BM MNCs Research Articles

Granulocytes Negatively Regulate Secretion of Transforming Growth Factor β1 by Bone Marrow Mononuclear Cells via Secretion of Erythropoietin Receptors in the Milieu.

In my previous study, I demonstrated that bone marrow-derived mononuclear cells (BM MNCs) secrete copious amounts of Transforming Growth Factor β1 (TGFβ1) in response to erythropoietin (EPO). In this study, I investigated the principal cell type involved in the process. I found that a large percentage of various marrow cells, but not their mature counterparts present in the peripheral blood, express EPO-receptors (EPO-R). Cell depletion experiments showed that depletion of Glycophorin positive erythroblasts and CD41+ megakaryocytes - the prime suspects - did not affect the EPO-mediated TGFβ1 secretion by the BM MNCs. However, individual depletion of CD2+ T lymphocytes, CD14+ monocyte/macrophages, and CD19+ B cells affected the TGFβ1 secretion by EPO-primed MNCs: depletion of CD2+ cells had the most striking effect. Unexpectedly, and most interestingly, depletion of CD15+ granulocytes led to a significant increase in the TGFβ1 secretion by both naïve and EPO-primed BM MNCs, suggesting that these cells negatively regulate the process. Mechanistically, I show that the CD15+ cells exert this regulatory effect via secretion of both full-length and soluble EPO-R in the milieu. Overall my results, for the first time, unravel an in-built regulatory mechanism prevailing in the BM microenvironment that regulates the secretion of TGFβ1 by controlling EPO-EPO-R interaction.My data could be relevant in understanding the pathophysiology of several conditions associated with deregulated production of TGFβ1 in the marrow compartment.

In Utero Hematopoietic Cell Transplantation of Lineage- c-Kit- Sca-1- Cells Is Responsible for Tolerance Induction

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (BM MNC) results in mixed hematopoietic chimerism in the murine model. In contrast, highly enriched HSCs (LSKs: Lin- c-Kit+ Sca-1+ cells) do not engraft after allogeneic IUHCT. Tracking studies in the first 72 hours after IUHCT demonstrate relatively large numbers of donor cells in the fetal thymus after IUHCT of BM MNCs, but not after IUHCT of LSK cells. This suggests that a sub-population of BM MNCs are capable of homing to the thymus within a window of opportunity for tolerance induction and that this population is lost, with enrichment of HSC.The aim of this study is to identify the specific cell population within BM MNCs that is responsible for tolerance induction. To achieve this goal, we developed a practical model for determination of prenatal tolerance induction and tested subpopulations of BM MNC for their ability to induce tolerance.MethodsDonor BM MNCs and subpopulations were isolated from 6-week old C57BL/6TgN(act-EGFP)OsbY01) (B6GFP) mice. Lineage (CD3, B220, CD11b, Gr-1) or CD3 depletion of donor cells was performed using magnetic sorting (Auto-MACS Pro Separator) and anti-Biotin microbeads.To isolate the Lin- subpopulations, anti-biotin antibody was used to exclude Lin+ cells, and then the remaining cells were stained for c-Kit and Sca-1 markers.Donor subpopulations were generated by sorting using the FACSAria.IUHCT was performed at E14 in Balb/c fetuses via vitelline vein injection. Post-natal injections were performed at P0 through the facial vein. Donor cell engraftment was assessed in peripheral blood at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population).Thymi and livers of E16 fetuses were harvested and GFP cells were detected using a fluorescence stereomicroscope.Statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results are expressed as mean±SEM. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsTo develop the model for tolerance assessment we determined that injection of 10^7 CD3 depleted BM MNCs into Balb/c pups at P0 resulted in no engraftment. In contrast, IUHCT at E14 of 10^7 BM MNCs resulted in chimerism (13.2±2.01% chimerism) at 4 weeks with associated donor specific tolerance. Finally, P0 injection of 10^7 CD3 depleted BM MNCs into in mice that had received E14 injections of 10^7 BM MNCs resulted in engraftment with enhancement of chimerism at 4 weeks (38.02±3.67% in E14 plus P0 group vs 0.008±0.005 in P0 only group, p=0.0001).After establishing the model to test tolerance, we performed IUHCT of subpopulations of cells derived from BM MNCs, using the dose of each subpopulation found in 10^7 BM MNCs, to test their potential for tolerance induction. Mice that underwent IUHCT of 10^5 Lin- cells prior to the P0 injection of 10^7 CD3depleted BM MNCs had higher chimerism at 4 weeks than mice that received E14 injections of 10^5 Lin+ cells (9.843±0.79% vs 0.088±0.056%, p=0.0001).Thymuses were harvested from E16 fetuses that were injected at E14 with different subsets of the Lin- population (c-Kit+ Sca-1+, c-Kit- Sca-1+, c-Kit+ Sca-1-, c-Kit- Sca-1int, and c-Kit- Sca-1- cells). The only thymuses to show GFP cells at E16 were those that had received E14 Lin- c-Kit- Sca-1- cells. Mice that received Lin- c-Kit- Sca-1- cells at E14 prior to P0 injections showed chimerism at 4 weeks when compared to those that had received Lin- c-Kit+ Sca-1-, Lin- c-Kit+ Sca-1+, Lin- c-Kit- Sca-1int, or Lin- c-Kit- Sca-1hi cells at E14, in which chimerism was absent (6.755±0.95% vs 0.0%; p=0.02.)ConclusionWe have demonstrated that it is the Lin- c-Kit- Sca-1- subpopulation of BM MNCs that induces tolerance and prevents rejection of subsequent injections from the same donor. We have also shown that only the Lin- c-Kit- Sca-1- population migrates to the thymus in time for the induction of central tolerance. Furthermore, mouse fetuses that were given Lin- c-Kit- Sca-1- intravascularly at E14 were able to become chimeric with a postnatal CD3 depleted MNCs transplantation without immunosuppression or myeloablation. These findings suggest that a c-Kit- Sca-1- progenitor is responsible for tolerance induction, likely via common dendritic progenitors that home to the thymus. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhancing Proteasome-Inhibitory Activity and Specificity of Bortezomib By Endocytosis-Mediated Uptake of CD38 Targeted Nanoparticles in Multiple Myeloma

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. Due to its high expression on MM cells, CD38 is used as a marker for identification of MM cells, and there are several indications supporting the notion that CD38 plays significant roles in the progression of MM.We have fine-tuned the production of anti-CD38 chitosan NPs with a small size (50nm) to allow better penetration to tumors, and we showed that these NPs were stable at different storage conditions and periods. The chitosan NPs showed preferential drug release in MM tumor microenvironment compared to normal tissue microenvironment, and this release was pH dependent.The uptake of the anti-CD38 chitosan NPs to MM cells (cell lines and primary) was significantly higher than their uptake in normal cells (4-fold of BM MNCs, and 10-fold of PB MNCs) demonstrating that the anti-CD38 chitosan NPs are selective and specific to MM. BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest and apoptosis leading to cell death through inactivation of proliferative cell signaling pathways (MAPK, PI3K) and activation of pro-apoptotic pathways (PARP and Caspase-3). We also found that BTZ-loaded anti-CD38 NPs proved to be more efficacious than free BTZ and overcame CAM-DR and hypoxia-mediated drug resistance. The use of a 3DTEBM model, which recreate MM pathophysiology, corroborated the increase cytotoxicity of anti-CD38 chitosan NPs on MM cells relative to free drug and allowed to corroborated co-localization of the NPs in the MM cells.We found that BTZ-loaded NPs were significantly more potent in the proteasome activity inhibition than free BTZ and correlated with the boron content in MM cells treated with BTZ-loaded anti-CD38 NPs, which was 3-fold higher than in MM cells treated with BTZ as free drug. To confirm that the process involved in the internalization of the anti-CD38 chitosan NPs was endocytosis, we have shown that the NPs distribution in the cells co-localized with the distribution of endosomes and found that the anti-CD38 NPs were uptaken through clathrin-mediated and caveolae-mediated early endocytosis which progressed to late endocytosis and further delivery of the NPs content into the cells. Finally, we linked the internalization through endocytic pathways of anti-CD38 NPs to the enhanced proteasome activity inhibition of these NPs in MM cells. Treatment of MM cells with clathrin and caveolae-mediated early endocytosis, or late endocytosis inhibitors (but not macropinocytosis inhibitors) significantly reduced the proteasome inhibition of BTZ-loaded anti-CD38 chitosan NPs. When BTZ is administered as a free drug, it penetrated through passive diffusion into MM cells as well as in normal cells, and inhibition of the different endocytosis routes did not induce any change in its ability to inhibit the proteasome.In vivo studies showed that treatment with BTZ-loaded anti-CD38 targeted NPs (1mg/kg once a week) showed a markedly improved tumor growth inhibition, improved overall survival, and reduction of side effects compared to non-targeted NPs and free drug.In conclusion, anti-CD38 chitosan NPs showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through endocytosis, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. We are the first to report the enhancement of the efficacy and specificity of proteasome inhibitors due to endocytosis-driven delivery of NPs. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Glycomimetics: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cellatrix LLC: Other: Founder and owner; Verastem: Research Funding; Cell Works: Research Funding; Abbvie: Research Funding; Tioma: Research Funding; Cleave Bioscience: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Transforming growth factor-β boosts the functionality of human bone marrow-derived mesenchymal stromal cells.

Transforming growth factor β1 (TGFβ1) is a negative regulator of hematopoiesis, and yet, it is frequently found at the active sites of hematopoiesis. Here, we show for the first time that bone marrow-derived mononuclear cells (BM MNCs) secrete TGFβ1 in response to erythropoietin (EPO). We further show that human bone marrow-derived mesenchymal stromal cells (BMSCs) briefly exposed to the conditioned medium of EPO-primed MNCs, or purified TGFβ1, gain significantly increased hematopoiesis-supportive ability. Mechanistically, we show that this phenomenon involves TGFβ1-mediated activation of nitric oxide (NO) signalling pathway in the BMSCs. The data suggest that EPO-MNC-TGFβ1 could be one of the regulatory axes operative in the bone marrow microenvironment involved in maintaining the functionality of the resident BMSCs.

Induction of apoptosis and differentiation by Na/H exchanger 1 modulation in acute myeloid leukemia cells

We investigated the effect of the modulation of Na/H exchanger 1 (NHE1) on apoptosis, differentiation, and chemoresistance in acute myeloid leukemia (AML) cells to evaluate the possibility of NHE1 modulation as a novel therapeutic strategy for AML. The pHi of leukemia cell lines except KG1a was higher than that of normal bone marrow mononuclear cells (BM MNCs). Notably, in K562, cytarabine (AraC)-resistant OCI-AML2, and primary leukemia cells, pHi was significantly higher than that of normal BM MNCs. Western blotting and real-time quantitative PCR confirmed that the increased NHE1 expression was responsible for the higher pHi. Specifically, compared to CD34+CD38+ leukemia cells, the mean fluorescence intensity of NHE1 was significantly higher in CD34+CD38− leukemic stem cells. The out of range in pHi by treatment with an NHE inhibitor, the amiloride analogue 5-(N,N-hexamethylene) amiloride (HMA), or an NHE activator, phorbol 12-myristate 13-acetate (PMA), resulted in dose- and time-dependent inhibition of leukemia cell proliferation. PMA induced CD14+ differentiation of leukemia cells, whereas HMA induced cell cycle arrest at the G1 phase. HMA could induce apoptosis of leukemia cells even in AraC-resistant cells and showed an additive effect on apoptosis in AraC-sensitive cells. Our result revealed that AML cells prefer more alkalic intracellular moiety than normal BM MNCs following increased NHE1 expression and that NHE1 modulation can induce apoptosis and differentiation of AML cells. These findings imply that NHE1 is a potential target in cytotoxic or differentiation-induction treatment for AML.

The effects of bone marrow mononuclear cell transplantation on the quality of life of children with cerebral palsy

BackgroundQuality of life (QOL) is an important factor in evaluating the effectiveness of treatment in children with cerebral palsy (CP). The aim of this study was to evaluate the effects of autologous bone marrow mononuclear cells (BM MNCs) on the QOL of children with CP.MethodsFrom December 2015 to December 2016, 30 children with CP aged from 2 to 15 years received two intrathecal infusions of BM MNCs, one at baseline and the other 3 months later, at Vinmec International Hospital. The motor function and muscle tone of the patients were evaluated using the Gross Motor Function Measure (GMFM)-88 and Modified Ashworth Score, respectively. Their QOL was assessed at baseline and 6 months after the first BM MNC transplant using the Vietnamese version of the Cerebral Palsy Quality of Life Questionnaire for children (CP QOL-Child)–the parental proxy report, which comprises seven domains. Nineteen mothers (mean age: 32.9±4.9 years) and 11 fathers (mean age: 36.1±6.8 years) were invited to complete the CP QOL-Child assessment before and after the transplantations, Paired t-tests and multivariate regression analyses were used to evaluate the changes in QOL and GMFM scores and to identify the key factors correlated with the QOL score.ResultsSignificant changes were observed in the children’s gross motor function and muscle spasticity, as evidenced by the GMFM-88 total score, scores for each of its domains, the GMFM-66 percentile and the muscle tone (P < 0.001). Six months after the transplantations, the QOL scores of children with CP were markedly increased (P < 0.001) for all the domains, except for the domain of access to services. In the multivariate regression analysis, significant associations were found between higher age of children and higher QOL except for feeling about functioning and pain and impact of disability domains. Gross Motor Function Classification System (GMFCS) level was negatively correlated with the score of pain and impact of disability domain, while the GMFM-88 scores were positively correlated with the QOL in terms of feelings about functioning and family health domain (P < 0.05).ConclusionThe QOL of the children with CP was noticeably improved 6 months after BM MNC transplantation and was accompanied by improvements in gross motor function and muscle tone.Trial registrationClinicalTrials.gov Identifier: NCT02574923. Registered on October 14, 2015.

Abstract 1892: Discovery of targeted therapies for acute myeloid leukemia patients with NUP98-NSD1 and FLT3-ITD

Abstract NUP98-NSD1 fusion resulting from the t(5;11)(q35;p15.5) is a clinically significant indicator of adverse prognosis in AML. While the understanding of NUP98-NSD1-driven leukaemogenesis has increased over the years, these patients still suffer from highly progressive disease with no effective therapies. To address this challenge, we analyzed primary patient samples and experimental cell models by high-throughput drug screening and RNA sequencing. Primary samples included bone marrow mononuclear cells (BM MNCs) from three NUP98-NSD1/FLT3-ITD AML patients and ten healthy donors. Experimental cells included lineage-depleted balb/c BM cells transduced with chimeric NUP98-NSD1 and FLT3-ITD retroviruses alone or together, and Ba/F3 cells transduced with human-cloned NUP98-NSD1 lentivirus. The cells were seeded on 384-well plates containing up to 306 approved and investigational drugs in five concentrations over a 10,000-fold range and incubated for 72h at +37°C. Cell viabilities were measured using CellTiter-Glo®. Drug sensitivity scores (DSS) were calculated from area under the dose response curve and select DSS by subtracting DSS of mock-transduced parental cells from the experimental model. We discovered 14 drugs with highly significant mean DSS difference (p &amp;lt; 0.001) between patients and healthy controls. Further analysis revealed that only two of these drugs (dasatinib and navitoclax) had positive correlation between DSS and BM blast percentage (Pearson's R: 0.83-0.98) as well as higher mean DSS in the context of other AML patients with (n = 9) and without (n = 38) FLT3-ITD. Experimental balb/c cells expressing NUP98-NSD1 had significantly increased sensitivity to BCL2 inhibitors (n = 3) (p &amp;lt; 0.05), while cells co-expressing NUP98-NSD1 and FLT3-ITD had significantly increased sensitivity to FLT3 (n = 11) and MEK inhibitors (n = 6) in comparison to cells expressing FLT3-ITD alone. In pilot combination screen, select efficacy of FLT3 and MEK inhibitors was further increased in the dual positive cells by dasatinib (100 nM). In synergy screens, navitoclax-dasatinib and navitoclax-quizartinib combinations were highly synergistic in primary cells and balb/c cells co-expressing NUP98-NSD1 and FLT3-ITD. Gene expression analysis showed significant up-regulation of dasatinib target genes LCK and FGR, and navitoclax target gene BCL2A1 in the primary AML cells. Interestingly, BCL2A1 was also upregulated in the balb/c BM cells expressing NUP98-NSD1 alone or with FLT3-ITD, but not in cells expressing FLT3-ITD alone. Taken together, we have identified potential candidate drugs and drug combinations for t(5;11) positive AML. Although the sample size was small, our data suggests that further research into BCL2 inhibitors in combination with TKIs may eventually translate to better survival outlook of AML patients with t(5;11). Citation Format: Jarno Kivioja, Angeliki Thanasopoulou, Mika Kontro, Ashwini Kumar, Bhagwan Yadav, Muntasir Mamun Majumder, Kimmo Porkka, Juerg Schwaller, Caroline A. Heckman. Discovery of targeted therapies for acute myeloid leukemia patients with NUP98-NSD1 and FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1892.

Response of human normal and leukemia cells to factors released by amnion fragments in vitro.

Amnion is a membrane surrounding the embryo/fetus which determine growth factors and interleukins with angiogenic, immunogenic, and anti-inflammatory properties. The aim of the present study was to investigate the effects of conditioned culture medium from 24-h cultures of human amnion (hAM CCM) on migration and proliferation of human umbilical vein endothelial primary cells (HUVECs), freshly isolated bone marrow mononuclear cells (BM MNCs), and Jurkat leukemia cell line. Amnion membrane was freshly isolated from healthy placenta and its fragments cultured in vitro to produce hAM CCM. Members of the IGFBP protein family made up one third of all assayed proteins present in the hAM medium. The hAM CCM did not affect the proliferation rate of HUVECs or MNCs, but we observed more intensive migration of those cells, and lower expression of CD31 surface antigen on HUVECs as compared to control cultures. In contrast, Jurkat cells did not respond to hAM CCM treatment by proliferation or mobility change. The conditioned medium from 24-h cultures of human amnion is easy to obtain and is a convenient source of various growth and other factors that may be useful in practical medicine.

Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia.Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells.We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein.These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. DisclosuresLevis:Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Integrin-Linked Kinase a Novel Therapeutic Target for Acute and Chronic Myeloid Leukemia

Introduction:Current treatment options as well as clinical efficacy are limited for acute myeloid leukemia (AML), Ph+ acute lymphoblastic leukemia (Ph+ ALL), and chronic myelogenous leukemia (CML). In response to the pressing need for more efficacious treatment approaches and strategies to override drug resistance in advanced stage AML, Ph+ ALL, and CML, we investigated the effects of inhibition of integrin-linked kinase (ILK) as a potentially novel and effective approach to treatment of these challenging malignancies. ILK is an intracellular adaptor and kinase that links the integrins, cell-adhesion receptors, and growth factors to a range of signaling pathways. It has been shown that inhibition of ILK expression and activity is anti-tumorigenic, which makes ILK an attractive target for cancer therapeutics. Compound 22 (Cpd22, N-Methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide) is a cell-permeable, tri-substituted pyrazol compound that acts as a potent and targeted ILK inhibitor. In the present study, we investigated ILK as a putative novel target for treatment of AML, Ph+ ALL, and CML, and Cpd22 as a potential novel anti-leukemia agent.Methods:Compound 22 (Cpd22) was purchased from EMD Millipore. The expression of ILK in AML (K052, NOMO-1, THP, MOLM14), CML (K562, Ku812F, LAMA84S and LAMA84R), and Ph+ ALL (SUP-B15) cell lines, primary patient cells, and human Bone Marrow Mononuclear Cells (BM MNCs) was analyzed by flow cytometry. AML, Ph+ ALL, CML cell lines and peripheral blood mononuclear cells (PBMCs) were cultured with ILK inhibitor, Cpd22 (0-1000 nM) for 3 days. Toxicity of Cdp22 (0-500 nM) toward BM MNCs and primary patient cells (CML, ALL, and AML) was also assessed. Cell lines and/or primary patient cells were analyzed for cell proliferation by MTT assay; cell cycle by DNA staining with PI and analyzed by flow cytometry; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with proliferation, cell cycle, and apoptosis was analyzed by western blotting. In addition, ILK knockdown of AML and CML cell lines was evaluated.Results:Normal BM MNCs showed ILK expression, but expression was lower than in cell lines and primary samples from AML, CML, and ALL. Cpd22 inhibited the proliferation of AML, ALL, and CML cell lines, while none of normal PBMC and normal BM MNCs controls were affected by the same concentration range after three days of treatment with Cpd22 as a single agent. ILK expression in primary AML patient cells correlated with efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. ILK expression in primary ALL patient cells also correlated with the efficacy of Cpd22: Specifically, the higher the ILK expression, the more sensitivity to Cpd22. We validated the target specificity of Cpd22 in CML cells by immuno-blotting and investigating inhibitor effects on signaling molecules downstream of ILK. Cpd22 potently suppressed the phosphorylation levels of Ser-473-Akt (pAKT) and another ILK substrate, pGSK-3.We observed cell cycle arrest in Cpd22-treated cells, specifically accumulation of apoptotic cells in subG1 and Annexin/PI staining showed a 3-fold increase in the fraction of apoptotic cells staining positive for Annexin and PI in Cpd22-treated cells. Immuno-blotting confirmed cell cycle arrest by decreased pRb and increased cell cycle inhibitor p27; and drug induction of apoptosis through the caspase intrinsic pathway by demonstrating increased cleavage of caspase-3 and caspase-9.The importance of ILK for the growth of leukemia cells was demonstrated by knockdown of ILK in AML and CML cell lines, which led to decreases in cell proliferation. These results suggest a partial dependency of acute and chronic myeloid leukemia cells on ILK for growth, and are consistent with results obtained with the ILK inhibitor, Cpd22.Conclusions:In conclusion, our results suggest that the ILK inhibition may be an effective treatment for AML, Ph+ ALL, and CML as a single therapy, with ILK expression levels positively correlating with the efficacy of ILK inhibition. The identification of ILK as a novel target for leukemia therapy warrants further investigation as a therapeutic approach that could be of potential clinical benefit in both acute and chronic myeloid leukemias. DisclosuresAzab:Verastem: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner; Cell Works: Research Funding.

A critical role of Src family kinase in SDF-1/CXCR4-mediated bone-marrow progenitor cell recruitment to the ischemic heart

The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP+ MNCs and eGFP+c-kit+ PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP+ and eGFP+c-kit+ cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment.

Impact of age on the efficacy of bone marrow mononuclear cell transplantation in experimental stroke

Bone marrow-derived mononuclear cells (BM MNC) have been effectively used to treat experimental stroke. Most of the preclinical trials have been performed in young and healthy laboratory animals, even though age and hypertension are major risk factors for stroke. To determine the influence of age on the properties of BM MNCs after cerebral ischemia, we compared the efficacy of aged and young BM MNC in an in vitro model of cerebral hypoxia and in an adapted in vivo model of stroke. Human BM MNCs were obtained from healthy young or aged donors and either co-cultured with rat hippocampal slices exposed to oxygen glucose deprivation (OGD), or transplanted intravenously 24 h after permanent middle cerebral artery occlusion in aged (18 months) spontaneously hypertensive rats (SHR). Efficacy was examined by quantification of hippocampal cell death, or respectively, by neurofunctional tests and MR investigations. Co-cultivation with young, but not with aged BM MNCs significantly reduced the hippocampal cell death after OGD. Transplantation of both young and old BM MNCs did not reduce functional deficits or ischemic lesion volume after stroke in aged SHR. These results suggest a significant impact of age on the therapeutic efficacy of BM MNCs after cerebral ischemia.

Phosphoprotein Profiling Predicts Response to Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukemia Patients

Abstract 4427Imatinib and other tyrosine kinase inhibitors (TKIs) are the standard first-line treatment for patients with chronic myeloid leukemia (CML) and have dramatically improved disease outcome. However, a proportion of patients fails to achieve optimal molecular response (major molecular response, MMR, or better) to TKI treatment and has a marked residual disease even after years of therapy start. The reasons for suboptimal imatinib response are unclear and these patients might benefit from treatment with more potent and broad spectrum 2nd generation TKIs (dasatinib and nilotinib) already from the diagnosis to prevent disease progression. By using a phosphoproteomic approach, we aimed to identify predictive markers for imatinib failure or suboptimal response, which could be used to guide treatment selection already at the time of diagnosis.The study consisted of 9 CML patients in chronic phase with optimal and 10 patients with suboptimal response to imatinib. Patients with suboptimal response had complete cytogenetic response, but no MMR after 18 months of therapy, whereas all patients with optimal response were at least in MMR at 18 months. 7 of 19 patients belonged to intermediate Sokal risk group (5/10 in suboptimal and 2/9 in optimal group) and 12 patients to low Sokal risk group. One additional failure patient without any cytogenetic response to any of the available TKIs was also analyzed (patient had M351T imatinib resistant mutation). Bone marrow mononuclear cells (BM MNC) were collected at the time of diagnosis prior to any TKI therapy and were analyzed for 46 kinase phosphorylation sites central in the pathogenesis of CML (R&D Human Phospho-Kinase Array Kit). BM MNCs from 3 healthy controls and the K562 Ph+ cell line were used as controls. Quantitative analyses of the phosphoproteins were performed by measurement of array spot densities with CellProfiler2.0.In K562 cell line, the protein phosphorylation was most prominent in pSTAT5b Y699 and only modest in pSTAT5a Y694. In diagnostic BM samples, pSTAT5b and pSTAT5a levels showed marked variability within individual patients. Src Y419 (P<0.01), c-Jun S63 (P<0.01), Yes Y426 (P<0.05), STAT3 Y705 (P<0.05), and AMPKα2 T172 (P<0.05) phosphorylation levels were significantly higher in CML patients compared to healthy controls.Patient with the M351T mutation without any cytogenetic response to any TKI revealed highly active state of HSP27 S78/S82, which was not detected in any other patient or in healthy control, revealing a candidate therapy target for this individual patientWhen median phosphorylation ratios of suboptimal vs. optimal patients' diagnostic samples were compared, STAT5b Y699 (ratio 2.3), PLCγ-1 Y783 (ratio 1.9), STAT5a Y694 (ratio 1.6) and Pyk2 Y402 (ratio 1.6) levels were increased in suboptimal group. Patients in intermediate Sokal risk group had significantly higher phosphorylation levels of ERK1/2 T202/Y204 T185/Y187 (P<0.01), CREB S133 (P<0.05), Akt S473 (P<0.05), JNK T183/Y185 T221/Y223 (P<0.05) and Lck Y394 (P<0.05).In conclusion, chronic phase CML patients had a distinct phosphoprotein pattern in BM, which differed from healthy controls and from the K562 cell line. Our phospohoproteomic approach revealed the importance of novel signaling pathways not previously discovered in CML such as the anti-apoptic HSP27 pathway. Furthermore, phosphoprotein pattern of suboptimally responding patients differed already at the time of diagnosis from optimally responding patients. In particular, high levels of phosphorylated STAT5b may herald a suboptimal imatinib response and warrant choosing a 2nd generation TKI as the first-line of therapy. Disclosures:Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Cilostazol enhances integrin‐dependent homing of progenitor cells by activation of camp‐dependent protein kinase in synergy with Epac1

Recruitment and adhesion of exogenous endothelial progenitor cells (EPCs) or endogenously mobilized bone marrow mononuclear cells (BM MNCs) to the sites of ischemia is an important focus of cell therapy. This study sought to determine whether cilostazol enhances integrin-dependent homing of progenitor cells both in vitro and in vivo. In the in vitro experiments with human umbilical cord blood (HUCB)-derived EPCs, cilostazol (10 μM) stimulated up-regulation of integrins β1, α1, and αv as well as 8-pCPT-2'-O-Me-cAMP (100 μM; 8-pCPT, Epac activator). Cilostazol and 8-pCPT significantly enhanced migration and adhesion of HUCB EPCs to a fibronectin-coated plate and endothelial cells, which were inhibited by KT5720 (PKA inhibitor, 1 μM) and GGTI-298 (Rap1 inhibitor, 20 μM). Cilostazol stimulated Epac1 expression and up-regulated the active Rap1, as did 8-pCPT, and they were suppressed by KT5720 (P < 0.001) and GGTI-298 (P < 0.001). 8-pCPT increased p-CREB expression and stimulated PKA activity, which was inhibited by KT5720, Rp-cAMPS, and GGTI-298. In addition, N(6)-benzoyl-cAMP (100 μM) increased Rap1 GTP expression, as did 8-pCPT; they were suppressed by Rp-cAMPS and GGTI-298. The in vivo experiments showed that cilostazol (30 mg/kg/day, orally for 7 days) significantly enhanced the integrin β1 expression in the molecular layer and up-regulated homing of BM MNCs to the injured molecular layer with increased capillary density in mouse brain subjected to transient forebrain ischemia (n = 6, P < 0.001). In conclusion, cilostazol stimulated integrin expression and enhanced migration and adhesion of progenitor cells through cooperative activation of PKA and Epac signals; such activity may improve the efficacy of cell therapy for ischemic disease.

The Vent-Like Homeobox Gene VENTX Promotes Human Myeloid Development and Is Highly Expressed In Acute Myeloid Leukemia

AbstractAbstract 739Recent studies suggest that a variety of regulatory molecules active in embryonic development such as clustered and non-clustered homeobox genes play an important role in normal and malignant hematopoiesis. Since it was shown that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, it is of particular interest to examine the expression profile and function of its only recently discovered human homologue VENTX in hematopoietic development. Expression of the VENTX gene was analyzed in normal human hematopoiesis and AML patients samples by microarray and qPCR. To test the impact of the constitutive expression of VENTX on human progenitor cells, CD34+ cord blood (CB) cells were retrovirally transduced with VENTX or the empty control vector and analyzed using in vitro and in vivo assays. So far we and others have not been able to identify a murine Xenopus xvent gene homologue. However, we were able to document the expression of this gene by qPCR in human lineage positive hematopoietic subpopulations. Amongst committed progenitors VENTX was significantly 13-fold higher expressed in CD33+ BM myeloid cells (4/4 positive) compared to CD19+ BM lymphoid cells (5/7 positive, p=0.01). Of note, expression of VENTX was negligible in normal CD34+/CD38− but detectable in CD34+ BM human progenitor cells. In contrast to this, leukemic CD34+/CD38− from AML patients (n=3) with translocation t(8,21) showed significantly elevated expression levels compared to normal CD34+ BM cells (n=5) (50-fold higher; p≤0.0001). Furthermore, patients with normal karyotype NPM1c+/FLT3-LM− (n=9), NPM1c−/FLT3-LM+ (n=8) or patients with t(8;21) (n=9) had an >100-fold higher expression of VENTX compared to normal CD34+ BM cells and a 5- to 7.8-fold higher expression compared to BM MNCs. Importantly, lentivirus-mediated long-term silencing of VENTX in human AML cell lines (mRNA knockdown between 58% and 75%) led to a significant, reduction in cell number compared to the non-silencing control construct (>79% after 120h). Suggesting that growth of human leukemic cell lines depends on VENTX expression in vitro. As we observed that VENTX is aberrantly expressed in leukemic CD34+ cells with negligible expression in normal counterparts, we assessed the impact of forced VENTX gene expression in normal CD34+ human progenitor cells on the transcription program. Gene expression and pathway analysis demonstrated that in normal CD34+ cells enforced expression of VENTX initiates genes associated with myeloid development (CD11b, CD125, CD9,CD14 and M-CSF), and downregulates genes involved in early lymphoid development (IL-7, IL-9R, LEF1/TCF and C-JUN) and erythroid development such as EPOR, CD35 and CD36. We then tested whether enforced expression of VENTX in CD34+ cells is able to alter the hematopoietic development of early human progenitors as indicated by gene expression and pathway analyses. Functional analyses confirmed that aberrant expression of VENTX in normal CD34+ human progenitor cells induced a significant increase in the number of myeloid colonies compared to the GFP control with 48 ± 6.5 compared to 28.9 ± 4.8 CFU-G per 1000 initially plated CD34+ cells (n=11; p=0.03) and complete block in erythroid colony formation with an 81% reduction of the number of BFU-E compared to the control (n=11; p<0.003). In a feeder dependent co-culture system, VENTX impaired the development of B-lymphoid cells. In the NOD/SCID xenograft model, VENTX expression in CD34+ CB cells promoted generation of myeloid cells with an over 5-fold and 2.5-fold increase in the proportion of human CD15+ and CD33+ primitive myeloid cells compared to the GFP control (n=5, p=0.01). Summary: Overexpression of VENTX perturbs normal hematopoietic development, promotes generation of myeloid cells and impairs generation of lymphoid cells in vitro and in vivo. Whereas VENTX depletion in human AML cell lines impaired their growth.Taken together, these data extend our insights into the function of human embryonic mesodermal factors in human hematopoiesis and indicate a role of VENTX in normal and malignant myelopoiesis. Disclosures:No relevant conflicts of interest to declare.

E0194 Src family kinase SFK is essential for recruitment of bonemarrow progenitor cells to the ischaemic myocardium

BackgroundThe G protein-coupled receptor CXCR4 and its ligand stromal–cell derived factor 1 (SDF-1) play an important role in directing progenitor cells (PC) homing to ischaemic tissue. The Src family protein...

Generation of Induced Pluripotent Stem Cells by Efficient Reprogramming of Adult Bone Marrow Cells

Reprogramming of somatic cells provides potential for the generation of specific cell types, which could be a key step in the study and treatment of human diseases. In vitro reprogramming of somatic cells into a pluripotent embryonic stem (ES) cell-like state has been reported by retroviral transduction of murine fibroblasts using four embryonic transcription factors or through cell fusion of somatic and pluripotent stem cells. Here we show that mouse adult bone marrow mononuclear cells (BM MNCs) are competent as donor cells and can be reprogrammed into pluripotent ES cell-like cells. We isolated BM MNCs and mouse embryonic fibroblasts (MEFs) from Oct4-GFP transgenic mice, fused them with ES cells, or infected them with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc. Fused BM MNCs formed more ES-like colonies than did MEFs. Infected BM MNCs gave rise to induced pluripotent stem (iPS) cells, although transduction efficiencies were not high. It was more efficient to pick up iPS colonies as compared with MEFs. BM-derived iPS (BM iPS) cells expressed ES cell markers, formed teratomas, and contributed to chimera mice with germ line development. Clonal analysis revealed that BM iPS clones had diversity, although some clones were found to be genetically identical with different phenotypes. Our findings imply that BM MNCs have potential advantages to generate iPS cells for the clinical application.

Cell Lines and “Knock-in” Mice Bearing FLT3/ITD Mutations Exhibit Elevated Repair Errors Generated through Alternative DNA Double Strand Break Repair Pathways: Implications for Genomic Instability.

Abstract 3237Poster Board III-174Activating mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor occur in approximately 30% of acute myeloid leukemia (AML) patients and, at least for internal tandem duplication (ITD) mutations, are associated with poor prognosis. We previously reported that FLT3/ITD mutations are associated with increased reactive oxygen species (ROS) production which leads to increased DNA double strand breaks (DSBs) and decreased repair fidelity, that may in part explain aggressive AML in FLT3/ITD patients. While the DNA-PK and Ku-dependent non homologous end-joining (NHEJ) pathway is one of the main routes for repairing DSBs in normal mammalian cells, recent reports have suggested that an alternative and less well defined NHEJ pathway (Alt NHEJ) involving DNA ligase IIIαa and PARP1, characterized by DNA sequence microhomology at the repair junctions, may play a role in the generation of deletions and translocations that can lead to cancer progression. Here, we report that in hematopoetic progenitor cells expressing FLT3/ITD (Ba/F3/ITD), the frequency of errors and the size of deletions during DSB repair is increased compared with the Ba/F3 control cells. Moreover, these data are confirmed in bone marrow mononuclear cells (BM MNCs) from FLT3/ITD “knock-in” mice, compared with WT control cells. Strikingly, we show that Ku proteins (Ku70 and Ku86), key components of the main NHEJ pathway, are down-regulated in FLT3/ITD-positive cell line and primary mouse BM MNCs. Concomitantly, DNA ligase IIIαa, a component of Alt NHEJ pathway is up-regulated in FLT3/ITD-expressing cells and plays a role in abnormal DSB repair. Importantly, after treatment with a FLT3 inhibitor, AML (MOLM-14, MV4-11) cells containing FLT3/ITD mutations demonstrate a reduction of misrepair and deletion size, and down-regulation of DNA ligase IIIαa, suggesting that FLT3 signaling regulates the pathways by which DSBs are repaired. Thus, therapy to inhibit FLT3/ITD may lead to repair that reduces repair errors and genomic instability. DisclosuresNo relevant conflicts of interest to declare.

Correction of Fanconi Anemia Group A in Primary Human and Murine Hematopoietic Progenitors with a Clinical Lentiviral Vector

Fanconi Anemia (FA) is a rare genetic disorder of DNA repair that typically manifests with bone marrow failure. Allogeneic stem cell transplant is the only cure known for the bone marrow disorder. Unfortunately, many patients do not have matched family donors, and alternative donor transplant has been associated with considerable morbidity and mortality. For individuals without an appropriate donor, ex vivo genetic modification of autologous stem cells is a potential therapeutic strategy. However, one of the major hurdles in gene therapy for this condition is the increased sensitivity of FA stem cells to free-radical induced DNA damage during ex vivo culture and manipulation. To minimize this damage we have developed a brief transduction procedure for lentivirus vector mediated gene transfer for Fanconi Anemia complementation group A (FancA), and evaluated this method in bone marrow progenitors from patients with FA, and murine progenitors from FancA deficient mice. The lentiviral vector, RSCPFancA-sW (FancA-sW), was specifically developed for clinical studies, and has a synthetic wpre which has been modified for safety (does not express a partial woodchuck hepatitis virus X protein open reading frame) and most of the 3′ untranslated region of the FancA gene was removed. FancA −/− mice were preconditioned with cyclophosphamide (120 mg/kg) prior to injection of transduced syngeneic bone marrow. After two subsequent rounds of the same dose of cyclophosphamide, 3/5 mice demonstrated mitomycin C (MMC) resistance in cells from bone marrow or spleen via colony assay. Two mice had peripheral blood marking with the vector seen by quantitative PCR. In an initial mouse experiment using similar conditions, the estimated transduction efficiency by colony PCR was determined to be 31% with the average number of integrations per cell determined to be 1.8 by LAM-PCR. Bone marrow mononuclear cells (BM MNCs) from a patient with FancA were also transduced with FancA-sW. Very few colonies were obtained on methylcellulose, although cell clusters were observed in MMC-treated cultures derived from FancA-sW transduced BM MNCs, while no clusters or colonies formed from cells transduced with the control vector. In an attempt to increase progenitor survival by reducing oxidative stress, primary human FA bone marrow was transduced and placed in 5% oxygen in the presence of 1 nM NAC (N-Acetyl-L-Cysteine). Cells plated in methylcellulose under these conditions had 2–3 fold increased colony formation compared to conditions without NAC or in 21% oxygen (P < 0.03). FancA-sW transduced BM MNCs from a second FancA patient were plated under these conditions formed colonies and numerous cell clusters and demonstrated resistance to MMC compared to GFP transduced controls (P < 0.007). Furthermore, transduced BM MNCs in culture had increased survival in culture when exposed to MMC ( P < 0.004). In summary, a lentiviral vector with a functional FancA transgene was developed which achieves phenotypic correction of human and murine hematopoietic progenitors with the brief period of prestimulation and transduction that would be required to maintain survival of FA cells ex vivo in culture. Reduction of oxidative stress may enhance the viability and engraftment of fragile FA stem cells.

Safety of MGMT Gene Transfer into Hematopoietic Progenitors: A Phase I Clinical Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG Plus Alkylating Agent Treatment of Advanced Malignancies.

The G156A mutation of the DNA repair gene O6-methylguanine DNA-methyltransferase (MGMT) confers hematopoietic cell resistance to O6-benzylguanine (BG) and the DNA-alkylating agents BCNU and temozolomide (TMZ). We conducted a Phase I clinical trial utilizing MGMT-mediated chemoprotection to improve treatment of advanced solid tumors. Here we report the safety and feasibility of this approach. Autologous CD34+ cells were transduced ex vivo with a retroviral vector MFG- G156A MGMT (K. Cornetta, NGVL at Indiana U.), then reinfused into non-myeloablated patients. Eight patients were accrued to the study and 6 patients were infused with a mean of 7.3 × 105 cells/kg obtained post transduction. Of the 6 patients who received cells, all received one cycle of chemotherapy (BG + BCNU or TMZ) 3 days prior to cell infusion and 3 patients received a second cycle of either BG + BCNU or BG + TMZ 4–6 weeks post infusion. No patient experienced grade 2 or greater infusion related toxicity, and one patient went off study prior to receiving cells due to neurological toxicity during cytokine administration. All patients who received G156A MGMT+ cells went off study due to progressive disease, and survival ranged from 2 weeks to 9 months post infusion. No patient showed evidence of replication competent retrovirus (RCR) when assessed for the presence of the viral envelope by quantitative PCR for GALV sequences (performed by the IU Vector Production Facility). The mean vector copy number in post transduction cells (n= 7 patients) was 0.29 copies per genome by quantitative PCR (Q-PCR), with a mean CFU transduction efficiency of 24%, and a mean of 9% CD34 cells expressing the G156A MGMT transgene by flow cytometry. Retroviral insertion sites in pre-infusion CFUs from 2 patients analyzed by linear amplification-mediated (LAM)-PCR were polyclonal, with an average of 1.4 retroviral insertions per positive CFU. Transgene positive cells were detected in vivo in 3 patients. In the first patient, after BG and BCNU at 6 weeks, the transgene was detected in 1 of 100 BM CFU by Q-PCR and one insertion site was detected in this clone by LAM-PCR. The patient went off study at 8 weeks, then received TMZ alone (300 mg/m2/day for 5 days). At 16 weeks, the transgene was detected at low frequency in BM MNCs and granulocytes by Q-PCR and in BM granulocytes as an internal band in LAM-PCR, although no other insertions sites were detectable. In a second patient, the transgene was detected in 2 of 30 BM CFU by PCR and in BM granulocytes and MNCs by LAM PCR at 5 weeks. In a third patient treated with BG + TMZ 4 weeks post cell infusion, 3 of 50 PB CFU were transgene positive by Q-PCR at week 28. These data demonstrate that infusion of retroviral MGMT transduced hematopoietic cells is safe and are the first to show emergence and low level persistence of transduced mutant G156A MGMT cells after nonmyeloablative conditioning in humans. Our future studies include using a lentiviral vector to achieve better levels of gene transfer and reduced risk of insertional mutagenesis.