NOD-scid IL2Rγnull Mice Research Articles

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model.

It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo. [89Zr] Keytruda (groups=2; NSG-ctl (control) and hNSG-nblk (non-blocking), n=3-5, 3.2±0.4MBq/15-16μg/200μl) and [64Cu] Keytruda (groups=3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n=4, 7.4±0.4MBq /20-25μg/200μl) were administered in mice. PET-CT scans were performed over 1-144h ([89Zr] Keytruda) and 1-48h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g±SD) of 3.8±0.4 ([89Zr] Keytruda), and 6.4±0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p=0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144h ([89Zr] Keytruda) and 48h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively. Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

Demonstration of Rapid Functional Myeloid Cell Recovery after Infusion of a Universal Donor Ex Vivo Expanded Progenitor Cell Therapy As a Bridging Graft Source

Cord blood transplant (CBT) recipients are known to be at risk for delayed engraftment, resulting in an increased risk of morbidity and mortality post transplant. To overcome delayed engraftment, several groups have developed methods to expand ex vivo cord blood stem/progenitor cells (HSPC) which are under clinical evaluation. The majority of these expansion methods require identification of a patient specific cord blood donor as the source material for expansion, resulting in delays in the time to transplant and inherently carry a risk of product failure. In contrast, we have developed an off-the-shelf, universal donor ex vivo expanded cord blood (CB) derived HSPC product intended for use as a transient graft source which has been demonstrated to significantly reduce the incidence of documented bacterial infections in both transplant and non-transplant settings.1,2 Donor chimerism studies conducted weekly in the first month post transplant confirm that the initial early (days 0-14) myelomonocytic engraftment is derived largely from our universal donor graft. Herein, we now demonstrate that the these rapidly engrafting myelomonocytic cells generated from the universal donor graft source are mature and functionally intact human myeloid cells that can fight infectious organisms.CBT recipients enrolled on a phase II myeloablative CBT trial were included in these ancillary studies in which we evaluated the functional capacity of newly generated myeloid cells in peripheral blood. A flow cytometry-based assay which allowed quantitation of both phagocytosis and O2-dependent killing (oxidative burst) in myeloid cells was used. Strikingly, both monocytes (CD14+) and granulocytes (CD15+) in patients’ blood displayed similar frequencies of phagocytosis and O2-dependent killing of Staphlococcus aureus at day 7 (90.3%±2.2% phagocytosis and 88.9±5.2% O2-dependent killing n=2) when more than 95% of myeloid cells were from the expanded cell product compared to day 14 (69±13.2% phagocytosis and 94±2% O2-dependent killing, n=2) when more than 99% of cells were from a non-manipulated CB unit as a result of immunologic rejection by the T cell replete CB unit. These findings provide strong evidence that de novo generated myeloid cells from expanded HSPCs are as functionally competent as myeloid cells de novo generated from non-expanded CB.To better study the functionional properties of myeloid cells derived in vivo from rapidly repopulating expanded CB HSPCs, we transplanted either 20,000 non-expanded (NE-HSPC) CD34+ CB cells or their expanded progeny (E-HSPC) into sub-lethally irradiated NOD-scid IL2rγnull (NSG) mice. At day 7 after transplantation mice transplanted with E-HSPC showed 40-fold higher human engraftment in the bone marrow than mice transplanted with NE-HSPC (28.3 ± 1% vs 0.7±0.1%, n=3, p<0.001). Remarkably, the monocytes and granulocytes from their bone marrow showed a similar phagocytic potential to that of the monocytes and granulocytes of mice receiving NE-HSPC (60.4±3.2% vs 69.6±3.2%, n=3, p=0.06). Moreover, the frequency of phagocytosis in the myeloid cells isolated from the lungs of mice receiving E-HSPC was 7-fold higher than in the lungs of mice receiving NE-HSPC. It has been well documented that E-HSPC when infused alone, also contribute to long term engraftment in NSG mice, and therefore at 22 weeks after transplantation, the frequency of phagocytosis in monocytes and granulocytes isolated from the bone marrow of mice receiving E-HSPC remained similar to that in the bone marrow of mice receiving NE-HSPC for Staphlococcus aureus (55.1 ±1.9% vs 43.8%±7%, n=5, p=0.15), Escherichia coli (50.8±2% vs 49 ±8.3%, n=5, p=0.83) and Zymosan (43.7%±3 vs 49.9%±9.2%, n=5, p=0.54) indicating the continued generation of functional myeloid cells from long term repopulating cells.We demonstrate for the first time that ex vivo expanded CB HSPCs rapidly give rise to functional myelomonocytic cells in vivo in patients and immunodeficient mice. This study validates that our universal donor off-the-shelf, expanded CB HSPC cell product is a valuable resource for patients undergoing myeloablative CBT, and further warrants its widespread use in a non-transplant setting as a supportive "myeloid bridge" to mitigate treatment-related morbidity and mortality.1. Delaney C. et al. Lancet Haematol. 2016 Jul;3(7):e330-92. Summers C. et al. Blood 2014 124:3860 DisclosuresDelaney:Nohla Therapeutics: Employment.

Disruption of Wnt/β-Catenin Signaling Exerts Antileukemia Activity and Sensitizes with Tyrosine Kinase Inhibitors in FLT3 Mutated AML In Vivo

Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient.Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice.Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib).Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. DisclosuresKonopleva:Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

Small Molecule ST2 Inhibitors Cause Reduction of Soluble ST2 and Improve Gvhd and Survival In Vivo

Activation of the membrane-bound Suppression of Tumorigenecity 2 (mST2) by Interleukin-33 (IL-33) in T cells leads to type-2 (Th2) and Foxp3+ regulatory (Tregs) immune responses. The mST2/IL-33 axis is not engaged when a ST2 splice isoform containing only the ST2 ectodomain and acting as a decoy receptor, called soluble ST2 (sST2), sequesters IL-33. Clinically, elevated plasma sST2 has been reported and used as a prognostic biomarker in cardiac allo-rejection, inflammatory bowel disease, and graft-versus-host disease (GVHD). We hypothesized that releasing IL-33 from circulating sST2 may augment the mST2/IL-33 axis activation to modulate the Th1 and Th2/Tregs responses for treating immune related diseases. This is supported by our study of a ST2 antibody in the GVHD models (Zhang et. al., Sci. Trans. Med. 2015). Advantages of small-molecule therapies over biologics include easier administration especially by oral, superior tissue penetration, modifiable pharmacokinetic properties including half-life, and lower manufacturing cost. Here, we present the proof-of-concept of this approach using three small molecule ST2 inhibitors we discovered recently. Our data show two inhibitors cause reduction of sST2, alleviate GVHD and improve survival in two in vivo GVHD models.Our high throughput screening (HTS) using the AlphaLISA assay and computational analysis led to discover three classes of small molecules inhibiting ST2 binding to IL-33 which were confirmed by the functional cell-based HEK-Blue assay. Seven compounds showing low toxicity at 66 uM to peripheral blood mononuclear cells (PBMC) were selected for the dose escalation toxicity study in mice. Six of them showed no toxicity up to 20 mg/kg in 13 days while five of them can be tolerated at 40 mg/kg. The HTS workflow is summarized in Fig. 1.Three compounds (named iST2-1-3) were evaluated in the in vivo GVHD disease models. The first model is the transplantation of human PBMC to the NOD/SCID/IL2rgnull (NSG) mice one day after the mice received 300 cGy total body irradiation (TBI). Compounds were injected via the intraperitoneal route to the mice the day before transplantation and continued daily at their respective IC50 dosages for 21 days. At Day 14, both iST2-1 and iST2-2 caused reduction of IFNg+ CD4+ T cells and increase of Foxp3+CD4+ Tregs population in the human CD45+CD4+ cells extracted from the gastrointestinal (GI), the main GVHD target organ, compared with the DMSO control (Fig. 2). More profound effect exerted by iST2-1 was observed at Day 21. Plasma concentrations of human IFNg and sST2, indicators of Th1 response, showed the highest reduction in the iST2-1 treatment group. While the hsST2 plasma concentration is maintained at < 30 pg/ml throughout 28 days in the iST2-1 treatment group, escalation of hsST2 up to 180 pg/ml was found in the DMSO control and iST2-3 treated group. Plasma hIFNg was at least 50% lower in the iST2-1 treated group at day 14 and 21. The GVHD scores and survival of NSG mouse models paralleled the changes of GI T cell populations and systemic cytokines data; iST2-1 treatment showed decreased GVHD score to 3 from 6 in the DMSO control and improvement of survival rate to 45 % on day 35 whereas all mice in the DMSO control group were dead.The second model is the minor mismatched model of allogeneic hematopoietic cell transplantation (HCT) from B6 to C3H.SW mice. We observed similar trends of IFNg+ reduction and Foxp3+ increase in CD4+ Tcells from the GI (Fig. 3). Plasma sST2 and IFNg concentrations were greatly reduced on day 14 in the iST2-1 treated group compared with DMSO control (50 versus 10 ng/ml for sST2 and 350 versus 100 pg/ml for IFNg).Finally, we have collected the Small Angle X-ray Scattering data to construct structural models between iST2-1 and sST2 to guide the inhibitor optimization. The CRISP-Cas9 technology was also used to obtain sST2 deficient B6 mice. Allo-HCT using these donor sST2 deficient T cells will delineate the specific mode of action by our ST2 inhibitors on mST2 and sST2. Our findings reveal iST2-1 is an attractive small molecule ST2 inhibitor that can be used to study the mST2/IL-33 axis in disease models and further developed as therapeutics to treat GVHD. [Display omitted] [Display omitted] [Display omitted] DisclosuresPaczesny:Viracor-IBT Laboratories: Patents & Royalties.

Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma

Background aimsNatural killer (NK) cells can rapidly respond to transformed and stressed cells and represent an important effector cell type for adoptive immunotherapy. In addition to donor-derived primary NK cells, continuously expanding cytotoxic cell lines such as NK-92 are being developed for clinical applications. MethodsTo enhance their therapeutic utility for the treatment of B-cell malignancies, we engineered NK-92 cells by lentiviral gene transfer to express chimeric antigen receptors (CARs) that target CD19 and contain human CD3ζ (CAR 63.z), composite CD28-CD3ζ or CD137-CD3ζ signaling domains (CARs 63.28.z and 63.137.z). ResultsExposure of CD19-positive targets to CAR NK-92 cells resulted in formation of conjugates between NK and cancer cells, NK-cell degranulation and selective cytotoxicity toward established B-cell leukemia and lymphoma cells. Likewise, the CAR NK cells displayed targeted cell killing of primary pre-B-ALL blasts that were resistant to parental NK-92. Although all three CAR NK-92 cell variants were functionally active, NK-92/63.137.z cells were less effective than NK-92/63.z and NK-92/63.28.z in cell killing and cytokine production, pointing to differential effects of the costimulatory CD28 and CD137 domains. In a Raji B-cell lymphoma model in NOD-SCID IL2R γnull mice, treatment with NK-92/63.z cells, but not parental NK-92 cells, inhibited disease progression, indicating that selective cytotoxicity was retained in vivo. ConclusionsOur data demonstrate that it is feasible to generate CAR-engineered NK-92 cells with potent and selective antitumor activity. These cells may become clinically useful as a continuously expandable off-the-shelf cell therapeutic agent.

Clinical-scale expansion of CD34+ cord blood cells amplifies committed progenitors and rapid scid repopulation cells

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.

Identification of a Potential Antimalarial Drug Candidate from a Series of 2-Aminopyrazines by Optimization of Aqueous Solubility and Potency across the Parasite Life Cycle.

Introduction of water-solubilizing groups on the 5-phenyl ring of a 2-aminopyrazine series led to the identification of highly potent compounds against the blood life-cycle stage of the human malaria parasite Plasmodium falciparum. Several compounds displayed high in vivo efficacy in two different mouse models for malaria, P. berghei-infected mice and P. falciparum-infected NOD-scid IL-2Rγnull mice. One of the frontrunners, compound 3, was identified to also have good pharmacokinetics and additionally very potent activity against the liver and gametocyte parasite life-cycle stages.

A mouse model for ulcerative colitis based on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells from affected individuals.

ABSTRACTAnimal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in the UC-affected population nor can they be used to test the efficacy of inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGFβ1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFNγ might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase the translation of animal and clinical studies.

Abstract 2332: Patient-derived tumor xenografts in humanized NSG-SGM3 mice: a new immuno-oncology platform

Abstract Humanized mice engrafted with tumors enable in vivo investigation of the interactions between the human immune system and human cancer. We have recently found that humanized NOD-scid IL2Rγnull (NSG) mice bearing patient-derived xenografts (PDX) allow efficacy studies of check-point inhibitors. Next generation NSG strains include triple transgenic NSG mice expressing human cytokines KITLG, CSF2, and IL-3 (NSG-SGM3). Here we provide a direct comparison of check-point inhibitors evaluation in NSG and NSG-SGM3 mice engrafted with CD34+ human hematopoietic progenitor cells (HPCs) from the same donor and implanted with PDX tumors. Corroborating earlier studies, reconstitution of human immune system in the blood was faster and more robust in NSG-SGM3 compared to NSG recipients throughout the course of the study (18 weeks). Human CD45+ cells reached 25% of total blood cells at week 4 in hu-NSG-SGM3 mice and at week 9 in hu-NSG mice. A majority of blood hCD45+ cells in hu-NSG-SGM3 at week 4 were CD33+ myeloid cells. Circulating hCD3+ T cells reached 10% at week 9 and included regulatory T cells (Tregs), consistent with earlier studies. Hu-NSG mice displayed comparable hCD3+ T cells in the blood only at 12-15 weeks and did not contain Tregs. PDX tumors were then engrafted into partially HLA-matched hu-NSG-SGM3 mice at 9 weeks post engraftment. Two PDX models previously shown to respond to anti-PD1 therapy in hu-NSG mice, BR1126 and LG1306, were used. Treatment with the anti-PD-1 receptor antibody pembrolizumab (Keytruda) significantly reduced tumor growth in both models. Thus, PDX-bearing hu-NSG-SGM3 mice might serve as a new and improved platform for preclinical immuno-oncology efficacy studies. Citation Format: Li-Chin Yao, Mingshan Cheng, Minan Wang, Jacques Banchereau, Karolina Palucka, Leonard Shultz, Carol Bult, James G. Keck. Patient-derived tumor xenografts in humanized NSG-SGM3 mice: a new immuno-oncology platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2332.

IL-15 enhances the anti-tumor activity of trastuzumab against breast cancer cells but causes fatal side effects in humanized tumor mice (HTM).

Cancer immunotherapy has been shown to enhance established treatment regimens. We evaluated the potential reinforcing effect of IL-15 in trastuzumab treated humanized tumor mice (HTM) which were generated by concurrent transplantation of neonatal NOD-scid IL2Rγnull mice with human hematopoietic stem cells (HSC) and HER2 positive breast cancer cells (metastasizing SK-BR-3, solid tumor forming BT474).We found that trastuzumab treatment efficacy mainly depends on the immediate anti-tumorigenic cellular effect which is significantly enhanced by tumor interacting immune cells upon cotransplantion of HSC. However, trastuzumab treatment caused elevated CD44 expression on tumor cells that metastasized into the lung and liver but did not hinder tumor cell dissemination into the bone marrow. Moreover, in a number of SK-BR-3-transplanted animals disseminated CD44high/CD24low tumor cells lost trastuzumab sensitivity. Concerning the FcγRIIIa polymorphism, trastuzumab treatment efficiency in HTM was higher in mice with NK-cells harboring the high affinity FcγRIIIa compared to those with low affinity FcγRIIIa. In contrast, IL-15 caused the strongest NK-cell activation in heterozygous low affinity FcγRIIIa animals. Although IL-15 enhanced the trastuzumab mediated tumor defense, an unspecific immune stimulation resulted in preterm animal death due to systemic inflammation. Overall, treatment studies based on “patient-like” HTM revealed critical and adverse immune-related mechanisms which must be managed prior to clinical testing.

530. Lentiviral Vector Particles Pseudotyped with Wild-Type Baboon Endogenous Retrovirus (BAEV) Glycoprotein Outperform VSV-G Particles in Transducing Human CD34+ Cells Isolated from Cytokine-Mobilized Peripheral Blood or Bone Marrow

Lentiviral vector gene therapy for hematopoietic disorders caused by single gene mutation or deficiency has demonstrated success in clinical trials. However, efficient gene delivery is still challenging, requiring high multiplicity of infection (MOI) to achieve average vector copy numbers of at least 1 per cell. Conditions utilizing high MOI result in improved transduction rates and adequate copy number but these benefits can coincide with potential insertional mutagenesis. Attempting to achieve optimal gene transfer at reduced MOI, we directly compared self-inactivating (SIN) lentiviral vector particles encoding eGFP under control of a murine stem cell virus (MSCV) promoter/enhancer pseudotyped with envelope glycoproteins for BaEV or vesicular stomatitis virus (VSV). BaEV proteins were either full-length (BaWT) or lacked the terminal R-peptide (BaR-less), a version previously reported to improve infectious titer (TU, transducing units per mL) using transient production procedures. Using a standardized four-plasmid transient production protocol, cells transfected with R-less expression plasmids produced syncitia; an issue that resolved using BaWT. Titers were significantly higher for virus packaged with BaWT compared to R-less (BaR-less: 3.6 × 105 ± 6.6 × 104 N=12; BaWT 2.4 × 106 ± 4.5 × 105; n=10; p<0.001) determined by flow cytometry analysis of GFP expression in transduced HEK-293T cells. However, these values were 10-to 20-fold less than those observed for VSV-G (3.5 × 107 ± 4.5 × 106). Human CD34+ hematopoietic stem/progenitor cells isolated from bone marrow (BM) or cytokine-mobilized peripheral blood (mPB) of healthy donors were prestimulated and transduced overnight on retronectin-coated plates with BaWT particles at an MOI of 0.5, 1, 2, or 4. The following day, cells were either plated in methylcellulose to assess colony-formation (CFU), maintained in liquid culture (Bulk), or transplanted into NOD-scid IL2Rgnull (NSG) mice pre-conditioned with busulfan (n=10 mice/1×106 cells each). Gene transfer efficiency was gauged by expression of GFP following flow cytometry analysis of cells maintained in culture or visual inspection of colonies growing in methylcellulose using an inverted fluorescence microscope after 6 or 12 days, respectively. Transduction efficiency increased with viral MOI reaching peak levels using an MOI of 2 (Bulk, 93% GFP+; CFU, 98% GFP+). Six NSG mice survived to 14 weeks post-transplant and demonstrated engraftment of CD45+ cells ranging from 19 to 54% (39 ± 13%; mean ± SD) and GFP marking ranging from 32 to 59% (41 ± 10%) determined by flow cytometry. Finally, we compared GFP-encoding particles pseudotyped with VSV-G or BaWT normalizing for levels of p24 quantified by ELISA (BaWT, 1155.0 pg/ml, 3.3 × 106 TU; VSV-G, 1164.9 pg/ml, 5.1 × 107 TU) to account for differences in calculated titer. Prestimulated mPB CD34+ cells were transduced overnight and placed into methylcellulose or transplanted into 15 NSG mice each. Methylcellulose colonies were analyzed for GFP after 12 days of growth (BaWT, 95.6%; VSV-G, 76.6%). Transplanted mice will be analyzed at 16-18 weeks post-transplant to measure engraftment and GFP expression. We believe the stability of producer cells expressing BaWT, the high efficiency of transduction at low MOI, and the ability to transduce adequate numbers of cells with unconcentrated virus supernatant warrant continued optimization of transient production methods and development of a stable producer cell line utilizing BaWT envelope as an alternative to VSV-g.

Establishment of a humanized lupus model

Abstract Systemic lupus erythematosus (SLE), a systemic autoimmune disease, involves mild to severe manifestations in multiple organs. Currently, lupus-prone mouse models are used to understand the mechanisms of SLE development and discover new therapies. However, due to differences between rodents and humans, such as physiological development, disease progression, and response to therapies and drugs, many mechanisms and therapies discovered in mice may not be efficiently transferred from murine lupus models to SLE patients. Here we present a humanized mouse model of lupus as a new technology to bridge the gap between rodent models and patients. In our model, which involves vigorous selection of donors, we engraft peripheral blood mononuclear cells (PBMCs) from SLE patients into NOD/SCID/IL2Rgnull (NSG) mice to build a human-specific disease-based immune system in the mice. To avoid graft versus host disease, we stimulate PBMCs before transferring them into either female or male NSG mice. This method enhances the survival and maintenance of the engrafted cells. Human T cells and B cells that are critical to lupus development can be detected in the peripheral blood and spleen of humanized mice with no sex preference two weeks after engraftment. The model responds well to dexamethasone treatment with reduced proteinuria and decrease of spleen weight, indicating that our model could be used for preclinical studies for SLE. The therapeutic window can be as long as 3 weeks. This model allows for mechanistic studies and testing of potential therapies of SLE directly on human immune system in vivo without putting human life at risk.

Neither epithelial nor mesenchymal circulating tumor cells isolated from breast cancer patients are tumorigenic in NOD-scid Il2rgnull mice.

The quantitative evaluation of circulating EpCAM+ tumor cells (CTCs) in the peripheral blood of breast cancer patients provides an independent predictor of risk of progression in patients with metastatic disease. The present study investigated the tumorigenic potential of CTCs from cryopreserved mobilized leukapheresis products obtained from three metastatic breast cancer patients in remission. Cells were immunomagnetically separated if they expressed either the epithelial cell surface marker EpCAM, or CD90, a mesenchymal stromal cell marker associated with tumorigenic stem-like cancer cells. Cells were injected into the mammary fat pads of NOD-scid Il2rgnull mice. The injection of very large numbers of CTCs (0.3–1.5×106 CTCs per site, 20 sites per sample) in an optimized xenograft model did not result in the establishment of human-derived tumor xenografts. Four orders of magnitude fewer cells of the same CD90+ phenotype, but obtained from metastatic breast cancer pleural effusions, were highly tumorigenic in the same model system. These results favor the interpretation that circulating tumor cell load does not directly bear on metastatic potential, and that tumorigenic circulating breast cancer cells in patients with metastatic breast cancer are exceedingly rare. Furthermore, the CD44+/CD90+ phenotypic signature indicative of tumorigenicity in cells separated from metastatic or primary breast tumors does not have the same significance in circulating tumor cells.

Inhibition of DYRK1A Stimulates Human β-Cell Proliferation.

Restoring functional β-cell mass is an important therapeutic goal for both type 1 and type 2 diabetes (1). While proliferation of existing β-cells is the primary means of β-cell replacement in rodents (2), it is unclear whether a similar principle applies to humans, as human β-cells are remarkably resistant to stimulation of division (3,4). Here, we show that 5-iodotubercidin (5-IT), an annotated adenosine kinase inhibitor previously reported to increase proliferation in rodent and porcine islets (5), strongly and selectively increases human β-cell proliferation in vitro and in vivo. Remarkably, 5-IT also increased glucose-dependent insulin secretion after prolonged treatment. Kinome profiling revealed 5-IT to be a potent and selective inhibitor of the dual-specificity tyrosine phosphorylation–regulated kinase (DYRK) and cell division cycle–like kinase families. Induction of β-cell proliferation by either 5-IT or harmine, another natural product DYRK1A inhibitor, was suppressed by coincubation with the calcineurin inhibitor FK506, suggesting involvement of DYRK1A and nuclear factor of activated T cells signaling. Gene expression profiling in whole islets treated with 5-IT revealed induction of proliferation- and cell cycle–related genes, suggesting that true proliferation is induced by 5-IT. Furthermore, 5-IT promotes β-cell proliferation in human islets grafted under the kidney capsule of NOD-scid IL2Rgnull mice. These results point to inhibition of DYRK1A as a therapeutic strategy to increase human β-cell proliferation.

Inhibition of TGF-β Signaling Promotes Human Pancreatic β-Cell Replication.

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16INK4a, resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rgnull mice. These data reveal a novel role for TGF-β signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-β signaling to promote human β-cell replication.

Artemisinin-derived dimer ART-838 potently inhibited human acute leukemias, persisted in vivo, and synergized with antileukemic drugs.

Artemisinins, endoperoxide-containing molecules, best known as antimalarials, have potent antineoplastic activity. The established antimalarial, artesunate (AS), and the novel artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838) inhibited growth of all 23 tested acute leukemia cell lines, reduced cell proliferation and clonogenicity, induced apoptosis, and increased intracellular levels of reactive oxygen species (ROS). ART-838 was 88-fold more potent that AS in vitro, inhibiting all leukemia cell lines at submicromolar concentrations. Both ART-838 and AS cooperated with several established antileukemic drugs and newer kinase inhibitors to inhibit leukemia cell growth. ART-838 had a longer plasma half-life than AS in immunodeficient NOD-SCID-IL2Rgnull (NSG) mice, remaining at effective antileukemic concentrations for >8h. Intermittent cycles of ART-838 inhibited growth of acute leukemia xenografts and primagrafts in NSG mice, at higher potency than AS. Based on these preclinical data, we propose that AS, with its established low toxicity and low cost, and ART-838, with its higher potency and longer persistence in vivo, should be further developed toward integration into antileukemic regimens.

TALEN-mediated genetic inactivation of the glucocorticoid receptor in cytomegalovirus-specific T cells

AbstractCytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity. This group of patients remains the highest clinical risk group for recurrent and problematic infections. Here, we address this unmet clinical need by genetic disruption of the glucocorticoid receptor (GR) gene using electroporation of transcription activator–like effector nuclease (TALEN) messenger RNA. We demonstrate efficient inactivation of the GR gene without off-target activity in Streptamer-selected CMV-specific CD8+ T cells (HLA-A02/NLV peptide), conferring resistance to glucocorticoids. TALEN-modified CMV-specific T cells retained specific killing of target cells pulsed with the CMV peptide NLV in the presence of dexamethasone (DEX). Inactivation of the GR gene also conferred resistance to DEX in a xenogeneic GVHD model in sublethally irradiated NOD-scid IL2rγnull mice. This proof of concept provides the rationale for the development of clinical protocols for producing and administering high-purity genetically engineered virus-specific T cells that are resistant to the suppressive effects of corticosteroids.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Patient-Derived Xenografts Are Faithful Genomic and Phenotypic Models of Primary Leukemia and Respond to the IL3 Receptor Targeting Agent SL-401 In Vivo

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive acute leukemia/lymphoma recently classified as a malignant transformation of plasmacytoid dendritic cells (pDCs) and a subtype of acute myeloid leukemia (AML). BPDCN has no standard treatment and a poor prognosis, with median survival <1 year. A significant roadblock to better understanding BPDCN is a lack of adequate model systems. We generated patient-derived xenografts (PDX) of BPDCN in NOD/Scid/IL2rgnull (NSG) mice. Bone marrow or peripheral blood cells involved by BPDCN blasts (CD45 low, CD123 high, HLA-DR high, CD3 neg) were transplanted into irradiated NSG recipients. Nine of 16 BPDCNs caused lethal leukemia involving blood, spleen, and bone marrow 2-6 months after transplantation. All nine BPDCN PDXs were serially transplantable. Flow characterization of each patient's BPDCN and corresponding xenograft revealed no major differences in BDCA2, BDCA4, FCeR1, ILT7, or cytoplasmic TCL1 staining. All samples maintained high expression of the human interleukin-3 (IL3) receptor (IL3Ralpha/CD123), a hallmark feature of BPDCN. To further characterize BPDCN pathogenesis we performed whole transcriptome sequencing (RNA-seq) on sorted blasts from 11 patients and on normal pDCs isolated from 4 healthy donors. These were compared to RNA-seq in six PDXs. The spectrum of mutations in BPDCN transcriptomes overlapped with that seen in other hematologic malignancies, particularly myeloid disorders, and was similar to reported DNA mutations in BPDCN, including in ASXL1, CTCF, IDH2, NRAS, RUNX1, STAG2, TET2, and TP53. Particularly striking was the presence of a canonical mutation in an RNA splicing factor in 7 of 11 cases (SRSF2 P95H/L/R in four, ZRSR2 R295* and gene locus deletion in two, and SF3B1 K666N in one). Known oncogenic mutations in the original disease were retained in the PDXs, including all splicing factor mutations, with the exception of an IDH2 R140Q that was lost in one PDX. BPDCN PDXs grouped together in unsupervised clustering of expression profiles, distinct from AML and ALL PDXs in an analysis of 134 models from the DFCI Public Repository of Xenografts (http://PRoXe.org). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways associated with differentially expressed genes between primary BPDCNs and non-malignant pDCs revealed signatures related to dendritic cell activation, cell cycle, and apoptosis. In addition, 3 of the top 11 sets were genes involved in mRNA processing, mRNA splicing, and processing capped intron-containing pre-mRNAs (all FDR<1e-6). To test the efficacy of BPDCN-targeted therapy using primary human leukemias in vivo, we performed a pre-clinical trial in NSG mice using SL-401, a recombinant biologic consisting of a fusion protein of IL3 and diphtheria toxin. Three independent BPDCN xenografts were injected into 20 NSG mice each, and followed by weekly peripheral blood monitoring for human CD45 and CD123. When leukemia burden reached >0.5% in at least half of the mice in the cohort, animals were randomized to receive SL-401 at 100 ug/kg or vehicle intraperitoneally daily for 5 days. Two mice in each group were sacrificed at day 7 for response assessment, and peripheral blood was followed weekly in the remaining mice for evidence of progression (>5% human CD45/CD123-positive cells). 7 days after treatment, mice receiving SL-401 had dramatic reductions in BPDCN in the peripheral blood, spleen, and bone marrow (0.31% vs 37.6% in marrow of SL-401 vs vehicle). SL-401 prolonged progression-free survival in all BPDCNs tested (12 vs 48 days, P<0.0001 by log-rank test). At the time of progression after SL-401, relapsing mice were re-randomized to receive a 2nd and in some cases 3rd cycle of SL-401 or vehicle. Repeated treatment in mice that progressed after SL-401 resulted in second and third peripheral blood remissions. All PDXs responded to SL-401 including those with and without splicing factor and TP53 mutations. CD123 expression was maintained at high levels on all SL-401 treated BPDCNs even after repeated cycles. Primary xenografts of BPDCN are faithful models of the human disease, maintain genetic and transcriptomic characteristics of the original tumor, and respond to multiple courses of IL3-DT in vivo, suggesting that they provide a valuable resource to study disease biology and response/resistance to targeted therapy. DisclosuresChen:Stemline Therapeutics, Inc.: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics, Inc.: Research Funding.

A Photosensitizer Verteporfin Has Light-Independent Anti-Leukemic Activity for Ph-Positive Acute Lymphoblastic Leukemia and Synergistically Works with Dasatinib in Vivo

Despite of great improvement of treatment outcome by ABL kinase inhibitors, Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is still intractable disease, and novel therapeutic agents are anticipated. We recently developed a high-throughput drug screening system using patient-derived xenograft (PDX) cells. PDX cells highly maintain phenotypes of primary malignant cells, such as heterogeneity of cancer cells, slower growth rate than cell lines, and microenvironment dependency. Drug screening by PDX cells can pick up anti-tumor reagents with new mechanisms that were overlooked by conventional cell-line based screenings. Here, we discovered verteporfin, an approved drug for macular degeneration, as a candidate for novel therapeutic agent of Ph+ ALL.We established PDX of 3 Ph+ ALL patients (PhLO, PhLK, and PhLH) by transplanting primary Ph+ ALL cells into NOD/SCID/IL-2Rgnull (NOG) mice. We developed a high-throughput drug screening system using one of these PDX cells (PDX screening) and screened the library of 3440 compounds containing approved drugs and pharmacologically active reagents. The profile of drugs selected by PDX screening was quite different from that by the screening using Ph+ ALL cell line (Cell line screening). Verteporfin was selected by PDX screening, whereas it did not demonstrate very high anti-leukemic effect in Cell line screening.We confirmed the anti-leukemic effect of verteporfin ex vivo using the 3 PDX cells and ALL-1, the cell line used in the Cell line screening. All 3 PDX cells were more sensitive to verteporfin than ALL-1 (EC50: PhLO cells, 228 nM; PhLK cells, 1.8 µM; PhLH cells; 395 nM; ALL-1, 3.93 µM). In addition, combined use of verteporfin and dasatinib, an ABL kinase inhibitor used for the treatment of Ph+ ALL, showed synergistic growth suppression of PhLO cells. Combination index (CI) values calculated by combination index algorism were less than 1.0 in most combinations of 16 data points (CI mean, 0.73; CI range, 0.28-1.34).Furthermore, the mechanism of action of verteporfin was intensively investigated. In combination with red light irradiation, verteporfin induces apoptosis of tumor cells through production of reactive oxygen species (ROS), which is known as photodynamic therapy. We revealed that verteporfin produced ROS light-independently in PhLO cells and induced their apoptosis. Verteporfin-induced apoptosis was inhibited by the addition of reduced glutathione to the culture medium, suggesting the large involvement of ROS production in the verteporfin-induced apoptosis.Finally, we assessed the in vivo effect of verteporfin. NOG mice transplanted with PhLO cells were treated with vehicle, verteporfin (140mg/kg/day), dasatinib (20mg/kg/day), or both of them from day 22 to day 28. Dasatinib was intraperitoneally injected and verteporfin was administered by continuous subcutaneous injection using osmotic pumps because of its very short half-life. We measured the ratios of leukemic PDX cells in bone marrow and spleen on day 28. Both single therapies by verteporfin and dasatinib significantly reduced the leukemia cell ratios in spleen and the combination therapy of them further reduced leukemia cells in spleen (Figure A). In bone marrow, both single therapies demonstrated weaker anti-leukemic effect than in spleen, but the combination therapy showed significantly enhanced effect, indicating the synergistic effect between them in vivo (Figure B). These results indicate the promisingness of verteporfin as a new anti-leukemic reagent and PDX screening as a new strategy for the development of anti-cancer drug. [Display omitted] DisclosuresNaoe:Pfizer Inc.: Research Funding; Toyama Chemical Co.,LTD.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Patents & Royalties, Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co.,LTD: Patents & Royalties; Celgene K.K.: Research Funding; Astellas Pharma Inc.: Research Funding. Kiyoi:Yakult Honsha Co.,Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; MSD K.K.: Research Funding; Eisai Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Teijin Ltd.: Research Funding; Japan Blood Products Organization: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Biomarker and Gene Profiling Analyses in Acute Myeloid Leukemia during Treatment with Hedgehog Signaling Inhibitor

Background: Cumulative evidence suggests that dormant self-renewing leukemic stem cells (LSCs) contribute to acute myeloid leukemia (AML) propagation and relapse by evading conventional chemotherapies that target cycling cells. Therefore, there is a great need to find innovative therapies for AML that eliminate LSCs by targeting their specific properties. Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in maintenance of the LSC populations in several experimental systems. PF-0444913 (PF-913) is a novel oral small molecule inhibitor that selectively binds and targets Smoothened (SMO), a membrane protein regulating the Hh pathway. Treatment with PF-913 has shown promising results regarding safety, tolerability, and early signs of efficacy in the early phase study of hematologic malignancies, including AML (Jamieson, et al. ASH, 2011). However, the detailed mode of action and biomarkers remain to be elucidated in AML therapy with the Hh pathway inhibitors.Methods: We used AML cell lines and patient-derived primary AML cells and bone marrow cells in PF-913 treatment. Using RQ-PCR assays, we examined the change of gene expressions in the canonical Hh pathway molecules. We carried out comprehensive gene set enrichment analysis (GSEA), and pathway analysis during PF-913 treatment both in the pre-clinical experimental systems and the clinical setting (bone marrow blast-rich mononuclear cells derived from AML patients during the PF-913 mono-therapy). We also examined cell cycle analyses using flow-cytometory and Ki-67 immuno-staining assays.Results: In primary AML cells, the Hh signaling pathway was activated more in CD34+ cells than in CD34- cells. In vitro treatment with PF-913 induced a minimal cell death-accompanied decrease of the quiescent (Hoechst-33342low/Pyronin-Ylow) cell population. Through the serial transplantation immunodeficient NOD/SCID/IL2rgnull (NOG) mouse model xenotransplanted with primary AML cells, in vivo treatment with PF-913 attenuated leukemia initiation potential in AML cells (0% human-CD45+ cells in bone marrow cells derived from the 2nd recipient mice), while limiting reduction of tumor burden in the primary xenotransplanted recipient mice. In the NOG mouse model and the ex vivo culturing system of AML cell lines, GSEA revealed that PF-913 treatment induced effects on the self-renewal signatures and the cell-cycling regulations associated with LSC-like properties. Moreover, GSEA revealed that PF-913 treatment in the clinical setting modulated the LSC-like signature, the cell-cycling regulation signature, and the chemokine activity signature in the AML bone marrow cells. Ki-67 immuno-staining of bone marrow derived from AML patients showed that PF-913 treatment temporarily increased cell-cycling status during shorter periods of treatment. We also examined the pluripotency factor, Nanog expression in bone marrow cells derived from AML patients during the PF-913 therapy, based on the previous report that downstream effectors in the Hh pathway, GLI1 and GLI2, directly bind to the Nanog promoter and that the GLI-Nanog axis promotes stemness and growth in several cancers. Change of Nanog transcripts was closely associated with the GLI-target genes in bone marrow blast-rich mononuclear cells derived from AML patients during the PF-913 therapy. Furthermore, by backing to the pre-clinical experimental systems, we are investigating more detailed mechanisms of Nanog regulation during PF-913 treatment and more rationale methods of combination treatment with PF-913 and other candidate drugs (such as conventional chemo-drugs and other type of molecular targeted inhibitors).Conclusion: Gene profiling analyses revealed that treatment with Hh signaling inhibitor, PF-913 modulates self-renewal and cell-cycling signatures in AML, and Nanog transcript can be a responsive biomarker during the therapy.Our findings imply that PF-913 treatment can improve AML therapy through sensitizing dormant LSCs to chemotherapy and overcoming residual LSC-like diseases in the bone marrow microenvironment. DisclosuresMinami:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Naoe:Astellas Pharma Inc.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Toyama Chemical CO., LTD.: Research Funding; Celgene K.K.: Research Funding. Minami:Novartis: Honoraria, Other: Member of IDMC , Research Funding.