Methylcellulose Culture Research Articles

NKG2D-CAR Transduced Primary Natural Killer Cells Efficiently Target Multiple Myeloma Cells

IntroductionImmunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity.Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer.The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients.MethodsThe activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells.ResultsIn vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target).In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1).ConclusionsIt is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. DisclosuresRio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

Mettl3 Mediated m6A Modification Is Essential in Fetal Hematopoiesis

N6-methyladenosine (m6A) is the most abundant RNA modification, with key roles in RNA metabolism and regulation of gene expression. Recent studies have elucidated a role for m6A in normal hematopoiesis and myeloid malignancies. Constitutive deletion of the m6A methyltransferase Mettl3 in zebrafish enforces endothelial identity during the endothelial-to-hematopoietic transition, thus precluding normal developmental emergence of HSPCs. Studies conducted in human CD34+ cells, as well as human and murine AML cell lines have suggested that loss of METTL3 results in resolution of differentiation blockade, and impaired engraftment in murine transplantation assays. However, the effects of METTL3 deletion on hematopoietic stem cells in the context of an intact hematopoietic hierarchy in vivo have not yet been extensively characterized.To study the effects of Mettl3 deletion on the hematopoietic system in vivo, we generated Vav-Cre METTL3-/- (VCM3-/-) mice. Deletion of Mettl3 resulted in embryonic lethality, evidenced by skewing of Mendelian ratios at birth. Occasional stillborn VCM3-/- pups were smaller than wildtype littermates, and exhibited pallor, pancytopenia, and dramatically reduced marrow cellularity. To study the effects of Mettl3 deletion on embryonic hematopoiesis, we isolated fetal liver (FL) at embryonic day 14.5 for analysis. At E14.5, expected Mendelian ratios were preserved. Western blot and qPCR confirmed loss of Mettl3 expression in VCM3-/- mice. Reduction of total m6A levels in VCM3-/- mice was confirmed by ELISA.Flow cytometry for hematopoietic markers demonstrated a significant increase in the total number and frequency of Lin-Sca+c-Kit+ (LSK) cells in E14.5 VCM3-/- FL, with an increase in the frequency of HPC-1 (CD48+CD150-) and HPC-2 (CD48+CD150+) cells. To determine the function of VCM3-/- FL cells, we performed colony forming and transplantation assays. VCM3-/- FL cells demonstrated reduced colony forming ability in methylcellulose culture, and colonies that did arise were morphologically abnormal. VCM3-/- FL cells were also deficient in hematopoietic rescue assays, with all lethally irradiated recipient mice dying by day 14 post-transplant. Transplantation of CFSE labeled cells confirmed that absence of Mettl3 -/- hematopoiesis was not attributable to a homing defect. Competitive transplantation of VCM3-/- FL with Pep3b bone marrow similarly resulted in almost total loss of peripheral blood and bone marrow VCM3-/- engraftment, whereas mice transplanted with VCM3+/+ FL maintained chimerism at 8 weeks. Interestingly, VCM3-/- FL cells and FL LSK cells displayed no differences in apoptotic rate or cell cycle.To determine the mechanism underlying the observed phenotypes, we first performed RNA sequencing of VCM3+/+ and VCM3-/- FL LSK. Mettl3 deletion resulted in the increased expression of 701 transcripts, and reduced expression of 1395 transcripts. Gene ontology (GO) analysis revealed that upregulated genes were enriched for mitochondrial function, ribosome and ribonucleoprotein complex proteins and downregulated genes for cell adhesion and developmental processes. M6A RNA modification affects mRNA stability and translation. To determine the effect of m6A depletion on the hematopoieitic stem and progenitor cell proteome we are in the process of validating changes in protein levels of select genes essential in hematopoiesis.Previously, studies have demonstrated that deletion of METTL3 in human CD34+ hematopoietic cells and AML cell lines promote myeloid differentiation. Interestingly, we see a similar depletion of myeloid progenitors in VCM3-/- FL, with an increased percentage of mature myeloid CD11b+ cells. However, these results also coincide with an increased fraction of LSK HSPCs at E14.5. Interestingly, this resembles the METTL3 knockout phenotype in embryonic stem cells, which results in a reinforced naïve pluripotent state with impaired differentiation. Our ongoing studies seek to determine the role of m6A in FL HSC maintenance and differentiation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Small Molecule Stimulants of Hematopoietic Stem Cells and Their Mode of Action

Agents that can improve the function and/or numbers of hematopoietic progenitor and stem cells (HSC) are of great importance for the treatment of bone marrow failures of different etiology. However, except for the hematopoietic growth factors, which lead to significant depletion HSC via simultaneous differentiation and some anabolic steroids, the list of capable agents that can improve the function or numbers of HSC is very short. However, such drugs would have a tremendous range of application from ex vivo expansion, to bone marrow regeneration in aplastic anemia (AA) post chemotherapy, HSC transplantation or to improve the function of normal HSC in aging.While performing a multidrug screen for the agents that could simultaneously decrease senescence and overcome proliferative block in pre-senescent cells, we have identified two compounds violuric acid (VA) and 1-naphthoquinone-2-monoxime (N2N1). Both compounds considerably extended the replicative life span (RLS) of normal cells. We applied these drugs to stromal/mesenchymal cells obtained from healthy bone marrows, primary human normal dermal fibroblasts, progeroid primary cells derived from the patients diagnosed with Werner or Bloom syndromes and small panel of cancer cell lines (SKM-1, K562, KG-1, THP-1). Both compounds, in dose dependent manner prolonged the RLS of replicatively pre-aged cells. On an average, 10-15 additional population doublings (PD) were achieved after addition of N2N1 at 1μM. VA treatment has added 8-10 extra population doublings. If compared with untreated controls that can propagate up to 45-50 PD, the treatment with VA or N2N1 adds from 16 to 30% increase in replicative life span. To compare, the effect of rapamycin (1nM) on human fibroblasts showed the RLS increase ranged from 5 to 10%. Treatment with both VA and N2N1 results in restoration of cell cycle progression, decreased activity of SAβG, down-regulation of p16, p21 and γH2A.X and, up-regulation of lamin B1 protein. Treatment with both compounds resulted in maintenance of normal telomere length.In term of HSC these agents in vivo increased the performance of HSC in competitive repopulation assay. Bone marrow cells were isolated from mice (CD45.1) treated with the vehicle or experimental drugs for three weeks. After that, they were mixed with the equal number of competitor bone marrow cells CD45.2 and injected into irradiated CD45.2 host animals. In three weeks, we observed a substantial domination of CD45.1 cells over CD45.2 in experimental groups, while control (vehicle) group exhibited equal representation of both genotypes. In vitro, treatment with VA or N2N1 contributed to prolonged availability of HSC in serial replating CFU assays in methylcellulose and long-term culture initiating cell (LTC-IC) assays. Addition of VA or N2N1 to the short-term cultures (7-14 days) of normal bone marrow cells in a medium containing a cocktail of growth factors (Il6, IL3, FLT3L, TPO, SCF) resulted in maintenance and growth of HSC or progenitors. Gated on lymphocyte sub-population, treated with N2N1 or VA samples revealed ~0.3%±0.02 or 0.2% ±0.02 of CD34+, CD45+ cells correspondingly, while control samples had 0.08% of these cells (the result of three independent experiments). Most importantly, we observed colonies formation, after application of these drugs to the bone marrow isolated from the patients diagnosed with severe AA. Further studies also indicated that these agents do not promote growth of leukemic cell.Analysis of mechanism of action showed that VA and N1N2 function as redox co-factors in oxidations of NAD(P)H. VA transfers electrons non-enzymaticly from NAD(P)H to oxidized glutathione or peroxides. N2N1 is a redox co-factor for the NAD(P)H dehydrogenase (quinone) 1 (NQO1) and together they move electrons from NAD(P)H to cytochrome c or CoQ10. As such, we presented here a comprehensive prove that pharmacologic manipulation of redox balance controlled by glutathione or NQO1 activity via redox catalysts can ameliorate the detrimental consequences of HSC loss during normal aging by interfering with direct ROS mediated signaling or attenuating collateral ROS mediated damages. [Display omitted] DisclosuresMaciejewski:Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Multilineage-differentiating stress-enduring (Muse)-like cells exist in synovial tissue.

Cartilage regeneration is a promising therapy for restoring joint function in patients with cartilage defects. The limited availability of autologous chondrocytes or chondrogenic progenitor cells is an obstacle to its clinical application. We investigated the existence and chondrogenic potential of synovial membrane-derived multilineage-differentiating stress-enduring (Muse)-like cells as an alternative cell source for cartilage regeneration. Cells positive for stage-specific embryonic antigen-3 (SSEA-3), a marker of Muse cells, were isolated from the synovial membranes of 6 of 8 patients (median age, 53.5 years; range 36-72 years) by fluorescence-activated cell sorting. SSEA-3-positive cells were cultured in methylcellulose to examine their ability to form Muse clusters that are similar to the embryoid bodies formed by human embryonic stem cells. Muse clusters were expanded and chondrogenic potential of M-cluster-derived MSCs examined using a pellet culture system. Chondrogenic differentiation was evaluated by proteoglycan, safranin O, toluidine blue and type II collagen staining. To evaluate the practicality of the procedure for isolating Muse-like cells, we compared chondrogenic potential of M-cluster derived MSCs with expanded cells derived from the clusters formed by unsorted synovial cells. Synovial membranes contained SSEA-3-positive cells that after isolation exhibited Muse-like characteristics such as forming clusters that expressed NANOG, OCT3/4, and SOX2. In the pellet culture system, cell pellets created from the M-cluster-derived MSCs exhibited an increase in wet weight, which implied an increase in extracellular matrix production, displayed metachromasia with toluidine blue and safranin O staining and were aggrecan-positive and type II collagen-positive by immunostaining. Unsorted synovial cells also formed clusters in methylcellulose culture, and the expanded cell population derived from them exhibited chondrogenic potential. The histological and immunohistochemical appearance of chondrogenic pellet created from unsorted synovial cell-derived cells were comparable with that from M-cluster-derived MSCs. Muse-like cells can be isolated from the human synovial membrane, even from older patients, and therefore may provide a source of multipotent cells for regenerative medicine. In addition, the cluster-forming cell population within synovial cells also has excellent chondrogenic potential. These cells may provide a more practical option for cartilage regeneration.

Development of Postmeiotic Cells In Vitro from Spermatogonial Cells of Prepubertal Cancer Patients.

Aggressive chemotherapy in childhood often results in testicular damage and consequently jeopardizes future fertility. The presence of spermatogonial cells (SPGCs) in the testes of prepubertal cancer patient boys (PCPBs) can be used to develop future strategies for male fertility preservation. In the present study, we examined the presence of SPGCs in testes of chemotherapy-treated PCPBs and their ability to develop spermatogenesis in vitro using a three-dimensional culture system. Seven testicular biopsies were obtained from chemotherapy-treated PCPBs and one from a patient with β-thalassemia major. Isolated testicular cells were cultured in a methylcellulose culture system (MCS)-containing StemPro enriched with growth factors for 5-15 weeks. The presence of premeiotic, meiotic, and postmeiotic cells was examined by immunofluorescence staining and/or reverse transcription-polymerase chain reaction (RT-PCR) analysis. We observed SPGCs in the examined testicular biopsies. Isolated testicular cells cultured in MCS developed into colonies and contained premeiotic, meiotic, and postmeiotic cells. Furthermore, we identified sperm-like cells that had developed from testicular cells of a PCPB. Our results demonstrate, for the first time, the presence of biologically active SPGCs in testicular biopsies of chemotherapy-treated PCPBs and their capacity to develop in vitro to different stages of spermatogenesis, including the generation of sperm-like cells. This study may open the way for new therapeutic strategies for fertility preservation of PCPBs and for azoospermic patients.

Human Bronchial Epithelial Cell-derived Factors from Severe Asthmatic Subjects Stimulate Eosinophil Differentiation.

Activated bronchial epithelial cells (BEC) release various alarmins, including thymic stromal lymphopoietin (TSLP), that drive type 2 inflammation. We hypothesize that BEC-derived factors promote in situ eosinophil differentiation and maturation, a process that is driven by an IL-5-rich microenvironment in asthmatic airways. To assess the eosinophilopoietic potential of epithelial-derived factors, eosinophil/basophil colony forming units (Eo/B-CFU) were enumerated in 14-day methylcellulose cultures of blood-derived nonadherent mononuclear cells incubated with BEC supernatants (BECSN) from healthy nonatopic controls (n = 8), mild atopic asthmatics (n = 9), and severe asthmatics (n = 5). Receptor-blocking antibodies were used to evaluate the contribution of alarmins. Modulation of the mRNA expression of transcription factors that are crucial for eosinophil differentiation was evaluated. BECSN stimulated the clonogenic expansion of eosinophil progenitors in vitro. In the presence of IL-5, Eo/B-CFU numbers were significantly greater in cocultures of BESCN from severe asthmatics compared with other groups. This was attenuated in the presence of a TSLP (but not an IL-33) receptor-blocking antibody. Recombinant human TSLP (optimal at 100 pg/ml) stimulated Eo/B-CFU growth, which was significantly enhanced in the presence of IL-5 (1 ng/ml). Overnight culture of CD34+ cells with IL-5 and TSLP synergistically increased GATA-binding factor 2 and CCAAT/enhancer-binding protein α mRNA expression. The eosinophilopoietic potential of factors derived from BEC is increased in severe asthma. Our data suggest that TSLP is a key alarmin that is produced by BECs and promotes in situ eosinophilopoiesis in a type 2-rich microenvironment.

Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells.To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro.Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage−Sca-1+Kit+(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing.In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs.Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation.Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis. DisclosuresTakaori-Kondo:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

An unexpected protein interaction promotes drug resistance in leukemia

The overall survival of patients with acute myeloid leukemia (AML) is poor and identification of new disease-related therapeutic targets remains a major goal for this disease. Here we show that expression of MPP1, a PDZ-domain-containing protein, highly correlated with ABCC4 in AML, is associated with worse overall survival in AML. Murine hematopoietic progenitor cells overexpressing MPP1 acquired the ability to serially replate in methylcellulose culture, a property crucially dependent upon ABCC4. The highly conserved PDZ-binding motif of ABCC4 is required for ABCC4 and MPP1 to form a protein complex, which increased ABCC4 membrane localization and retention, to enhance drug resistance. Specific disruption of this protein complex, either genetically or chemically, removed ABCC4 from the plasma membrane, increased drug sensitivity, and abrogated MPP1-dependent hematopoietic progenitor cell replating in methylcellulose. High-throughput screening identified Antimycin A as a small molecule that disrupted the ABCC4–MPP1 protein complex and reversed drug resistance in AML cell lines and in primary patient AML cells. In all, targeting the ABCC4–MPP1 protein complex can lead to new therapies to improve treatment outcome of AML, a disease where the long-term prognosis is poor.

TGF-β inhibitors stimulate red blood cell production by enhancing self-renewal of BFU-E erythroid progenitors

AbstractBurst-forming unit erythroid progenitors (BFU-Es) are so named based on their ability to generate in methylcellulose culture large colonies of erythroid cells that consist of “bursts” of smaller erythroid colonies derived from the later colony-forming unit erythroid progenitor erythropoietin (Epo)–dependent progenitors. “Early” BFU-E cells forming large BFU-E colonies presumably have higher capacities for self-renewal than do “late” BFU-Es forming small colonies, but the mechanism underlying this heterogeneity remains unknown. We show that the type III transforming growth factor β (TGF-β) receptor (TβRIII) is a marker that distinguishes early and late BFU-Es. Transient elevation of TβRIII expression promotes TGF-β signaling during the early BFU-E to late BFU-E transition. Blocking TGF-β signaling using a receptor kinase inhibitor increases early BFU-E cell self-renewal and total erythroblast production, suggesting the usefulness of this type of drug in treating Epo-unresponsive anemias.

E35, a Novel Analog of Emodin, Selectively Eliminates Acute Leukemia Stem and Progenitor Cells

Leukemia stem cells (LSCs) play central roles in the pathogenesis of acute leukemia and contribute to both disease initiation and relapse. Thus, identification of agents to eradicate LSCs is an important priority. Our previous studies have demonstrated that emodin, an extracted natural compound, might exert anti-leukemic effects in vitro. To improvethe bioavailability and solubility of emodin, we designed and synthesized a family of novel emodin analogs. Among them, E35 has demonstrated greater bioactivity. In this study, we investigated whether E35 may selectively eliminate acute leukemia stem and progenitor cellsand the underlying mechanism involved. We isolated CD34+ cells from leukemic stem-like cell line KG1a and resistant myeloid cell line HARs. Results demonstrated that E35 affected the viability of CD34+ leukemia cells with the average IC50 value of 15.33 ± 3.88 μM in KG1a cells and 9.12 ± 0.08 μM in HARs, respectively. We then performed the biological studies in primary leukemia specimens from 33 acute leukemia patients. All of the primary leukemia cells were sensitive to E35 with the average IC50 value of 13.86 ± 9.35 μM. Treatment with 16 μM and 32 μM E35 resulted in viability of 41.68 ± 3.90% (n = 33) and 16.63 ± 3.09% (n = 21) for overall primary leukemia cells, and 29.68 ± 4.18% (n = 17) and 15.98 ± 6.71% (n = 7) for CD34+ leukemia cells, respectively. However, both normal mononuclear cells and CD34+ cells obtained from healthy donors were not significantly affected by E35. The viability at 16 μM was 79.43 ± 3.73% (n = 6) for overall healthy cells and 88.64 ± 5.00% (n = 4) for CD34+ hematopoieticcells. Around 65% normal hematopoieticcells remained alive even though treated with E35 as high as 32 μM. E35 strongly induced robust apoptosis of LSCs from KG1a and HARs. After exposure to 16 μM E35 for 24 hours, the percentages of apoptotic LSCs in KG1a and HARs significantly increased up to 4.7 and 2.0 folds compared with those of without treatment groups (P < 0.01, n = 3), respectively. The percentage of CD34+ Annexin V+ cells from primary leukemia specimens following E35 treatment was also significantly greater than that in untreated group (27.29 ± 6.29% vs 10.08 ± 2.15%; P < 0.05, n = 15). Consistently, E35 stimuli raised the percentage of CD34+ CD38- LSCs undergoing apoptosis from 19.79 ± 4.91% to 28.36 ± 7.14% (n = 14). Notably, E35 did not trigger apoptosis in normal hematopoietic stemcells on comparison with control group (P > 0.05, n = 5). Western Blotting results showed that activation of Caspase-family proteins (eg, pro-caspase and PARP cleavage) and inhibiton of Akt/mTOR signaling pathway (eg, p-Akt, p-4E-BP1 and p-p-70S6K blockage) might be key steps toward LSC-specific apoptosis by E35. To determine whether E35 targets functionally defined leukemia stem and progenitor cells, colony assays and NOD/SCID xenotransplantation were performed. Primary AML cells versus normal cells were treated with E35 for 18 hours, followed by plating in methylcellulose culture or transplanting into Busulfan preconditioned NOD/SCID mice. Normal hematopoietic cells with E35 treatment did not affect myeloid colony formation relative to untreated control (n = 6). In contrast, E35 treatment strongly inhibited the ability of leukemia cells to form colonies (P < 0 .0001, n = 7). Similarly, primary leukemic cells with with E35 treatment led to a significant decrease in stem cell activity, with reduction engraftment levels from 19.47 ± 4.77 % to 2.92 ± 0.34 % (P < 0.01, n = 7). In contrast, the engraftment levels of E35 preconditioned normal cells were not statistical significant different from those of healthy cells without treatment (28.10 ± 5.30% vs 31.10 ± 6.88%, n = 4). These data demonstrate that E35 specifically ablates leukemic but not normal hematopoietic progenitor- and stem-cell function. In conclusion, E35 has the potential to selectively eradicate primitive leukemic cells, indicating it is a novel therapeutic candidate against hematologic cancers. DisclosuresNo relevant conflicts of interest to declare.

The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy.Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation.Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. DisclosuresNo relevant conflicts of interest to declare.

689. The Content of the More Immature Hematopoietic Stem Cells (HSCs) Is Dependent from the Underlying Genetic Diseases: Consequence on Transduction Efficiencies

Ex vivo gene transfer into hematopoietic CD34+ cells provides therapeutic benefits in primary immunodeficiencies. However, the CD34+ cell population is heterogeneous and is mainly composed of progenitors (CD34+lin-CD38+) and multilineage progenitors (CD34+lin-CD38-). This latter population can be divided in 3 subtypes regarding to the CD45RA and CD90 expression. CD45RA+ define cells with lymphoid potential, CD45RA-CD90- defined cells with myeloid potential and CD45RA-CD90+ define cells with long term repopulating capability (HSCs) which constitutes the relevant target for gene correction. We found that in bone marrow (BM) CD34+linneg, addition of CD133 helps to delimitate CD38neg population and we have analysed HSC, MLP and MPP population in BM from SCID-X1 (n=4), Wiskott Aldrich Syndrome (WAS) (n=2), Artemis (n=2), Chronic Granulomatous Disease (CGD) patients (n=3), sickle cell disease (SCD) (n=3) and age matched healthy controls (n=6).As expected, mean percentage of HSC represent 1% of CD34+ in healthy donors and similar results are obtained in CD34+ from SCID-X1 (1.76% ± 0.72), Artemis (1.98% ± 0.15), and SCD (2.16% ± 0.6) patients. On the contrary HSC percentage are lower in CD34+ from WAS (0.35% ± 0.15) and nearly undetectable in CGD patients. Moreover in multilineage progenitors, proportion of HSC, MLP and MPP varies from one pathology to another as shown in table1table1.Table 1Mean percentage of HSC, MLP and MPP in the linnegCD133+CD38neg CD34+ cellsHSCsMLPMPPHealthy Donor42%33%24%SCIDX137%45%28%WAS35%35%30%Artemis30%48%20%SCD36%19%44%Our aim was then to determine the ability of vectors used for gene therapy to transduce these different progenitors. We first analysed transduction of bulk CD34+ and HSCs by SIN retroviral vector for IL2RG in 2 SCIDX-1 patients enrolled in the clinical trial. After transduction, the 3 cell types were purified by flow cytometry and transduction determined in CFU after 14 days of culture in methyl cellulose in order to avoid detection of non-integrated vector. We show that in both patients, transduction efficiency was lower in HSCs than in bulk CD34+ cells. We were not able to recover enough CFU to determine VCN from MPP fraction and as expected, no CFU were obtained from MLP fraction. These results will be extended to transduction of CD34+ from WAS and SCD patients and efforts will be concentrated on transduction protocols in order to improve transduction of HSCs. Nevertheless low HSCs contents observed in CD34+ from CGD patients also represents a major difficulty to obtain long term reconstitution after gene therapy.

686. Forced Fetal-Globin Reactivation Mediated by the TALE-ldb1 Fusion Protein

Sickle cell disease (SCD) and thalassemia are very severe inherited disorders of the red cell and are amongst the world's most common genetic diseases. Thalassemia is caused by a serious lack of normal β-globin production in red cells whereas, sickle cell disease reflects a mutant hemoglobin that produces protein aggregates. The production of fetal-globin (HbF) may compensate for the reduction of β-globin and also it functions to inhibit sickling by participating in formation of mixed tetramers including both the γ and βS chain. Thus the induction of HbF holds tremendous promise to ameliorate the clinical symptoms of β -thalassemia and sickle cell disease (SCD). For this reason, the adult-to-fetal hemoglobin switch has been an area of long-standing interest among hematologists and basic scientists. The recent revolution in gene editing technology has provided new opportunities for high efficiency fetal-globin gene activation and provides strong potential for clinical benefit. Recently, it was reported that zinc finger artificial proteins that bind to the ? globin gene promoter could reactivate fetal-globin expression, via tethering the globin enhancer LCR to the ? globin gene promoter. We have designed Transcriptional Activator Like Effectors (TALE) targeting the ? globin gene promoters to reactivate ? globin gene expression. The EF1 short promoter was used initially to drive TALE gene expression. The TALEs were fused with a ldb1 dimerization domain, followed by a T2A GFP cassette. The various elements were then assembled into a lentiviral vector. To prevent deletions caused by repeats of TALEs during the lentivirus packing process, we changed the TALE's DNA coding sequence by using codon-optimized DNA for their expression. Both PCR and Southern blot analysis confirmed that TALEs remained as full-length intact DNA without deletion. We employed HUDEP-2 (human umbilical cord blood derived erythroid progenitor cells) to examine the function of TALEs with respect to fetal-globin gene reactivation. Quantitative RT-PCR was performed to determine the relative mRNA level of fetal-globin which was normalized to the alpha-globin mRNA content. Three of eight TALEs were found to activate fetal-globin expression in HUDEP-2 cells with increases in mRNA levels from several fold to more than 400-fold. Furthermore, we also observed a symmetrical reduction of β-globin mRNA by more than 50%. The fetal-globin level determined by HPLC was up to 40% of total globin, compared to 0.4% in control cells. To define the ability of the vector to transduce primitive CD34 cells, we performed methylcellulose culture after transduction of CD34 cells with TALE encoding lentiviral vectors at an MOI of 5. Individual red colonies were picked up to determine the vector copy number (VCN) by quantitative real time-PCR. Forty to fifty percent of the colonies were positive for vector insertion. In the future we are planning to do human CD34 cell erythroid differentiation to evaluate how the vector activates fetal-globin expression in red cells. To promote strong expression of TALE in the erythroid linage, we have replaced the EF1 short promoter with strong MND promoter or one erythroid specific ankrin promoter.

Mice with a Mutation in the Mdm2 Gene That Interferes with MDM2/Ribosomal Protein Binding Develop a Defect in Erythropoiesis.

MDM2, an E3 ubiquitin ligase, is an important negative regulator of tumor suppressor p53. In turn the Mdm2 gene is a transcriptional target of p53, forming a negative feedback loop that is important in cell cycle control. It has recently become apparent that the ubiquitination of p53 by MDM2 can be inhibited when certain ribosomal proteins, including RPL5 and RPL11, bind to MDM2. This inhibition, and the resulting increase in p53 levels has been proposed to be responsible for the red cell aplasia seen in Diamond-Blackfan anemia (DBA) and in 5q- myelodysplastic syndrome (MDS). DBA and 5q- MDS are associated with inherited (DBA) or acquired (5q- MDS) haploinsufficiency of ribosomal proteins. A mutation in Mdm2 causing a C305F amino acid substitution blocks the binding of ribosomal proteins. Mice harboring this mutation (Mdm2C305F), retain a normal p53 response to DNA damage, but lack the p53 response to perturbations in ribosome biogenesis. While studying the interaction between RP haploinsufficiency and the Mdm2C305F mutation we noticed that Mdm2C305F homozygous mice had altered hematopoiesis. These mice developed a mild macrocytic anemia with reticulocytosis. In the bone marrow (BM), these mice showed a significant decrease in Ter119hi cells compared to wild type (WT) littermates, while no decrease in the number of mature erythroid cells (Ter119hiCD71low) was found in the spleen, which showed compensated bone marrow hematopoiesis. In methylcellulose cultures, BFU-E colonies from the mutant mice were slightly reduced in number and there was a significant reduction in CFU-E colony numbers in mutant mice compared with WT controls (p < 0.01). This erythropoietic defect was abrogated by concomitant p53 deficiency (Trp53ko/ko). Further investigation revealed that in Mdm2C305F animals, there was a decrease in Lin-Sca-1+c-Kit+ (LSK) cells, accompanied by significant decreases in multipotent progenitor (MPP) cells (p < 0.01). Competitive BM repopulation experiments showed that donor BM harboring the Mdm2C305F mutation possessed decreased repopulation capacity compared to WT BM, suggesting a functional stem cell deficit. These results suggest that there is a fine tuned balance in the interaction of ribosomal proteins with the MDM2/p53 axis which is important in normal hematopoiesis.

ASXL1 Is a Key Regulator for Erythroid Development and Asxl1 Loss Impairs Erythropoiesis In Vivo

Objective:Recently, ASXL1 mutations are found in a spectrum of numerous of myeloid malignancies, including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). ASXL1 mutation is a poor prognostic markers. Recently, we reported that Asxl1+/- mice developed myelodysplastic syndrome (MDS)-like disease and MDS/MPN. Regardless of the existence of defective erythroid lineage maturation and anima in patients with MDS or MPN, the role of ASXL1 in erythropoiesis remains unveiled.Methods:Asxl1+/- mice were utilized for hematopoietic analysis, including peripheral blood counts, blood smear, morphology, flow cytometry and histology. To assess the erythroid phenotypes in vivo, blood smears were subjected to acridine staining for reticulocyte counts. The percentage of reticulocytes and erythroid cell differentiation in peripheral blood and bone marrow were evaluated flow cytometrically following thiazole orange and CD71/Ter119 staining. Colony-forming cells (CFU) and in vitro erythroid differentiation assays were performed with methylcellulose (EPO, SCF) and liquid (EPO, transferrin) cultures of Lin-Kit+ cells of BM, spleen and fetal liver cells. In addition, the nuclear fractions were prepared from cultured erythoblasts of WT and Asxl1+/- mice and subjected to western blotting analyses to determine the histone levels of H3K27me3. RNA-Seq was performed to survey the differentially expressed genes in LK cells of WT and Asxl1+/- mice. Real time PCR was performed to confirm the alteration of erythroid development related gene.Results:Adult Asxl1+/- mice were anemic. High frequencies of immature and dysplastic (tear drop) red blood cells were observed in the peripheral blood of Asxl1+/- mice. Flow cytometric analysis revealed an increased percentage of pro-erythroblasts and basophilic erythroblasts populations in peripheral blood and bone marrow cells compared to that of WT controls, suggesting a blockage of erythroid maturation in Asxl1+/- mice. In vitro methylcellulose cultures showed a dramatic decrease of BFU-E and CFU-GEMM in the bone marrow of Asxl1+/- mice compared to WT controls. Western blot analysis showed that Asxl1+/- erythroblast cells exhibited decreased global levels of H3K27me3 and qPCR of Asxl1+/- erythroblast cells revealed altered expression of genes involved in development and homeostasis of erythroid lineage, including Gata1, Ldb1, Cdc42, and Zfpm1.Conclusion:Our study demonstrates that loss of Asxl1 results in blockage of erythroid cell maturation, indicating that ASXL1 is a key regulator for erythroid development and Asxl1 loss impairs erythropoiesis in vivo. DisclosuresNo relevant conflicts of interest to declare.

The Bone Marrow "Mystery Population" of Stem Cells 20 Years Later - a Puzzle Solved?

Background. Almost 20 years ago, a "mystery" population of small stem cells with many of the phenotypic characteristics attributed to resting hematopoietic stem cells was identified in murine bone marrow (BM) (Stem Cells 1998, 16, 38-48). These cells expressed high levels of Sca-1, H-2K, and CD38 and low levels of Thy-1.1; they expressed CD45 antigen but were lineage-negative (lin-) for other hematopoietic markers. These cells incorporated only low levels of Rh123 and were resistant to the cytotoxic effects of 5-fluorouracil. The only phenotypic characteristic that distinguishes these cells from Sca-1+, Lin-, CD45+ Thy-1.1low long-term-reconstituting hematopoietic stem cell population is the lack of c-kit expression. In sum, this "mystery" population of small Sca-1+, lin-, c-kit- but CD45+ stem cells do not respond to hematopoietic growth factors in vitro, form in vivo spleen colonies, or reconstitute lethally irradiated mice. With our discovery of Sca-1+ Lin- CD45- very small embryonic-like stem cells (VSELs) in murine bone marrow (BM) (Leukemia 2006, 20, 857-869), we became interested in this "mystery" population of stem cells. VSELs, like the "mystery" population, are c-kit - and, if freshly isolated from BM, do not show any hematopoietic activity in standard in vitro and in vivo assays. In order to become specified to hematopoiesis, they need to be expanded over an OP-9 stromal support (Exp Hematol 2011;39:225-237).Hypothesis. Since (1) very small CD45- VSELs can be specified in OP-9 co-cultures into long-term reconstituting CD45+ HSCs, (2) the size of the "mystery" population is intermediate between VSELs and HSCs, and (3) VSELs and HSCs differ in cell surface receptor expression, we hypothesized that the "mystery" population is a missing developmental intermediate between VSELs and HSCs.Materials and Methods. Multicolor FACS analysis was employed to compare size and expression of surface markers between murine BM HSCs, the unknown population of stem cells, and VSELs. Next, the populations of small Sca-1+ H2-K+ lin- c-kit+ CD38+/- CD45+ cells (HSCs), smaller Sca-1+ H-2K+ lin- c-kit- CD38+ CD45+ cells (the "mystery" population), and very small in size Sca-1+ H-2K+ lin- c-kit- CD38+/- CD45- cells (VSELs) were purified by FACS from BM (Figure 1) and tested for in vitro colony formation. All these cell populations were primed/expanded over OP-9 support and subsequently evaluated for their hematopoietic potential after passaging in consecutive methylocellulose cultures (passages 1-4). RQ-PCR analysis was employed for detection of pluripotency marker expression as well as hematopoietic gene expression.Results. We found that, in contrast to HSCs, neither freshly sorted stem cells from the "mystery" BM population nor, as expected, VSELs grew hematopoietic colonies in standard methylcellulose cultures. This was also an important step in excluding contamination of our sorted populations with clonogenic cells. We also found that, while VSELs highly expressed Oct-4, this transcription factor was expressed at very low levels in the "mystery" population and was not detectable in HSCs. The most important observation was that the "mystery" population of stem cells became specified in OP-9-supported cultures into clonogenic HSPCs, and this specification occurred faster than the delayed specification of VSELs. VSELs first became enriched for HSPCs after acquiring CD45 antigen expression.Conclusions. Based on the results presented, we propose that the "mystery" population in murine BM is a population of stem cells intermediate between the most primitive population of BM-residing stem cells (VSELs) and the population of stem cells already specified to lympho-hematopoietic development (HSCs). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Definitive Erythro-Myeloid Progenitors (EMPs) Emerge in the Myb-/- Embryo and Retain the Capacity to Differentiate into Macrophages

In the adult, the proto-oncogene Myb critically regulates both the maintenance of hematopoiesis and the differentiation of several hematopoietic lineages. Myb-/- mouse embryos die by embryonic day (E) 15 with severe anemia due to the absence of definitive erythropoiesis (Mucenski et al., Cell, 1991). Similarly, zebrafish embryos lacking myb do not express adult globin genes, have a reduction in other mature hematopoietic lineages by 48 hours post fertilization, and maintain a bloodless phenotype through adulthood (Soza-Ried et al., PNAS, 2010). These and other data have led to the concept that Myb-/- embryos entirely lack definitive hematopoiesis.In both mouse and zebrafish embryos, the first definitive hematopoietic potential arises as a hematopoietic stem cell (HSC)-independent wave of erythro-myeloid progenitors (EMPs). EMPs emerge in the murine yolk sac beginning at E8.25, partially overlapping with an earlier wave of primitive hematopoietic progenitors. We previously demonstrated that EMPs are multipotent progenitors, and are the major source of definitive erythroid potential in the early fetal liver, prior to the colonization of adult-repopulating HSCs (McGrath et al., Blood, 2011). Recently, we identified a unique cell surface phenotype that facilitates the prospective isolation of murine definitive EMPs, distinguishing them from primitive hematopoietic progenitors and maturing populations of megakaryocytes and macrophages in the yolk sac (McGrath et al., Cell Reports, 2015). We detected expression of Myb in sorted EMPs, suggesting that Myb may regulate the emergence and/or differentiation of EMPs. We tested this hypothesis by assessing the emergence, hematopoietic potential and expansion capacity of EMPs, compared with other maturing primitive hematopoietic lineages, in Myb-/- mouse embryos. Consistent with the proposed Myb-independence of the earlier wave of primitive progenitors, we observed normal numbers of maturing macrophages in E9.5 Myb-/- yolk sacs. Interestingly, E9.5 Myb-/- yolk sacs also contained normal numbers of immunophenotypic EMPs. These EMPs were present in hemogenic endothelial-derived clusters expressing Runx1, similar to littermate controls, suggesting that Myb is dispensable for EMP emergence from hemogenic endothelium.We next assessed the differentiation capability of Myb-/- EMPs in vitro. E9.5 Myb-/- yolk sacs lacked high proliferative colony-forming potential (HPP-CFC), a hallmark of immature definitive hematopoietic progenitors. In addition, both definitive erythroid and granulocyte colony-forming potential were absent in methylcellulose cultures of sorted Myb-/- EMPs, in contrast to littermate controls. Surprisingly, however, sorted Myb-/- EMPs gave rise to macrophage progenitors in colony-forming assays, and CD11b+ F4/80+ macrophages in differentiation cultures. These data indicate that Myb is not required for the differentiation of primary definitive EMPs into macrophages. Analysis of Myb-/- fetal liversalso confirmed the presence of F4/80+ macrophages. While these fetal liver macrophages have been previously proposed to be of primitive hematopoietic origin, our data raise the possibility that they may also be derived from EMPs.Further analysis of in vitro differentiation cultures demonstrated an inability of sorted Myb-/- EMPs to proliferate when compared with normal littermates, although these cultures still generated small numbers of macrophages. It is not yet clear whether this reduction in proliferation is due solely to the loss of differentiation of multiple hematopoietic lineages, or is also due to defective maintenance or expansion of EMPs. However, consistent with a role for Myb in continued emergence and/or expansion of EMPs, we observed a reduction in the total number of EMPs by E10.5 in yolk sacs of Myb-/- embryos compared with normal littermates. Taken together, these data indicate that Myb is a critical regulator not only of HSCs, but also of HSC-independent definitive hematopoietic progenitors (EMPs). While Myb is dispensable for the initial emergence of EMPs, it is required for their subsequent differentiation into erythroid and granulocyte lineages. Surprisingly, the persistence of EMPs, while reduced, may provide a source of definitive macrophages in Myb-/- embryos. DisclosuresNo relevant conflicts of interest to declare.

A Wilms Tumor 1 (WT1) Mutation Causes Myelodysplastic Syndrome in a Knock-in Mouse Model, and a Mixed Myelodysplastic/Myeloproliferative Neoplam in Double Knock-in Mice with WT1 and FLT3/ITD Mutations

Background: WT1 is a zinc finger transcriptional regulator and acts as a tumor suppressor gene in various cell types. WT1 mutations are reported in approximately 10% of both adult and pediatric patients with acute myeloid leukemia (AML), and at a lower frequency in patients with myelodysplastic syndome (MDS). Reported mutations consist of insertions, deletions or point mutations, and are thought to alter WT1 DNA-binding ability and result in a loss of function. WT1 mutations are associated with FLT3/ITD mutations in AML, suggesting possible leukemogenic cooperativity, and yet WT1 mutations have been independently associated with treatment failure and a poor prognosis. Recently, a physical interaction demonstrated between WT1 and TET2 suggests a common functional pathway, and explains the mutual exclusivity of these mutations in AML. Despite these observations, the functional contribution of WT1 mutations in hematologic malignancies is not entirely understood. To our knowledge, we are the first to describe here a hematologic phenotype in a WT1 mutant mouse model and in a novel WT1 mutant x FLT3/ITD crossbred mouse model.Methods: Knock-in WT1 mutant mice are heterozygous for missense mutation R394W in the DNA-binding domain, which has been described in cases of human AML. Mice with a heterozygous 18-bp ITD knocked into the FLT3 gene were crossbred with the WT1 mutant mice, and Kaplan-Meier survival analysis was performed across genotypes. CBCs and BM cytospin morphology from moribund mutant mice were compared to wild type controls. To create a transplant model, 2e6 whole BM cells from each genotype were injected into lethally irradiated congenic mice. Competitive transplants were performed by injecting a 1:1 ratio of CD45.1 wild type (control) cells with CD45.2 WT1 mutant or wild type (test) cells into lethally irradiated C45.1 recipients.Results: We noted an expansion of lineage negative cells and various progenitor cell compartments in WT1 mutant (WT1mut) BM relative to wild type (wt); including the megakaryocyte-erythroid progenitor (MEP) compartment. WT1mut BM cells from two-month old mice showed an increased ability to serially replate in methylcellulose culture compared to wt BM cells, demonstrating aberrantly enhanced self-renewal capacity.WT1mut mice demonstrated a trend towards an inferior late survival compared to wt in survival analysis, and several moribund WT1mut mice were found to have anemia and erythrodysplasia. Most ITD mice developed a fatal myeloproliferative neoplasm (MPN), as previously described. Interestingly, double mutant mice (WT1mut+ITD) had an inferior survival compared to ITD (p <0.001) or WT1mut alone, and BM from moribund WT1mut+ITD mice demonstrated features of both erythrodysplasia and myeloproliferation, diagnostic of MDS/MPN. We then transplanted BM from each genotype into lethally irradiated congenic mice. WT1mut BM transplant recipients showed a trend toward inferior survival compared to wt BM recipients. Strikingly, all evaluable WT1mut recipient mice developed MDS in this model; manifested as decreased hemoglobin (p = 0.03), clear erythrodysplasia, and a decreased myeloid:erythroid (M:E) ratio. ITD BM recipients developed MPN as expected, and the majority of WT1mut+ITD BM recipients developed MDS/MPN. Finally, in a competitive transplant model, WT1mut BM cells exhibited an initial engraftment disadvantage, followed by a increasing engraftment advantage over wt BM cells at later time points - further supportive data that WT1mut BM causes dysfunctional hematopoiesis, a hallmark of MDS.Conclusion: BM cells from mice with the leukemogenic WT1 mutation R394W demonstrate enhanced self-renewal of hematopoietic progenitor cells, which could potentially prime cells for leukemic transformation upon acquisition of cooperative events. Importantly, transplanted mice with WT1 mutant BM consistently develop MDS, manifested as anemia and erythrodysplasia and contributing to a trend in decreased survival. Mice with both this WT1 mutation and a FLT3/ITD mutation develop a mixed MDS/MPN phenotype, which is a discrete diagnostic entity, and results in a more aggressive disease and inferior survival to mice with ITD mutations alone. These data provide new and important insights into the aberrant functional effects of WT1 mutations on hematopoiesis, and are the first to characterize the hematopoietic phenotype of a WT1 mutation in vivo. DisclosuresNo relevant conflicts of interest to declare.

O4.2 - Development of novel small molecule MerTK and Flt3 tyrosine kinase inhibitors for treatment of acute leukemia

ABSTRACT Background: Studies investigating the potential utility of novel small molecule dual MerTK and Flt3 inhibitors (MRX TKIs1) as therapy for AML and ALL will be presented. Methods: MerTK and Flt3 phosphorylation were measured using enzymatic and cell-based assays. Induction of apoptosis was determined using flow cytometry and colony-forming potential was assessed in methylcellulose or soft agar cultures. Orthotopic xenografts were generated in NOD/Scid/IL-2 gamma mice using a luciferase-expressing derivative of the 697 B-ALL cell line, the Molm14 AML cell line, or a Molm14 derivative with the FLT3-ITD D835Y or F691L mutation. Orthotopic patient-derived xenografts (PDX) were generated from primary AML samples. Inhibition of MerTK in bone marrow leukemia cells was assessed by immunoblot. Disease burden was measured using bioluminescence imaging or flow cytometry and median survival was determined. Results: MRX TKI inhibited MerTK and Flt3 with nanomolar potency and had limited off-target activity against 300 other kinases. MRX TKI decreased pro-survival signaling, induced apoptosis, and reduced colony-forming potential in leukemia cells. In B-ALL xenografts, MRX TKI inhibited MerTK phosphorylation in leukemic blasts leading to a dose-dependent reduction in tumor burden and a 2- to 3-fold increase in survival. MRX TKI also increased sensitivity to methotrexate, a chemotherapy currently used for treatment of ALL, resulting in reduced tumor burden and a 50% increase in survival compared to either agent alone. In a MerTK-expressing AML PDX, treatment with MRX TKI resulted in disease regression in blood, spleen, and bone marrow and a 2-fold increase in survival. MRX TKI was similarly effective in xenograft models of FLT3-ITD AML. Importantly, MRX TKI has activity against clinically-relevant FLT3 mutants that mediate resistance to current FLT3 TKIs. Conclusions: The very high potency, relative selectivity, oral bioavailability, target inhibition, and therapeutic efficacy mediated by MRX TKIs in MerTK and FLT3-dependent murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of acute leukemia.

Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta).

In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.