edu. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-0320 www.aacrjournals.org 6171 Cancer Res 2008; 68: (15). August 1, 2008 Research Article American Association for Cancer Research Copyright © 2008 on February 21, 2013 cancerres.aacrjournals.org Downloaded from DOI:10.1158/0008-5472.CAN-08-0320

O6-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against alkylating agents that generate, among other lesions, O6-alkylguanine in DNA (collectively termed O6-alkylating agents [O6AA]). The defense is highly important, since O6AA are common environmental carcinogens, are formed endogenously during normal cellular metabolism and possibly inflammation, and are being used in cancer therapy. O6AA induced DNA damage is subject to repair, which is executed by MGMT, AlkB homologous proteins (ABH) and base excision repair (BER). Although this review focuses on MGMT, the mechanism of repair by ABH and BER will also be discussed. Experimental systems, in which MGMT has been modulated, revealed that O6-methylguanine (O6MeG) and O6-chloroethylguanine are major mutagenic, carcinogenic, recombinogenic, clastogenic and killing lesions. O6MeG-induced clastogenicity and cell death require MutSα-dependent mismatch repair (MMR), whereas O6-chloroethylguanine-induced killing occurs independently of MMR. Extensive DNA replication is required for O6MeG to provoke cytotoxicity. In MGMT depleted cells, O6MeG induces apoptosis almost exclusively, barely any necrosis, which is presumably due to the remarkable ability of secondarily formed DNA double-strand breaks (DSBs) to trigger apoptosis via ATM/ATR, Chk1, Chk2, p53 and p73. Depending on the cellular background, O6MeG activates both the death receptor and the mitochondrial apoptotic pathway. The inter-individual expression of MGMT in human lymphocytes is highly variable. Given the key role of MGMT in cellular defense, determination of MGMT activity could be useful for assessing a patient's drug sensitivity. MGMT is expressed at highly variable amounts in human tumors. In gliomas, a correlation was found between MGMT activity, MGMT promoter methylation and response to O6AA. Although the human MGMT gene is inducible by glucocorticoids and genotoxins such as radiation and alkylating agents, the role of this induction in the protection against carcinogens and the development of chemotherapeutic alkylating drug resistance are still unclear. Modulation of MGMT expression in tumors and normal tissue is currently being investigated as a possible strategy for improving cancer therapy.

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesone- phros are hypothesized to colonize the fetal liver, but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here, we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach, we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1, there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos, while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos, culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5, Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired interaction with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis.

Ectopic delivery of HOXB4 elicits the in vitro and in vivo expansion of hematopoietic stem cells (HSC) although the mechanism is still unknown. We have previously shown that overexpression of HOXB4 attenuates the TNFα signaling pathway at the transcriptional level (Schiedlmeier et al., 2007, PNAS). TNFα is expression is induced at high levels in vivo after irradiation used in bone marrow (BM) transplantation preparative regimens. Since HSC and progenitors (P) derived from Fanconi anemia (FA) knockout mice are hypersensitive to the action of inhibitory cytokines such as TNFα, we chose the Fancc−/− mouse as a model to study the physiologic effects of HOXB4 on TNFα sensitivity of HSC/P and the relationship of these effects to the engraftment defect of FA HSC. Competitive repopulating assays were used to evaluate the effect of HOXB4 overexpression upon engraftment of Fancc−/− BM. Control (CON, expressing eGFP only) transduced Fancc−/− BM demonstrated 80-fold lower engraftment compared with wild type (WT) eGFP+ BM at 26 weeks post-transplant (Table 1). In marked contrast, HOXB4 transduced (HOXB4+) Fancc−/− BM showed a 26-fold higher level of engraftment compared with CON transduced Fancc−/− BM and a 2-fold higher level of engraftment compared with Fancc−/− cells corrected with a FANCC-expressing vector. Fancc−/− BM co-transduced with both vectors (expressing HOXB4 and FANCC) further increased the level of engraftment to that of WT eGFP+ BM suggesting a synergistic correction of the FA HSC engraftment defect. To determine the potential role of TNFα signaling in these effects, we directly assessed the impact of HOXB4 expression on the response of Fancc−/− BM to treatment with TNFα. Fancc−/− BM cells transduced with the CON eGFP vector demonstrated >70% reduction in colony formation upon treatment with 10ng/ml TNFα (Table 2), while Fancc−/− BM overexpressing HOXB4 showed no significant inhibition of CFU at 10ng/ml TNFα compared to either untreated cells or to treated WT BM transduced with eGFP CON vector. Additionally, in vitro treatment of eGFP CON transduced Fancc−/− BM with 100ng/ml TNFα resulted in a 21±5% decrease in lineage-, Sca-1+, c-Kit+ (LSK) cells within 24 hrs. In contrast, similarly treated HOXB4+ Fancc−/− demonstrated a 6±9% increase in LSK cells (p<0.01 compared to Fancc−/− eGFP CON). Hence HOXB4 completely protects Fancc−/− HSC/P against the inhibitory effects of TNFα. In order to further define the mechanism through which HOXB4 attenuates TNFα signaling, we determined the level of expression of the TNFα receptors, TNFR1 and TNFR2, on transduced Fancc−/− BM by flow analysis. Fancc−/− LSK BM transduced with eGFP CON or FANCC expressing vectors had equivalent expression of TNFR1 and TNFR2 compared to WT eGFP CON transduced BM (Table 3). In contrast, Fancc−/− LSK cells transduced HOXB4 demonstrated a >25% reduction in the number of cells that stained positive for either TNFR1 or TNFR2. In addition, there was a >35% decrease in the MFI of staining for TNFR1 in HOXB4+ Fancc−/− LSK compared to other groups. In summary, ectopic HOXB4 protects Fancc−/− and WT HSC/P from the inhibitory effects of TNFα. Since HOXB4 expression also protects WT cells from TNFα treatment (data not shown), we propose that HOXB4 enhances engraftment by protecting HSC from the elevated TNFα levels, which is a result of conditioning regimens applied to transplant recipients. We suggest that this mechanism reveals a novel target for the pharmacologic manipulation of HSC during engraftment, thus avoiding the potential adverse effects of constitutive HOXB4 overexpression.

Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted pre-clinical model defined to assess their safety, in particular their risk related to insertional mutagenesis. In the murine pre-clinical study presented here, 40 test and 10 control mice were transplanted with ex vivo manipulated bone marrow cells to assess the long-term effects of the transduction of hematopoietic cells with the retroviral vector MSCV-MGMT(P140K)wc. Test mice had significant gene marking 8-12 months post-transplantation with an average of 0.93 vector copies per cell and 41.5% of peripheral blood cells expressing the transgene MGMT(P140K), thus confirming persistent vector expression. Unexpectedly, six test mice developed malignant lymphoma. No vector was detected in the tumor cells of five animals with malignancies, indicating that the malignancies were not caused by insertional mutagenesis or MGMT(P140K) expression. Mice from a concurrent study with a different transgene also revealed additional cases of vector-negative lymphomas of host origin. We conclude that the background tumor formation in this mouse model complicates safety determination of retroviral vectors and propose an improved study design that we predict will increase the relevance and accuracy of interpretation of pre-clinical mouse studies.

Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

T cell acute lymphoblastic leukemia (T-ALL) is frequently associated with overexpression of the oncogenes LMO2 and SCL(TAL1) which are normally down regulated following the double negative stage of T cell development. Our goal is to decipher the molecular and cellular mechanisms leading to the onset of LMO2 associated T-ALL. We were able to isolate a complex containing the transcription factors LMO2, SCL(TAL1) and E47 from primary human T-ALL cells with proven aberrant expression of LMO2 and SCL(TAL1) by applying immunoprecipitation and Western blotting techniques. This protein complex regulates the transcription of a truncated form of RALDH2 (retinaldehyde dehydrogenase) in T-ALL cells as shown by gene transcription profiling in conjunction with RT-PCR and siRNA approaches. To monitor the effect of LMO2 expression on T cell development and leukemogenesis, lethally irradiated mice (C57BL/6) were transplanted with bone marrow cells that had been transduced with a retrovirus carrying LMO2 as the transgene. One year later, 88% of the cells in the thymus expressed LMO2 and a shift towards CD3−/CD44+/CD25+ cells was observed (an 88% increase compared to normal thymocytes), suggesting a differentiation block caused by LMO2 leading to an accumulation of immature T cells. To test and identify cooperating genes in T-ALL development, bone marrow cells of LMO2 double transgenic mice in which tet-inducible LMO2 is controlled by a thymic specific promoter, were retrovirally transduced with SCL(TAL1). So far, none of the control animals, transplanted with bone marrow cells transduced with a vector only containing EGFP, developed T-ALL. However, six out of the seven test animals developed T-ALL exhibiting enlargement of the spleen, liver and thymus between seven and nine months after transplantation. Organs and blood of the diseased animals were infiltrated with T-ALL cells of the immature phenotype CD8+/CD4+ in five cases and of the CD3−/CD44+/CD25+ phenotype in one case. This indicates that the differentiation block caused by a lack of down-regulation of LMO2 and SCL(TAL1) in maturing T cells leads to a block in T cell differentiation and precedes T-ALL. These models will be used to examine the involvement of other cooperating genes in T-ALL development as well as downstream target genes of LMO2/SCL(TAL1), such as RALDH2, in the onset of T-ALL. We conclude that aberrant expression of LMO2 in T cells leads to a block in T cell maturation and, in conjunction with up-regulation of secondary genes like SCL(TAL1), triggers deregulation of genes in immature T cells leading to impaired T cell development and the onset of T-ALL. The described model will help to identify cooperating genes in LMO2 associated T-ALL as well as the chain of events leading to malignancy.

Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc.LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140Kwc vector on recombinant fibronectin CH296 and transplanted into 40 lethally irradiated (11.75 Gy) C57BL/6 recipient test mice (10 controls received mock-transduced cells). Titer measured on non-hematopoietic cells led to an unintended high MOI on the transplanted cells of 4. The animals were observed for up to 12 months. Gene marking was determined by quantitative PCR and by intracellular staining of the human MGMT transgene product.All mice were found to have significant gene marking in the peripheral blood with 0.1-2 vector copies per cell. The majority of the animals (80%) demonstrated more than 40% peripheral blood cells expressing human MGMT protein 8-12 months post transplantation, thus confirming persistent vector expression. Unexpectedly, 5 test mice have been diagnosed with malignant lymphoma. None of the control mice have been found to have developed malignancies, although the full pathologic evaluation is pending for 5 of the 8 remaining control animals. Laser capture microdissection (LCM) of tumor cells with subsequent quantitative PCR detected no vector in the tumor cells of any of the 5 animals with malignancies, whereas vector was consistently detected in non-malignant hematopoietic tissue.These results indicate that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140Kwc gene transfer as a causative factor for development of malignancies. A new murine study is initiated to distinguish host vs. donor cells, use a lower irradiation dose, include equal numbers of control animals, avoid 5-FU and utilize a transduction protocol with a MOI similar to the clinical protocol. Retroviral gene therapy vectors expressing the MGMTP140K transgene have been shown to protect hematopoietic cells from toxicity associated with a combined cancer treatment using 6-benzylguanine (6-BG) and an alkylating agent such as Temozolomide (TEM). In a Phase I gene transfer trial, high grade astrocytoma patients having poor prognoses using standard therapies, will undergo escalating dose treatments with 6-BG and TEM following MGMTP140K gene transfer into autologous hematopoietic stem cells. Although retroviral vectors are one of the most widely used vehicles for gene transfer, there is no uniformly accepted preclinical model defined to assess their safety, and, in particular their risk related to insertional mutagenesis. This study was designed as a murine pre-clinical study to assess the long term effects of the transduction of hematopoietic cells with the retroviral vector to be used in the clinical trial, MSCV-MGMTP140Kwc. LDBM cells from 5-FU treated C57BL/6 donors were transduced with ecotropic MSCV-MGMTP140Kwc vector on recombinant fibronectin CH296 and transplanted into 40 lethally irradiated (11.75 Gy) C57BL/6 recipient test mice (10 controls received mock-transduced cells). Titer measured on non-hematopoietic cells led to an unintended high MOI on the transplanted cells of 4. The animals were observed for up to 12 months. Gene marking was determined by quantitative PCR and by intracellular staining of the human MGMT transgene product. All mice were found to have significant gene marking in the peripheral blood with 0.1-2 vector copies per cell. The majority of the animals (80%) demonstrated more than 40% peripheral blood cells expressing human MGMT protein 8-12 months post transplantation, thus confirming persistent vector expression. Unexpectedly, 5 test mice have been diagnosed with malignant lymphoma. None of the control mice have been found to have developed malignancies, although the full pathologic evaluation is pending for 5 of the 8 remaining control animals. Laser capture microdissection (LCM) of tumor cells with subsequent quantitative PCR detected no vector in the tumor cells of any of the 5 animals with malignancies, whereas vector was consistently detected in non-malignant hematopoietic tissue. These results indicate that the malignancies were not caused by insertional mutagenesis or MGMTP140K expression. Low numbers of control animals may explain the failure to observe malignancies in this group; however, further studies are required to exclude MSCV-MGMTP140Kwc gene transfer as a causative factor for development of malignancies. A new murine study is initiated to distinguish host vs. donor cells, use a lower irradiation dose, include equal numbers of control animals, avoid 5-FU and utilize a transduction protocol with a MOI similar to the clinical protocol.

Ras-related Rho GTPases regulate actin cytoskeletal organization, adhesion, gene transcription, and cell-cycle progression. The Rac subfamily of Rho GTPases and Cdc42 has been shown to play essential roles in hematopoietic stem cell (HSC) engraftment and mobilization. Here, we study the role of RhoA, a related Rho GTPase, in HSC functions. Using retrovirus-mediated gene transfer of a dominant-negative (DN) mutant of RhoA (RhoAN19), we demonstrate that down-regulation of RhoA activity resulted in increased HSC engraftment and self-renewal as measured by competitive repopulation and serial transplantation assays. However, overexpression of RhoAN19 resulted in decreased migration toward SDF-1alpha and alpha(4)beta(1)- and alpha(5)beta(2)-integrin-mediated adhesion of hematopoietic progenitor cells in vitro. Low RhoA activity was associated with higher proliferation rate of hematopoietic progenitor cells and increased cells in active phases of cell cycle, most likely via decreasing p21Cip/Waf expression and increasing cyclin D1 levels. Thus, reducing RhoA activity by optimizing the balance between adhesion/migration and proliferation/self-renewal results in a net increase in HSC engraftment. This mechanism could provide a novel therapeutic target to enhance HSC therapies.

The hematopoietic-specific Rho GTPase, Rac2, regulates a variety of cellular functions including cell shape changes, motility, integrin-dependent adhesion, and apoptosis. In the study reported here, we demonstrate that wild-type (WT) hematopoietic stem cells/progenitors (HSC/P) preferentially engraft in nonablated Rac2(-/-) bone marrow. In addition, primitive Rac2(-/-) HSC/P transplanted into lethally irradiated WT recipients showed a significant competitive defect compared with WT cells. These defects appeared to be related to HSC/P-intrinsic defective microenvironment interactions, since Rac2(-/-) cells showed less adhesion to the femur bone marrow density 1 (FBMD-1) stromal cell line, a lower frequency of cobblestone area-forming cells, and lower performance in long-term marrow cultures in vitro when compared with WT cells. In contrast, primitive Rac2(-/-) hematopoietic cells exhibited normal progenitor colony formation in semisolid medium in vitro and normal proliferation in the steady state in vivo when compared with WT cells. Taken together, these data suggest that Rac2(-/-) stem/progenitor cells exhibit abnormal interaction with the hematopoietic microenvironment, which leads to defective long-term engraftment.

Hematopoiesis originates in the mouse embryo at gastrulation with the formation of an initial wave of primitive erythrocytes in the yolk sac (YS) of the extra embryonic tissue. During embryonic development the anatomic site of hematopoiesis changes and hematopoietic stem cells (HSC) are postulated to migrate and seed successive embryonic sites. The origin of definitive hematopoiesis, which has been postulated to be either the YS or the PAS/AGM region, remains controversial, and both sites have previously been shown to contribute to the seeding of HSC/P to the fetal liver (FL). We utilized a genetic approach to study molecular pathways involved in the migration of definitive hematopoietic progenitor cells between the YS and the embryo (AGM and FL). We have recently shown that Rac1 regulates the engraftment phenotype of adult HSC/P (Cancelas et al. Nat Med 2005). In the present study, we induced deletion of Rac1 in a hematopoietic tissue-specific manner by crossing Rac1 flox/null mice with a Vav1-Cre transgenic mouse (Croker B et al. Immunity 2004). Using RosaLacZ reporter mice, we first documented the presence of Vav1Cre-induced recombination in mice in the extraembryonic tissue as early as E 7.5, and in an anatomical distribution concordant with emergence of primitive hematopoiesis. Flow cytometric analysis of single cells derived from the embryo body and from the YS of E 9.5 double transgenic (RosaGFP, Vav1Cre) embryos demonstrated complete recombination in definitive hematopoietic progenitors defined as CD41hiFlk1dim. These data demonstrate that Vav1Cre transgene can induce recombination of genomic flox sequences in both primitive as well as definitive hematopoietic cells early in embryonic development. In crosses of Vav1Cre, Rac1null/wt and Rac1flox/wt mice, no triple transgenic (Vav1Cre, Rac1flox/null) mice survived past E14.5 (0/29, p These data demonstrate that Rac1 regulates the migration of HSC/P from the YS into the blood and suggest that this migration is essential for subsequent seeding of both the embryo proper and the FL.