Murine Xenotransplant Model Research Articles

Isorhamnetin Regulates Programmed Death Ligand-1 Expression by Suppressing the EGFR-STAT3 Signaling Pathway in Canine Mammary Tumors.

Programmed death ligand-1 (PD-L1) is highly expressed in a variety of cancer cells and suggests a poorer prognosis for patients. The natural compound isorhamnetin (ISO) shows promise in treating cancers and causing damage to canine mammary tumor (CMT) cells. We investigated the mechanism of ISO in reducing PD-L1 expression in CMT cells. Clustered, regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) was used to mediate CD274 knockout in U27 cells. Then, monoclonal cells were screened and cultured. Nucleotide sequencing and expression of PD-L1 were detected. Additionally, we examined cell migration, invasion, and damage. Immunofluorescent staining of PD-L1 was examined in U27 cells. The signaling pathways were measured by Western blotting. Murine xenotransplantation models and murine immunocompetent allograft mammary tumor models were established to evaluate the effect of ISO therapy. Expression of Ki-67, caspase3, and PD-L1 were analyzed by immunohistochemistry. A pull-down assay was used to explore which proteins could bind to ISO. Canine EGFR protein was purified and used to detect whether it directly binds to ISO using a surface plasmon resonance assay. ISO inhibited the EGFR-STAT3-PD-L1 signaling pathway and blocked cancer growth, significantly increasing the survival rate of healthy cells. The cell membrane receptor EGFR was identified as a direct target of ISO. ISO could be exploited as an antineoplastic treatment of CMT by targeting EGFR to suppress PD-L1 expression.

Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis

AbstractSystemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor–independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Abstract 2863: Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies

Abstract Antitumor antibodies like Rituximab and Trastuzumab activate the patient’s immune system to engage malignant cells. Despite the advances achieved by their introduction in cancer treatment, an urgent medical need remains to improve and optimize the efficacy of antibody therapy. This may be achievable using the cytokine IL-15 which induces proliferation and activation of both, NK cells and CD8 T cells, and enhances their cytolytic capacity. So far, short half-life, poor accumulation at the tumor site and severe toxicity upon systemic application due to unspecific immune activation prevent the broad use of IL-15 for treatment of cancer patients (Conlon et al, JCO 2015). IL-15 predominantly acts as part of an immunological synapse with IL-15Rα expressed on antigen-presenting cells, which present IL-15 in trans to IL-15Rβγ receptor expressing cytotoxic lymphocytes. We here report on the generation of so called “modified immunocytokines” (MICs) which consist of an Fc-optimized (SDIE modification) CD19 or CD20 antibody for targeting of (malignant) B cells fused to an IL-15 mutant with abolished binding to IL-15Rα termed MIC19 or MIC20. The abrogation of IL-15Rα binding by the IL-15 mutation enables substitution of the physiological trans-presentation by target binding of the antibody, allowing for target antigen-restricted stimulation of the IL-15Rβγ on NK cells and CD8 T cells. Analyses of activation and toxicity with MIC19 compared to a CD19 immunocytokine with an unmodified IL-15 (IC19) or immunocytokines with unrelated specificity as control confirmed target antigen-restricted function of the MIC proteins. Killing of autologous and allogeneic CD19+/CD20+ target cells was clearly superior as compared to the CD19-directed Fc optimized antibody (Tafasitamab) and Rituximab that bear no cytokine activity. Compared to antibody treatment, the MIC proteins induced a pronounced and long-lasting proliferation of effector cells and were effective to treat leukemia in a xenograft (Nalm-6) leukemia mouse model. Anti-leukemic activity was further confirmed in a murine NSG xenotransplantation model using patient ALL cells and human NK cells as effectors. In summary, MIC constructs consisting of an Fc-optimized antibody and mutated IL-15 with deficient IL-15Rα binding induce target cell restricted activation and proliferation of NK cells, resulting in efficient anti-leukemic activity. Our MICs thus combine the benefits of Fc-optimized antibodies and IL-15 activity and constitute a promising novel treatment modality for B cell malignancies with low side effects that allow the application of truly effective doses. Citation Format: Ilona Hagelstein, Latifa Zekri, Boris Klimovich, Martina S. Lutz, Carsten Greve, Stefanie Müller, Melanie Märklin, Bastian Schmied, Gundram Jung, Helmut R. Salih. Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2863.

Chemotherapy-Free Targeted Anti-BCR-ABL+ Acute Lymphoblastic Leukemia Therapy May Benefit the Heart.

Simple SummaryRisk-adapted multiagent chemotherapy has led to a remarkable improvement in the life expectancy of patients with acute lymphoblastic leukemia (ALL). Nevertheless, in high-risk subgroups such as BCR-ABL+ ALL, relapse rates remain high without allogeneic hematopoietic stem cell transplantation, and the adverse effects of chemotherapy may cause acute and chronic cardiac complications or dysfunction. Here, we demonstrated that chemotherapy-free targeted therapies designed to optimize apoptosis induction in BCR-ABL+ ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery.Targeted therapies are currently considered the best cost–benefit anti-cancer treatment. In hematological malignancies, however, relapse rates and non-hematopoietic side effects including cardiotoxicity remain high. Here, we describe significant heart damage due to advanced acute lymphoblastic leukemia (ALL) with t(9;22) encoding the bcr-abl oncogene (BCR-ABL+ ALL) in murine xenotransplantation models. Echocardiography reveals severe cardiac dysfunction with impaired left ventricular function and reduced heart and cardiomyocyte dimensions associated with increased apoptosis. This cardiac damage is fully reversible, but cardiac recovery depends on the therapy used to induce ALL remission. Chemotherapy-free combination therapy with dasatinib (DAS), venetoclax (VEN) (targeting the BCR-ABL oncoprotein and mitochondrial B-cell CLL/Lymphoma 2 (BCL2), respectively), and dexamethasone (DEX) can fully revert cardiac defects, whereas the depletion of otherwise identical ALL in a genetic model using herpes simplex virus type 1 thymidine kinase (HSV-TK) cannot. Mechanistically, dexamethasone induces a pro-apoptotic BCL2-interacting mediator of cell death (BIM) expression and apoptosis in ALL cells but enhances pro-survival B-cell lymphoma extra-large (BCLXL) expression in cardiomyocytes and clinical recovery with the reversion of cardiac atrophy. These data demonstrate that therapies designed to optimize apoptosis induction in ALL may circumvent cardiac on-target side effects and may even activate cardiac recovery. In the future, combining the careful clinical monitoring of cardiotoxicity in leukemic patients with the further characterization of organ-specific side effects and signaling pathways activated by malignancy and/or anti-tumor therapies seems reasonable.

ENGRAFTMENT POTENTIAL OF CD34+ CELLS FROM SEVERE APLASTIC ANEMIA PATIENTS EXPANDED WITH UM171

Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34 + cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34 + cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34 + cells from patients with AA expanded with UM171 in a murine xenotransplantation model. CD34 + cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×10 4 UCB CD34 + cells or 8.250 AA patient CD34 + cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. We found that both progenies of UCB and AA BM CD34 + cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45 + cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded CD34 + cells. The enhanced engraftment capacity enabled by UM171 could allow for immune AA patients to be treated with a novel strategy, autologous transplantation of their own CD34 + expanded cells. The present study indicates that UM171 could be a promising candidate drug for treating patients with immune AA. More replicates (patients and controls) are required to firmly establish the benefit of UM171 in conferring repopulation potential to ex vivo expanded CD34 + cells from AA patients. In initial experiments, in vitro treatment with UM171 appears to increase the engraftment of CD34 + cells isolated from UCB and from the BM of immune AA patients. These results need to be confirmed in additional experiments.

E35 ablates acute leukemia stem and progenitor cells in vitro and in vivo.

Leukemia stem cells (LSCs) have critical functions in acute leukemia (AL) pathogenesis, participating in its initiation and relapse. Thus, identifying new molecules to eradicate LSCs represents a high priority for AL management. This work identified E35, a novel Emodin derivative, which strongly inhibited growth and enhanced apoptosis of AL stem cell lines, and primary stem and progenitor cells from AL cases, while sparing normal hematopoietic cells. Furthermore, functional assays in cultured cells and animals suggested that E35 preferentially ablated primitive leukemia cell populations without impairing their normal counterparts. Moreover, molecular studies showed that E35 remarkably downregulated drug‐resistant gene and dramatically inhibited the Akt/mammalian target of rapamycin signaling pathway. Notably, the in vivo anti‐LSC activity of E35 was further confirmed in murine xenotransplantation models. Collectively, these findings indicate E35 constitutes a novel therapeutic candidate for AL, potentially targeting leukemia stem and progenitor cells.

Artesunate induces mitochondria-mediated apoptosis of human retinoblastoma cells by upregulating Kruppel-like factor 6

Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Intravitreal chemotherapy achieves favorable clinical outcomes in controlling RB vitreous seeds, which are a common reason for treatment failure. Thus, a novel, effective and safe intravitreal chemotherapeutic drug is urgently required. The malaria drug artesunate (ART) recently demonstrated remarkable anticancer effects with mild side effects. The purpose of this study is to investigate the anti-RB efficacy, the underlying mechanism and the intraocular safety of ART. Herein, we verified that ART inhibits RB cell viability and induces cell apoptosis in a dose- and time-dependent manner. Microarray analysis revealed that Kruppel-like factor 6 (KLF6) was upregulated after ART treatment, and this was further confirmed by real-time PCR and western blot assays. Silencing of KLF6 expression significantly reversed ART-induced RB cell growth inhibition and apoptosis. Furthermore, ART activated mitochondria-mediated apoptosis of RB cells, while silencing KLF6 expression significantly inhibited this effect. In murine xenotransplantation models of RB, we further confirmed that ART inhibits RB tumor growth, induces tumor cell apoptosis and upregulates KLF6 expression. In addition, KLF6 silencing attenuates ART-mediated inhibition of tumor growth in vivo. Furthermore, we proved that intravitreal injection of ART in Sprague-Dawley (SD) rats is safe, with no obvious retinal function damage or structural disorders observed by electrophysiology (ERG), fundal photographs, fundus fluorescein angiography (FFA) or optical coherence tomography (OCT) examinations. Collectively, our study revealed that ART induces mitochondrial apoptosis of RB cells via upregulating KLF6, and our results may extend the application of ART to the clinic as an effective and safe intravitreal chemotherapeutic drug to treat RB, especially RB with vitreous seeds.

Optimized Induction of Mitochondrial Apoptosis By Combination Therapies with Venetoclax for Chemotherapy-Free Treatment of BCR-ABL+ Acute Lymphoblastic Leukemia in Preclinical Models

Background: BCR-ABL+ acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. BH3 mimetics induce mitochondrial outer membrane permeabilization (MOMP) linked to apoptosis induction by releasing BH3-only proteins BIM and/or BID from the anti-apoptotic factors BCL2, BCL-XL, MCL1, BCLW and BFL1. The BCL2-specific BH3 mimetic venetoclax (ABT-199) may provide an opportunity to improve pharmacotherapy of BCR-ABL+ ALL in particular for elderly patients not suitable for SCT.Aim: We aimed to rationally design optimized combination therapies for BCR-ABL+ ALL based on the molecular mechanisms of apoptosis induction by BH3 mimetics.Methods: We first biochemically characterized binding of BIM, a BH3 only activator of mitochondrial apoptosis, to BCL2, BCLXL and MCL1 and its release by BH3 mimetics in two BCR-ABL+ ALL cell lines. We next visualized and quantitated MOMP-induction by BH3 mimetics in viable cells. We then characterized the effects of dexamethasone and tyrosine kinase inhibitors (TKI) imatinib and dasatinib on BIM expression and calculated dose-effect combination indices (CI) for combination therapies in cell lines and two BCR-ABL+ ALL primograft models co-cultured with mesenchymal stem cells ex vivo. We finally used in vivo bioluminescence and survival analyses in murine xenotransplantation models to evaluate therapeutic efficacy in vivo.Results: In BCR-ABL+ BV173 and SUPB15 cells BIM but not BID binds to BCL2. BIM is rapidly released from BCL2 by venetoclax in a time and dose dependent manner. Release of BIM induces both MOMP (as defined by a decrease in mitochondrial membrane potential) and apoptosis (as defined by PARP cleavage and propidium iodide staining). Furthermore, BIM is strongly required for cytotoxicity of venetoclax, dasatinib and dexamethasone. Primary BCR-ABL+ ALL cells are more resistant against MOMP induction by venetoclax than BCR-ABL-negative ones, and BIM expression is reduced in these cells. Both, TKIs and dexamethasone augment BIM expression in BV173 and SUPB15 cells and act synergistically with venetoclax in cell lines and two BCR-ABL+ primografts (CI for the triple combination therapy of venetoclax, dexamethasone and dasatinib between 0.1 and 0.15, CI< 1.0 considered synergistic). Triple combinations with venetoclax, dexamethasone and TKIs efficiently attenuate leukemia progression in xenotransplantation models and, notably, the dasatinib- but not the imatinib-containing combination led to treatment- and leukemia-free long-term survival in a BCR-ABL+ mouse model.Conclusions: These data demonstrate efficacy of venetoclax in ALL. Although BCR-ABL inhibits venetoclax cytotoxicity, this inhibition can be overcome by triple combination therapy with venetoclax, dexamethasone and dasatinib. Since the triple combination therapy can be curative in preclinical xenotranplatation models clinical studies with oral chemotherapy-free regimens may be considered in particular for elderly patients not suitable for allogeneic SCT. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees.

Large-Scale Ex Vivo Generation of Human Red Blood Cells from Cord Blood CD34+ Cells.

The ex vivo generation of human red blood cells on a large scale from hematopoietic stem and progenitor cells has been considered as a potential method to overcome blood supply shortages. Here, we report that functional human erythrocytes can be efficiently produced from cord blood (CB) CD34+ cells using a bottle turning device culture system. Safety and efficiency studies were performed in murine and nonhuman primate (NHP) models. With the selected optimized culture conditions, one human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1% ± 6.2% and 50% ± 5.7% (mean ± SD) for CD235a+ cells and enucleated cells, respectively. The yield of erythrocytes from one CB unit (5 million CD34+ cells) could be, in theory, equivalent to 500 blood transfusion units in clinical application. Moreover, induced human erythrocytes had normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model. In NHP model, xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia. These findings suggested that the ex vivo‐generated erythrocytes could be an alternative blood source for traditional transfusion products in the clinic. Stem Cells Translational Medicine 2017;6:1698–1709

Targeted therapy for a subset of acute myeloid leukemias that lack expression of aldehyde dehydrogenase 1A1.

Aldehyde dehydrogenase 1A1 (ALDH1A1) activity is high in hematopoietic stem cells and functions in part to protect stem cells from reactive aldehydes and other toxic compounds. In contrast, we found that approximately 25% of all acute myeloid leukemias expressed low or undetectable levels of ALDH1A1 and that this ALDH1A1− subset of leukemias correlates with good prognosis cytogenetics. ALDH1A1− cell lines as well as primary leukemia cells were found to be sensitive to treatment with compounds that directly and indirectly generate toxic ALDH substrates including 4-hydroxynonenal and the clinically relevant compounds arsenic trioxide and 4-hydroperoxycyclophosphamide. In contrast, normal hematopoietic stem cells were relatively resistant to these compounds. Using a murine xenotransplant model to emulate a clinical treatment strategy, established ALDH1A1− leukemias were also sensitive to in vivo treatment with cyclophosphamide combined with arsenic trioxide. These results demonstrate that targeting ALDH1A1− leukemic cells with toxic ALDH1A1 substrates such as arsenic and cyclophosphamide may be a novel targeted therapeutic strategy for this subset of acute myeloid leukemias.

Safety and Efficacy of Megakaryocytes Induced from Hematopoietic Stem Cells in Murine and Nonhuman Primate Models.

Because of a lack of platelet supply and a U.S. Food and Drug Administration‐approved platelet growth factor, megakaryocytes have emerged as an effective substitute for alleviating thrombocytopenia. Here, we report the development of an efficient two‐stage culture system that is free of stroma, animal components, and genetic manipulations for the production of functional megakaryocytes from hematopoietic stem cells. Safety and functional studies were performed in murine and nonhuman primate models. One human cryopreserved cord blood CD34+ cell could be induced ex vivo to produce up to 1.0 × 104 megakaryocytes that included CD41a+ and CD42b+ cells at 82.4% ± 6.1% and 73.3% ± 8.5% (mean ± SD), respectively, yielding approximately 650‐fold higher cell numbers than reported previously. Induced human megakaryocytic cells were capable of engrafting and producing functional platelets in the murine xenotransplantation model. In the nonhuman primate model, transplantation of primate megakaryocytic progenitors increased platelet count nadir and enhanced hemostatic function with no adverse effects. In addition, primate platelets were released in vivo as early as 3 hours after transplantation with autologous or allogeneic mature megakaryocytes and lasted for more than 48 hours. These results strongly suggest that large‐scale induction of functional megakaryocytic cells is applicable for treating thrombocytopenic blood diseases in the clinic. Stem Cells Translational Medicine 2017;6:897–909

Drug-DNA Conjugated Gold Nanoparticles for the Treatment of Acute Myeloid Leukemia

INTRODUCTIONNovel therapies are needed for acute myeloid leukemia as only ~60% of children are cured despite maximally intensive cytotoxic chemotherapy. Functionalized gold nanoparticles (AuNP) are utilized for many biomedical applications and represent a potentially novel therapeutic approach in leukemia. We enhanced this technology by developing an AuNP system that selectively releases molecularly targeted drugs in leukemia cells. METHODSAuNPs were functionalized with short, double-stranded oligonucleotides with sequence complementarity to genes overexpressed in or unique to a leukemia cell (e.g. survivin or AML/ETO). Only the anti-sense oligonucleotide is covalently bound to the nanoparticle via a thiol linker. Thus, after entering leukemia cells, the endogenous targeted oncogene mRNA can bind to its complementary sequence on the AuNP and displace the non-covalently bound oligonucleotide which, in our system, is conjugated to the multi-tyrosine kinase inhibitor dasatinib. As a binary reaction, the amount of dasatinib-conjugated oligonucleotide released from the nanoparticle is directly proportional to both the presence and abundance of the complementary mRNA present in a cell. We evaluated AuNP uptake into multiple AML cell lines, as well as normal hematopoietic cells. The effect of dasatinib-AuNPs on dasatinib-sensitive leukemia cell lines was evaluated using proliferation assays, annexin V staining, and cell colony assays. Toxicity in T-cells and CD34+ cells was assessed with T-cell activation and p-SRC assays, respectively. RESULTSConjugation of dasatinib to an oligonucleotide complementary to a region of the survivin gene did not perturb its ability to inhibit SRC and c-KIT kinases in vitro. Leukemia cells demonstrate highly efficient AuNP uptake when cultured alone or with up to a 100-1000 fold excess of normal bone marrow cells. Treatment of K562 leukemia cells, containing a BCR/ABL translocation and high levels of survivin mRNA, with dasatinib-AuNPs resulted in dose-dependent p-SRC and p-CRKL inhibition. Furthermore, K562 cells also showed significantly impaired proliferation, increased apoptosis, and formed fewer colonies in methylcellulose. Conversely, normal T-cells and CD34+ cells, which express less survivin than leukemia cells, were significantly less affected by dasatinib-AuNPs than dasatinib alone as measured by T-cell activation assays and p-SRC levels, respectively. CONCLUSIONSThis method of using functionalized AuNPs to deliver and activate dasatinib in leukemia cells augments drug efficacy while minimizing toxicity and thus represents a novel therapeutic strategy for AML. Ongoing work is underway to characterize the mechanism of preferential AuNP uptake in leukemia cells and assessing in vivo activity using a murine xenotransplantation model of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies.

Turner syndrome is caused by complete or partial loss of the second sex chromosome and is characterized by spontaneous fetal loss in >90% of conceptions. Survivors possess an array of somatic and germline clinical characteristics. Induced pluripotent stem cells (iPSCs) offer an opportunity for insight into genetic requirements of the X chromosome linked to Turner syndrome. We derived iPSCs from Turner syndrome and control individuals and examined germ cell development as a function of X chromosome composition. We demonstrate that two X chromosomes are not necessary for reprogramming or maintenance of pluripotency and that there are minimal differences in gene expression, at the single cell level, linked to X chromosome aneuploidies. Formation of germ cells, as assessed in vivo through a murine xenotransplantation model, indicated that undifferentiated iPSCs, independent of X chromosome composition, are capable of forming germ-cell-like cells (GCLCs) in vivo. In combination with clinical data regarding infertility in women with X chromosome aneuploidies, results suggest that two intact X chromosomes are not required for human germ cell formation, qualitatively or quantitatively, but rather are likely to be required for maintenance of human germ cells to adulthood.

Ligand exchange on gold nanoparticles for drug delivery and enhanced therapeutic index evaluated in acute myeloid leukemia models.

Cancer chemotherapy is typically toxic. This problem could be addressed by using differences between cancer and normal cells for controlled delivery of drugs to cancer cells. One such difference is the ubiquitously elevated glutathione expression in cancer cells. We report a simple and versatile synthesis of water-soluble gold nanoparticles passivated with amine-containing molecules, which allow for controlled drug release via ligand exchange with bio-available glutathione. Taking methotrexate-passivated gold nanoparticles (Au:MTX) as an example, drug delivery and controlled release via glutathione-mediated ligand exchange was evaluated. Furthermore, the possibility of using Au:MTX to improve therapeutic index in acute myeloid leukemia (AML) models was examined invitro and invivo. Au:MTX exhibited cancer selectivity invitro. Au:MTX had an elevated potency toward an AML cell line THP-1 in a dosage range of 1-5 nM, and therefore an enhanced delivery of drug, whereas normal hematopoietic stem/progenitor cell (HSPC) growth was minimally affected by Au:MTX and MTX treatments within the same range of dosage. Invivo efficacy and safety of Au:MTX was evaluated in a murine xenotransplant model of primary human AML. Au:MTX treatment, compared to control groups including MTX-only and Au nanoparticle-only treatments, produced better leukemia suppression without added toxicity, indicating an enhanced therapeutic index.

High cytidine deaminase expression in the liver provides sanctuary for cancer cells from decitabine treatment effects

We document for the first time that sanctuary in an organ which expresses high levels of the enzyme cytidine deaminase (CDA) is a mechanism of cancer cell resistance to cytidine analogues. This mechanism could explain why historically, cytidine analogues have not been successful chemotherapeutics against hepatotropic cancers, despite efficacy in vitro. Importantly, this mechanism of resistance can be readily reversed, without increasing toxicity to sensitive organs, by combining a cytidine analogue with an inhibitor of cytidine deaminase (tetrahydrouridine). Specifically, CDA rapidly metabolizes cytidine analogues into inactive uridine counterparts. Hence, to determine if sheltering/protection of cancer cells in organs which express high levels of CDA (e.g., liver) is a mechanism of resistance, we utilized a murine xenotransplant model of myeloid cancer that is sensitive to epigenetic therapeutic effects of the cytidine analogue decitabine in vitro and hepato-tropic in vivo. Treatment of tumor-bearing mice with decitabine (subcutaneous 0.2mg/kg 2X/week) doubled median survival and significantly decreased extra-hepatic tumor burden, but hepatic tumor burden remained substantial, to which the animals eventually succumbed. Combining a clinically-relevant inhibitor of CDA (tetrahydrouridine) with a lower dose of decitabine (subcutaneous 0.1mg/kg 2X/week) markedly decreased liver tumor burden without blood count or bone marrow evidence of myelotoxicity, and with further improvement in survival. In conclusion, sanctuary in a CDA-rich organ is a mechanism by which otherwise susceptible cancer cells can resist the effects of decitabine epigenetic therapy. This protection can be reversed without increasing myelotoxicity by combining tetrahydrouridine with a lower dose of decitabine.

FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene is a gain-of-function mutation common in acute myeloid leukaemia (AML). It is associated with inferior prognosis and response to chemotherapy. Single base mutations at the FLT3 tyrosine kinase domain (TKD) also leads to a gain of function, although its prognostic significance is less well defined because of its rarity. The clinical benefits of FLT3 inhibition are generally limited to AML with FLT3-ITD. However, responses are transient and leukaemia progression invariably occurs. There is compelling evidence that leukaemia clones carrying both ITD and TKD mutations appear when resistance to FLT3 inhibitors occurs. Interestingly, the emergence of double ITD and TKD mutants can be recapitulated in vitro when FLT3-ITD+ leukaemia cell lines are treated with mutagens and FLT3 inhibitors. Furthermore, murine xenotransplantation models also suggest that, in some cases, the FTL3-ITD and TKD double mutants actually exist in minute amounts before treatment with FLT3 inhibitors, expand under the selection pressure of FLT3 inhibition and become the predominant resistant clone(s) during the drug-refractory phase. On the basis of this model of clonal evolution, a multipronged strategy using more potent FLT3 inhibitors, and a combinatorial approach targeting both FLT3-dependent and FLT3-independent pathways, will be needed to improve outcome.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Abstract 3876Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo.Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study.We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL.We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn’t observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing.To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells.Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001).Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. Disclosures:No relevant conflicts of interest to declare.

Combining Tetrahydrouridine with Decitabine Addresses Malignant Cell Sanctuary in the Liver, a Tissue That Expresses High Levels of Cytidine Deaminase,

Abstract 3509Non-cytotoxic DNA methyl-transferase 1 (DNMT1) depletion by decitabine induces p53-independent differentiation mediated cell cycle exit in acute myeloid leukemia (AML) cells and spares normal hematopoietic stem cells, providing a potentially important alternative to conventional apoptosis-based therapy. However, there are pharmacologic barriers that impede optimal clinical translation of pre-clinical observations. One major barrier is the enzyme cytidine deaminase (CDA), which drastically reduces the half-life of decitabine from many hours in vitro to a few minutes in vivo. This problem is significant since decitabine is S-phase specific in its mechanism of action, and for effective DNMT1 depletion, low peak levels but extended half-life are optimal. Furthermore, CDA is highly expressed in the liver and intestines, generating potential sanctuary sites from the effects of decitabine. Finally, upregulation of CDA expression is a potential mechanism by which AML cells may resist decitabine at the cellular level. To overcome the multiple CDA-mediated barriers to effective therapy, we evaluated the combination of very low dose decitabine with the CDA inhibitor tetrahydrouridine (THU) in a murine xenotransplantation model of aggressive human AML (THP1 AML cells – p53 and p16-null). Treatment groups (n=5 per group) received equal volume vehicle (PBS), low dose cytarabine (0.1 mg/kg subcutaneous [SC]) with (THU) (4mg/kg intraperitoneal [IP]), decitabine (0.1 mg/kg SC) with THU (4mg/kg IP), and decitabine alone (0.2 mg/kg SC). Median survival in decitabine (61 days) and THU-decitabine (70 days) treated mice was significantly extended compared to the PBS (38 days) and THU-cytarabine (50 days) treatment groups (Log Rank p=0.00156 (figure 1). In decitabine treated mice, eventual emergence of disease was concentrated in the liver, consistent with a sanctuary function of this organ. Combination of THU with decitabine largely eliminated liver infiltration by AML, reducing liver tumor load from average 5.2g in PBS treated mice to 0.8g in THU-Decitabine treated mice (t-test p<0.001, livers were harvested at different time-points corresponding to the Kaplan-Meier curve). In separate experiments without AML cell xenotransplantation, the non-cytotoxic, DNMT1 depleting character of the decitabine therapy was confirmed by hematologic and bone marrow evaluation for DNA damage by phospho-H2AX staining. In conclusion, combining THU with decitabine can address malignant cell sanctuary in the liver, an organ that expresses high levels of cytidine deaminase. The ability of this combination to address other aspects of CDA-mediated resistance to therapy is also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Chemical genomic screening reveals synergism between parthenolide and inhibitors of the PI-3 kinase and mTOR pathways

AbstractWe have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However, despite the activity of this agent, PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus, we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal, we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment, exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together, our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells.

High fidelity of phenotype and genotype in serial NOD/SCID pediatric acute lymphoblastic leukemia

Primary ALL cells are very difficult to culture in vitro and currently available cell lines only poorly reflect the heterogeneous nature of the disease. Many groups therefore use murine xenotransplantation models for primary pediatric ALL. But, to date it remains unclear as to what extent the characteristics of the original patient leukemia are retained over consecutive xenograft passages or whether there is selection for a clone with characteristics distinct from the bulk leukemia at diagnosis. We here address this question for 7 consecutive leukemias diagnosed in our clinic and directly transplanted onto unconditioned NOD/SCID-mice. After primary passage, clinical presentation, immunophenotype and gene expression profiles were highly similar to the diagnostic leukemia, with minor differences arising from the presence of normal human hematopoietic cells in primary patient material. Moreover, all characteristics analyzed remained stable after secondary and tertiary passages. The model precisely recapitulates the primary human leukemia in the mouse and thereby reflects the heterogeneity and inherent variability of different primary patient leukemia cells.