ALBERT

Description: T-cell and B-cell reconstitution was studied in nine patients who received fluorescence activated cell sorter (FACS)-sorted autologous CD34+ hematopoietic progenitor cells (HPC). The mean numbers of T cells (CD3+), B cells (CD19+) and CD34+ HPC administered to each patient were .004, .002, and 1.8 × 106 cells/kg, respectively. After high-dose myeloablative chemotherapy (busulfan, cyclophosphamide, etoposide) CD34+ HPC were infused and lymphoid reconstitution was monitored using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ T-cell receptor (TcR) sequences. Restoration of normal numbers of peripheral blood T cells and B cells among recipients of FACS-sorted CD34+ HPC was delayed compared to recipients of non-T-cell–depleted PBSC autografts. In both patient groups, the circulating T cells were primarily CD4−, CD8+, αβ TcR+, and CD45RO+, CD45RA− during the first 2 months after transplant. Subsequent increases in the frequency of CD45RA+ CD45RO− T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+hematopoietic progenitors to “naive” T cells. Analysis of the TcR repertoire after hematopoietic reconstitution demonstrated decreased diversity of Vβ TcR expression associated with global decreases in the absolute number of total peripheral blood T cells and most Vβ TcR+ subsets. Three of nine recipients of FACS-sorted CD34+ HPC demonstrated significant increases in the percentage of γδ+ peripheral T cells and CD5+ B cells at 3 to 9 weeks after transplantation, and all patients had transient oligoclonal expansions of T cells expressing specific Vβ TcR. Transplantation with highly purified CD34+ HPC results in reduced diversity of the peripheral T-cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.

Description: Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2−/− mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit+Sca-1+Lineage− (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2−/− KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2−/− mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2−/− KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2−/− bone marrow cells. We conclude that Fancd2−/− mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies.

Description: Abstract 504 The hematopoietic niche supports both normal and malignant hematopoiesis. We have evaluated the functional interactions between leukemic cells and the endothelial cell component of the hematopoietic niche. Highly enriched populations of circulating human acute myeloid leukemia (AML) cells were obtained by therapeutic leukapheresis. These primary AML cells rapidly homed to and associated with pre-existing vascular tubes in vitro. Co-culture of human lung microvascular endothelial cells (HLMVEC) with AML cells both inhibited AML apoptosis by ∼50% (p

Description: Hematopoiesis is tightly regulated by the niche and highly sensitive to a variety of genotoxic stressors. The mechanisms responsible for the repair and regeneration of hematopoietic stem cells (HSCs) are poorly understood. Endothelial cells (ECs) are an essential component of the bone marrow microenvironment that provide regenerative signals for HSCs after injury; however, the identity of these signals remain largely unknown. We utilized RNA sequencing and a bioinformatics approach to identify hepatocyte growth factor (HGF) as an endothelial-derived mediator of HSC regeneration. HGF gene expression was 7.6-fold higher in arterial-derived ECs (HAEC) that regenerate functional long-term HSCs (CD150+LineageloSca1+Kit+ cells; CD150+LSK cells) compared to venous ECs (HUVEC) that support only hematopoietic progenitors. Furthermore, the HSC-supportive HAECs secreted up to 25 pg/mL HGF into conditioned media, whereas HGF was undetectable in media conditioned by HUVEC. To directly test the role of HGF in HSC regeneration, we supplemented co-cultures of irradiated bone marrow cells and primary ECs with recombinant HGF. Addition of HGF was sufficient to augment CD150+LSK cell regeneration up to 20-fold by HUVEC. This effect was abolished by treatment with the Met-specific small molecular inhibitor, PF04217903. Importantly, neither the addition of HGF alone to irradiated hematopoietic cells nor spiking HUVEC-conditioned media with HGF improved HSC regeneration in the absence of an EC monolayer. Together, these results indicate that HGF activates a signaling loop in ECs that supports HSC regeneration. Disclosures No relevant conflicts of interest to declare.

Description: Previously, we showed that the co-repressor CoREST (Rcor1) is essential for the maturation of definitive erythroid cells in the mouse fetal liver. To elucidate Rcor1’s function in multilineage hematopoiesis in the adult, we conditionally deleted Rcor1 using Mx1-Cre. The loss of Rcor1 expression in hematopoietic cells led to the rapid onset of a severe anemia due to a complete block of erythropoiesis downstream of committed erythroid progenitors. By contrast, the production of megakaryocyte progenitors, megakaryocyte maturation and platelets were maintained. In the myelomonocytic lineages, although neutrophil differentiation was completely abrogated, the number of monocytic cells was significantly increased, resulting in a peripheral blood leukocytosis and monocytic infiltrations in the liver. Rcor1-deficient monocytes showed a 66% reduction in apoptosis and possessed ~100-fold more CFU-M activity than control cells. The CFU-M derived from Rcor1 deficient bone marrow could be serially replated up to 5 times; however, replating activity was entirely cytokine dependent. Defective myelomonocytic differentiation persisted following transplantation into wild type hosts for up to 12 months but did not progress to leukemia. To begin to understand at the molecular level how Rcor1 regulates monocyte expansion, we evaluated the expression levels of genes whose ectopic expression is associated with myeloid neoplasia. In Rcor1-deficient monocytes, Gata2, Meis1 and Hoxa9 were overexpressed by 8- to 200-fold. Taken together, these data demonstrate that Rcor1 is essential for both erythroid and myelomonocytic differentiation and that its loss of function causes significant myelodysplasia. Disclosures No relevant conflicts of interest to declare.

Description: Fanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.

Description: Substantial barriers exist to the engraftment of hematopoietic cells in mice after in utero transplantation. Although high levels of donor-derived hematopoiesis have been reported in SCID mice, the majority of chimeric recipients exhibit decreasing levels of donor cells over time. To directly test whether the natural killer cell and macrophage activity of the recipients represents a barrier to sustained engraftment, fetal NOD/SCID mice were injected on day 13.5 of gestation with an enriched congenic hematopoietic progenitor cell population. Forty-four percent of pups showed the presence of Ly5.1+ donor cells 4 weeks after transplantation. The mean number of donor-derived nucleated cells in the peripheral blood (PB) was 30%. Although the majority of circulating donor cells were lymphocytes, up to 15% expressed myelomonocytic markers. Serial PB samples from individual mice indicated that the percentage of circulating donor cells increased from 17% to 55% between 4 and 24 weeks. At 6 months posttransplantation, an increased frequency of multilineage, donor-derived cells was also observed in the bone marrow (BM) and the spleen of chimeric recipients. The engraftment of pluripotent hematopoietic stem cells was evaluated by transplanting BM from chimeric mice into irradiated congenic recipients. Irradiated secondary recipients also exhibited multilineage donor-derived hematopoiesis in the PB, BM, and spleen for up to 6 months. These results show that the in utero transplantation of lineage-depleted BM cells into NOD/SCID recipients produces a high frequency of sustained engraftment of allogeneic hematopoietic stem cells.

Description: Abstract 1700 Directed signaling between acute myelogenous leukemia (AML) cells and the bone marrow microenvironment plays an important role in leukemia cell survival. How leukemic blasts transform the homeostatic bone marrow microenvironment is not fully explained by known ligand-receptor interactions. Microvesicles (MV) are membrane-enclosed compartments that, depending on cell specific pathways, range in diameter from 50–100 nm (exosomes) to 100–1000 nm (microparticles) and are constitutively released into circulation in many tissues. Biogenesis has been found to be upregulated in a range of cancers. We hypothesized that MV-mediated transfer of signaling components by AML blasts may contribute to intercellular signaling in the leukemic niche. In an initial screen, we characterized MV that we recovered from the culture supernatant of multiple AML-derived cell lines, as well as AML cells from patients. Live-cell imaging of primary AML blasts using the exosomal pathway-specific dye N-Rh-PE revealed that at least a portion of these vesicles could be categorized as exosomes. Consistent with the non-random incorporation of molecular cargo observed in recent studies with solid tumors, we found the protein and RNA content of MV to be distinct from cellular lysates. In a focused, candidate target approach, our experiments showed that MV contain mRNA transcripts encoding the broadly relevant AML prognostic markers FLT3-ITD, NPM1, and IGF-1R. In both cell lines and cultured primary AML cells, insulin receptor (IR) and IGF-1R mRNAs were enriched up to ∼400-fold over GAPDH in MV versus cellular fractions. The IGF-1R receptor tyrosine kinase (RTK) transduces growth and survival signals, and the IGF-I signaling pathway has recently been identified as a potentially important candidate for molecularly targeted drug therapy for AML. Corroborative evidence for a role of MV and IGF-I signaling in shaping the leukemic microenvironment comes from the detection of human IGF-1R mRNA in cDNA prepared from mouse stromal cells (OP9) co-cultured for 48 hours with primary AML cells across a cell impermeable transwell barrier. To determine if the mRNA is transferred in a biologically significant quantity, we developed a plasmid-based quantitative PCR method to measure and compare transferred transcripts to steady state IGF-1R mRNA levels in the AML cell line HL-60. Results indicated that 0.3 HL-60 cellular equivalents of the human IGF-1R transcript were detected per OP9 cell following co-culture with primary AML cells. These data imply that transfer occurs at a level that may be functionally relevant. To further explore the potential role of MV in AML-stroma signaling, experiments are underway with HL-60 cells stably transfected with a pcDNA vector encoding the human IGF1R with a C-terminal FLAG tag. These HL-60:IGF1R-FLAG cells release MV that contain the IGF1R-FLAG transcript and will serve as a model to explore the downstream intracellular RTK signaling. In conclusion, we report for the first time the production of MV containing protein and disease-specific transcripts by AML blast cells, the selective enrichment in MV of disease-relevant transcripts, and direct cell-cell transfer of leukemia cell mRNAs to bystander cells. Our findings underscore the potential role of MV in shaping the AML microenvironment through lateral transfer of RTK mRNA transcripts. We propose a role for MV during disease progression and highlight their potential as highly sensitive, cell-free, and minimally invasive AML biomarkers. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1471 Introduction: A variety of prognostic molecular markers have been proposed for risk stratification of intermediate-risk AML, which includes patients with normal cytogenetics (CN) and patients with cytogenetic abnormalities other than the recurrent prognostically informative cytogenetic aberrations (CA). Acquired mutations of FLT3 ITD, NPM1, and CEBPA have well-established prognostic significance, and have been incorporated into consensus classification schemes. In addition, several gene expression markers (WT1, ERG, BAALC, and MN1) have been proposed as prognostic indicators, but with less extensive validation. Whether these new gene expression markers should be incorporated into proposed “molecular risk scores” for AML has not been well refined. In this study, we have determined the transcript levels of WT1, ERG, BAALC, and MN1 in AML patients, and correlated these quantitative levels with other validated clinical and laboratory prognostic indicators. Methods: Diagnostic blood or bone marrow samples were analyzed from 56 pre-treatment AML patients from our institution (48% male; median age 59). Metaphase cytogenetics showed 4 patients with inv(16), 4 with a monosomal karyotype, 25 with a CN karyotype and 23 with a CA karyotype (intermediate-risk cytogenetics). The expression levels of WT1, ERG, BAALC, and MN1 were quantified with TaqMan real-time PCR and normalized to ABL. The FLT3 ITD, NPM1, DNMT3A R882, CEBPA, and MLL PTD mutations were detected with either HRM, direct sequencing, and/or PCR fragment analysis. The WT1 SNP rs16754 was determined with a TaqMan genotyping assay. Results: In the 56 AML patients, the prevalence of the FLT3 ITD, NPM1, MLL-PTD, and DNMT3A R882 mutations were similar to previously reported values at 37%, 27%, 6%, and 13%, respectively. The frequency of patients carrying the minor allele of the WT1 SNP (combined heterozygous and homozygous) was 30%. Compared with their expression level in 29 normal control individuals, all four genes were significantly over-expressed in the AML patients, with mean RNA level differences of 1.98-log for WT1 (P