ALBERT

Description: The role of Gas6 in endothelial cell (EC) function remains incompletely characterized. Here we report that Gas6 amplifies EC activation in response to inflammatory stimuli in vitro. In vivo, Gas6 promotes and accelerates the sequestration of circulating platelets and leukocytes on activated endothelium as well as the formation and endothelial sequestration of circulating platelet-leukocyte conjugates. In addition, Gas6 promotes leukocyte extravasation, inflammation, and thrombosis in mouse models of inflammation (endotoxinemia, vasculitis, heart transplantation). Thus, Gas6 amplifies EC activation, thereby playing a key role in enhancing the interactions between ECs, platelets, and leukocytes during inflammation.

Description: In vivo selection is a key strategy for increasing contribution of gene-modified cells in hematopoietic stem cell gene therapy. The combination of DNA-alkylating agents such as temozolomide (TMZ) with O6-benzylguanine (BG) allows selection for cells expressing a drug-resistant O6-methylguanine-DNA-methyltransferase (MGMT) gene, resulting in hematopoietic repopulation with gene-modified cells in numerous mouse studies. TMZ is used clinically for treatment of glioma and other malignancies, with dose-limiting myelotoxicity, and has been used with BG in a phase I trial of drug-resistant MGMT gene transfer for chemoprotection in treatment of advanced solid malignancies. We report here that in transplants of lymphoma-prone LMO1 transgenic or retrovirus-transduced normal mouse marrow to study the effect of in vivo selection on insertional mutagenesis by retrovirus vectors in gene therapy, administration of BG+TMZ post-transplant increased the incidence of donor-derived T-cell lymphomas. In transplants of LMO1 transgenic mouse lineage-negative (Lin−) marrow progenitor cells, BG+TMZ significantly increased (p=0.0026) the incidence of lymphoma (87.5% incidence in mice surviving drug-induced hematologic toxicity; n=8) compared to mice without drug (41% incidence by day 315 post-transplant; n=17). Increased tumor incidence after BG+TMZ was observed both in transplants of untransduced LMO1 Lin− cells and in LMO1 cells transduced with an MFG gamma-retroviral vector encoding a drug-resistant P140K MGMT gene and a GFP reporter gene. Additionally, in transplants of normal C57BL/6 mouse Lin− cells transduced with the P140K-GFP retroviral vector, BG+TMZ significantly increased (p=0.0043) the tumor incidence (100% incidence by day 169 post-transplant in mice surviving drug toxicity; n=6) compared to mice without drug (0% incidence by day 315 post-transplant; n=9). This is a higher frequency of lymphomas than we have previous observed in numerous selection studies with MGMT transduction of normal mouse whole marrow. Administration of BG+TMZ following transplant of transduced C57BL/6 mouse cells resulted in an approximately 2-fold increase in vector-expressing cells in peripheral blood, compared to an approximately 57% decline in untreated mice, demonstrating in vivo selection of MGMT-expressing cells. However, in all cases of LMO1 or C57BL/6-derived lymphomas, the tumor cells were found to lack expression of the P140K-GFP vector, suggesting that cells expressing drug-resistant MGMT were protected from the mutagenesis caused by BG+TMZ, as well as from its hematologic toxicity. Despite the lack of transgene expression, retroviral vector DNA was detected by LAM-PCR in some of the LMO1-derived and all of the C57BL/6-derived lymphomas, suggesting that insertional mutagenesis by an integrated but non-expressing (or silenced) retroviral vector may have contributed to TMZ-associated tumorigenesis in some instances. Two cases of treatment-related secondary myelodysplastic syndrome and acute myeloid leukemia have been reported in the literature for glioma patients treated with TMZ, indicating a clinical risk of TMZ tumorigenesis in some cancer patients. Here we present evidence that TMZ can cooperate with a tumor-promoting transgene (in LMO1 marrow) or potentially with insertional mutagenesis (in gamma-retrovirus transduced C57BL/6 or LMO1 marrow) for hematopoietic transformation, and that hematopoietic cells not protected by drug-resistant MGMT expression can give rise to lymphomas in a majority of mice following TMZ treatment.

Description: Intact function of DNA repair gene is required for maintenance of genomic stability and long term survival of stem cells. We hypothesize that DNA-PKcs, a key factor for DNA double-strand break (DSB) repair, is critical for hematopoietic stem cell (HSC) function. Expression level of DNA-PKcs mRNA monitored by RT-PCR was high in kit+lin− and sca+lin− cells, low in sca+kit+lin− cells and not seen in lin+ cells, implying its role in highly proliferative progenitors. To assess the function of HSCs deficient in DSB repair, serial transplantation capacity of scid (DNA-PKcs−/−) BM cells into lethally irradiated recipients was compared to wildtype BM. Primary transplants of scid BM died after treatment with 2Gy irradiation 4 wks post-transplantation (n=3). In contrast, parental scid mice survived 3Gy irradiation, implying radiation hypersensitivity of scid BM cells after transplantation. No changes were found in the telomere length, cell cycle distribution and apoptosis between the wildtype and scid BM cells after primary transplantation. Scid BM cells failed to repopulate recipients after the third round of transplantation (n=8). To assess competitive repopulating capacity, mixtures of wildtype and scid cells were transplanted into lethally irradiated recipients. BM CFU of primary recipients were predominantly wildtype (8 mice for C3H background, total CFU=262; 5 mice for C56B/6 background, total CFU=336; n〉15 per mouse). Scid cells with two independent genetic backgrounds caused consistent repopulation defects, confirming repopulation defect is caused by DNA-PKcs deficiency. All five primary recipients with C56B/6 background was repopulated predominantly by wildtype CFU (wt CFU 93±5% vs. wt CFU of input; 60±31%, p

Description: Lentiviral vectors have been shown to infect non-dividing cells, including hematopoietic stem cell [HSC], and HIV lentiviral vector has been studied extensively in preclinical models. However low HIV lentiviral vector transduction efficiency compared to retroviral vectors, is seen in murine HSC, hampering transplantation and long-term expression of transgene in the recipients. Furthermore, concerns remain regarding the safety of HIV based vectors. Simian Immunodeficiency Viral [SIV] vectors could be safer since the parent virus does not cause disease in humans. However, to model this approach has been difficult because native SIV vectors do not transduce murine cells. We have generated a bicistronic SIV lentiviral SIN vector, containing MGMT and firefly luciferase genes linked by a self-cleavage FMDV 2A sequence. The SIV backbone was kindly provided by Dr. Donald Kohn (University of Southern California). The transgenes are controlled by the MND promoter, which has been shown to express well in murine hematopoietic stem cells. The vector was generated by cross-packaging SIV RNA with HIV-1 ΔR8.91 packaging plasmid and VSVG pseudotyped envelope (Ref. Retrovirology2005, 2:55). Unconcentrated viruses had an average titer of 1E+06 iu/ml, which was similar to HIV-1 lentiviral vectors. In vitro, HIV-1 cross-packaged SIV-mnd-MGMT-2A-Luc vector was able to transduce both human and murine cell lines with no reduction of expression for 10 weeks. In addition, this cross-packaged SIV vector was also able to transduce primary murine bone marrow cells from Balb/C mice with low MOI of 0.5 to 1. Transduced primary murine bone marrow cells maintained transgene expression during a 4 week culture. To analyze in vivo expression, Balb/C bone marrow cells were transduced for 48 hrs in cytokines with the HIV-1 packaged SIV vector and transplanted into irradiated recipients. We used bioluminescent imaging (BLI) to monitor the transgene expression and the dynamic engraftment of transduced murine bone marrow cells. At MOI of 0.5 or 5, transduction efficiencies in murine progenitor cells were 24.4% and 46.7% respectively by PCR of transgene from CFU colonies. Bioluminescent imaging indicated similar engraftment patterns of transduced bone marrow cells by HIV-1 lentiviral vector or cross-packaged SIV lentiviral vector, as early as day 5. Consistent BLI signals indicated sustained expression of transgene in SIV vector transduced bone marrow cells beyond 30 days. With this study, cross-packaged SIV SIN vector could be used as a potential gene transfer vector in both preclinical murine studies and perhaps in clinical trials.

Description: While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.

Description: Abstract 1455 Maintenance of hematopoietic stem cells (HSC) requires proper interaction between HSC and the bone marrow niche. DNA repair proteins, especially proteins involved in nonhomologous end joining (NHEJ), are critical for HSC maintenance. We hypothesize that NHEJ deficient HSCs have a BM niche occupancy defect. NHEJ pathway is the major mechanism for double strand break repair in mammalian cells, several proteins participate in the process. Ku70 recognizes and binds to the DNA DSB ends with Ku80, recruits DNA activated catalytic polypeptide (Prkdc) to form DNA dependent protein kinase complex, and further recruits Artemis to process the ends and DNA ligase 4 to rejoin the broken ends. NHEJ deficient cells are hypersensitive to irradiation, and NHEJ deficient mice display SCID phenotypes. We have previous shown that Ku70 deficient HSCs are defective in repopulation as well as BM niche occupancy. Prkdc is another key factor in the NHEJ pathway, and mice homologous for a spontaneous nonsense mutation (Prkdcscid, commonly referred to as scid) are characterized by an absence of functional T cells and B cells due to the inability of V(D)J recombination. Though scid mice has been widely used for human HSCs engraftment studies and the nonobese diabetic/severe combined immune deficiency (NOD/scid) xenotransplantation model is now the “gold standard” for assaying human HSC activity, the function and BM niche occupancy capacity of HSCs of scid mice origin has not been characterized. scid mice show comparable HSC frequency and similar apoptosis rates compared to HSC from WT mice. To assess scid HSC function in vivo, single and competitive serial transplantation was performed. BM from scid mice were able to reconstitute the myeloid lineage (Mac1+) but not lymphoid lineages (CD3+ or B220+) in primary recipients due to the deficiency in V(D)J recombination. Serial transplantation was hampered by a high frequency of donor-derived thymic lymphomas in primary recipients. Competitive repopulation assays between WT and scid BM cells showed that when scid BM were mixed with WT competitor BM at a 1:1 ratio, scid BM cells were completely outcompeted by the WT BM cells. To investigate the BM niche occupancy ability of scid HSCs, we used the hematopoiesis niche occupancy assay, in which 5×106 WT congenic BM cells were transplanted into WT and scid recipients without any recipient conditioning and evaluated for blood and marrow cell origins at 16–24 weeks. Remarkably, transplanted WT BM made a long-term multi-lineage (Mac1+, CD3+ and B220+) contribution to hematopoiesis as well as HSC engraftment in scid recipients, (18.4+ 3.8% Mac1+, all the CD3+, B220+ cells, and 8.2+ 2.6% of HSC are donor derived). By contrast, less than 1% stem cell engraftment occurred in WT recipients as expected. Thus, have a long term niche occupancy defect impacting hematopoiesis. NHEJ is involved in both DNA double strand break repair and V(D)J recombination in lymphocytopoiesis. To determine whether lymphocytopoiesis is required for HSC function and BM niche occupancy, RAG1 deficient mice were used in the competitive repopulation assay and the hematopoiesis niche occupancy assay. Our results showed that RAG1-/- HSCs competed with WT HSC did not have defects in competitive repopulation or BM niche occupancy. These data demonstrate that Prkdc, a key component of NHEJ, is required for HSC function. Prkdc deficiency in the HSC compartment results in loss of competitive repopulation ability and loss of long term BM niche occupancy. Together with our previous results obtained from Ku70-/- mice, we conclude that NHEJ is critical for HSC self-renewal and BM niche occupancy, a function independent of its role in lymphogenesis. These data also explains the nature of the conducive marrow niche environment of scid mice for xenotransplantation. Disclosures: No relevant conflicts of interest to declare.

Description: Although scid mice have been widely used for human HSC engraftment studies, the function of HSCs of scid mice has not been characterized. We hypothesized that the DNA repair defect of scid mice results in a stem cell defect that facilitates HSC engraftment. scid BM cells showed severely impaired repopulation potentials in the competitive repopulation assay. To assess the BM hematopoietic niche occupancy ability of scid HSC, WT BM cells were transplanted into scid mice without any conditioning and observed to achieve long-term engraftment. Furthermore, the defects of scid HSCs are independent of their inability to perform lymphopoiesis because a similar defect in hematopoietic niche occupancy was not observed with Rag1−/− recipients. These results demonstrate that scid HSCs are impaired in maintenance within the niche, which may explain the nature of the conducive marrow niche environment of scid mice for xenotransplantation.

Description: Aberrant activation of Rho guanine nucleotide exchange factors (RhoGEFs) is a chief mechanism driving abnormal activation of their RhoGTPase targets in cancer. Thus, small molecule inhibitor of RhoGEF activities can be used as a drug lead to treat leukemia and other malignancies. We have identified an active small molecule, IODVA1, in several xenograft mouse models of cancer including Ras-driven cancers. Here, we used cellular and mouse models of Ph+(BCR-ABL1) B-ALL to identify Vav3, the multi-domain tyrosine phosphorylation-dependent RacGEF as the target of the small molecule IODVA1. IODVA1 specifically reduces the proliferation and survival of p190-BCR-ABL but not of empty vector expressing human CD34+ blood cells. IODVA1 binds tightly to recombinant Vav3 (Kd= 400 nM) but not to Rac1 or to the RhoGEF LARG. In Ba/F3 cells expressing p190-BCR-ABL, IODVA1 inhibits Rac activation and signaling within minutes of exposure. The decrease in Rac activity is not due to the activation of p50GAP or RhoGDI1 and is accompanied by a decrease in the activity of the pro-survival effectors PAK, JNK, and 4EBP and an increase in pro-apoptotic BAD activity. IODVA1prevents leukemia-related death and eliminates the leukemia burden in a BCR-ABL-induced murine model with no apparent toxicity. It eradicates leukemic propagating activity assessed by serial transplantation. Most importantly, IODVA1 increases the survival of a mouse model of TKI-resistant p210-BCR-ABL1(T315I) B-ALL better than ABL1-TKI imatinib and eliminates leukemic burden. p210-T315 leukemic mice survive 45 days after treatment has ended. Vav3- and Rac1/Rac2-deficient leukemic cells do not respond to IODVA1 in colony formation assay consistent with Vav3 being IODVA1's target. A mouse model of BCR-ABL leukemia deficient in Vav3 also does not respond to IODVA1. Cells from PDX models representing pediatric ALL patients including Ph+ (BCR-ABL(T315I) were found to be highly sensitive to IODVA1 ex vivo. Based on the described mechanism of action, we propose that IODVA1 is an allosteric inhibitor of Vav3 that holds promise as an anti-leukemic agent. Disclosures Cancelas: Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Velico: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding.

Description: Xenoantibody production directed at a wide variety of T lymphocyte–dependent and T lymphocyte–independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell–independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell–deficient athymic recipient mice, that rapidly induced, T-cell–independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell–mediated IFN-γ production, nor NK-cell–mediated cytolytic killing of xenogeneic target cells. The T-cell–independent IgM xenoantibody response could be partially suppressed by CD40L blockade.