Identification and functional study of novel oligonucleotides: CpG Seq 13 and CpG Seq 19

Immunotherapy ◽  
2021 ◽  
Author(s):  
Hailing Liu ◽  
Shan Meng ◽  
Nan Yang ◽  
Jinqiu Chen ◽  
Huan Yao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Functional Study ◽  
Therapeutic Effects ◽  
Cpg Oligodeoxynucleotides ◽  
Immune Adjuvant ◽  
Acute Myeloid

Aim: This study explored new immunoadjuvants with stronger immune activity to enhance therapeutic effects against leukemia. Materials & methods: Whole blood and bone marrow of acute myeloid leukemia (AML) patients and healthy volunteers were collected. Isolated mononuclear cells were treated with two newly designed CpG oligodeoxynucleotides, CpG sequence 13 and 19, and known CpG oligodeoxynucleotides and analyzed via flow cytometry. Results: CpG Seq 13 and 19 possess strong immune activation and enhance the proliferation, degranulation and cytotoxicity of T cells. They also inhibit AML cell proliferation. When CpG Seq 13/19 are combined with anti-OX40 antibodies, the cytotoxicity of T cells on AML cells are further enhanced. Conclusion: CpG Seq 13 and 19 are strong immune adjuvant candidates for AML treatment.

scholarly journals Therapeutic Targeting of Mesothelin in Acute Myeloid Leukemia with Chimeric Antigen Receptor T Cell Therapy

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11 ◽  
Author(s):  
Quy Le ◽  
Sommer Castro ◽  
Thao T. Tang ◽  
Anisha Loeb ◽  
Amanda R. Leonti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cell Surface ◽  
Myeloid Leukemia ◽  
Car T Cells ◽  
Car T ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is one of the most highly refractory hematologic malignancies despite intensive combination chemotherapy and bone marrow stem cell transplantation. Lack of curative treatments is in large part due to our poor understanding of the disease biology and paucity of therapeutic targets. In an effort to identify actionable targets, we recently completed the largest genome, epigenome and transcriptome profiling of AML in nearly 3000 children and young adults. This discovery effort has led to the identification of a library of novel AML-restricted targets (high expression in AML, minimal-to-no expression in normal hematopoiesis) for therapeutic development. One such target was MSLN which encodes for mesothelin, a cell surface adhesion molecule that is highly expressed in 30-50% of AML cases in pediatric (Children Oncology Group) and adult (MD Anderson) cohorts and is entirely absent in normal bone marrow and peripheral blood CD34+ cells. MSLN expression in normal tissues is confined to mesothelial cells lining the pleura, pericardium, and peritoneum. Previous studies targeting MSLN in solid tumors have demonstrated clinical efficacy with minimal toxicities. Given that T cells genetically modified to express chimeric antigen receptors (CARs) are extremely effective at eradicating relapsed and refractory malignancy, we developed MSLN-directed CAR T cells for pre-clinical evaluation in AML. Methods: From primary patient samples, we verified MSLN expression by RT-PCR and confirmed mesothelin surface protein expression on leukemic blasts by flow-cytometry as well as detected soluble mesothelin in the plasma by ELISA. The VH and VL sequences from Amatuximab were used to create the scFv domain of the standard CAR (41-BB and CD3Zeta). For in vivo CAR T study, Nomo-1 cells, which express endogenous level of MSLN, and Kasumi-1 cells engineered to express MSLN with a lentivirus construct (Kasumi-1 MSLN+) were transplanted into NSG mice. Mock transduced MSLN-directed CAR T cells were infused 1 week (Nomo-1) and 2 weeks (Kasumi-1 MSLN+) following leukemic cell injection. Leukemic burden was measured by bioluminescence IVIS imaging weekly. For in vitro study, Nomo-1 cells were treated with GM6001 (50uM), a metalloprotease inhibitor, or DMSO control for 48 hr prior to evaluation of surface mesothelin by flow cytometry and soluble mesothelin in the culture supernatant by ELISA. Results: In vivo cytotoxicity of CAR T cells against Nomo-1 and Kasumi-1MSLN+ AML models demonstrated potent, target-dependent tumor killing. After 1- and 2-weeks post CAR T infusion, leukemic cells were eradicated in both Nomo-1 (p<0.0005, week 2, Figure 1A) and Kasumi-1 MSLN+ xenografts (p<0.005 at week 2, Figure 1B). Mesothelin undergoes shedding at the cell membrane as a result of ADAM17-mediated cleavage. Blocking ADAM17 activity with GM6001 in Nomo-1 cells led to increased cell surface mesothelin (Figure 1C) with a corresponding reduction in the shed form (Figure 1D), suggesting that GM6001 treatment stabilizes mesothelin on the cell surface. Furthermore, GM6001 treatment during co-culture of Nomo-1 and CAR T cells enhanced cytolytic activity of CAR T cells (Figure 1E). GM6001 treatment did not significantly impact cell viability of Nomo-1 cells in the absence of CAR T cells (data not shown). Conclusion: In this study, we demonstrate that mesothelin is a viable therapeutic target and a potential diagnostic biomarker in AML. We show that MSLN CAR T cells were highly effective in eliminating MSLN-positive AML cells in vitro and in vivo. Shedding contributes to the loss of mesothelin antigen and provides a source of soluble mesothelin that may interfere with antibody-based therapies, including CAR T cells. Modulating MSLN shedding by inhibiting ADAM17-mediated cleavage resulted in stabilized mesothelin and improved CAR T cell functionality. This work warrants further evaluation of MSLN CAR T cells to be tested in clinical trials for AML and demonstrates that inhibiting MSLN shedding is a promising approach to improve CAR T efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals 871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A912-A912
Author(s):  
Rebecca Moeller ◽  
Julian Scherer ◽  
Sadik Kassim
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Leukemic Stem Cells ◽  
Cd69 Expression ◽  
Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

scholarly journals Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia

Pharmaceutics ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1681
Author(s):  
Patrick Connerty ◽  
Ernest Moles ◽  
Charles E. de Bock ◽  
Nisitha Jayatilleke ◽  
Jenny L. Smith ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Sirna Delivery ◽  
Standard Of Care ◽  
Lipid Nanoparticles ◽  
Protein Product ◽  
Peripheral Blood Mononuclear ◽  
Acute Myeloid

Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Here, we describe the development of a new siRNA-loaded lipid nanoparticle for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Transcriptomic analysis of children with AML identified LINC01257 as specifically expressed in t(8;21) AML and absent in healthy patients. Using NxGen microfluidic technology, we efficiently and reproducibly packaged anti-LINC01257 siRNA (LNP-si-LINC01257) into lipid nanoparticles based on the FDA-approved Patisiran (Onpattro®) formulation. LNP-si-LINC01257 size and ζ-potential were determined by dynamic light scattering using a Malvern Zetasizer Ultra. LNP-si-LINC01257 internalization and siRNA delivery were verified by fluorescence microscopy and flow cytometry analysis. lncRNA knockdown was determined by RT-qPCR and cell viability was characterized by flow cytometry-based apoptosis assay. LNP-siRNA production yielded a mean LNP size of ~65 nm with PDI ≤0.22 along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment was able to successfully ablate LINC01257 expression which was accompanied by a significant 55% reduction in total cell count following 48 h of treatment. In contrast, healthy peripheral blood mononuclear cells (PBMCs), which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. This is the first report demonstrating the use of LNP-assisted RNA interference modalities for the silencing of cancer-driving lncRNAs as a therapeutically viable and non-toxic approach in the management of AML.

CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Blood ◽  
2002 ◽  
Vol 100 (6) ◽  
pp. 2132-2137 ◽  
Author(s):  
Carmen Scheibenbogen ◽  
Anne Letsch ◽  
Eckhard Thiel ◽  
Alexander Schmittel ◽  
Volker Mailaender ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Wilms Tumor ◽  
Proteinase 3 ◽  
Ifn Γ ◽  
Tumor Gene ◽  
Acute Myeloid

Abstract Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

The Growth Inhibition and Apoptosis Induction of Acute Myeloid Leukemia Blast Population by the Blocking IGF-IR Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4515-4515
Author(s):  
Si Sun ◽  
Yanli He ◽  
Xingbing Wang ◽  
Wei Liu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Pi3k Pathway ◽  
Leukemia Cell Line ◽  
Free System ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract The insulin-like growth factor-1receptor (IGF-1R) is overexpressed in a variety of tumors and has been associated with cancer development. Here, we analysis the IGF-IR expression on the bone marrow cells from 45 newly diagnosed patients with acute myeloid leukemia (AML) by flow cytometry. IGF-1R universally expressed on AML blasts and the leukemia cell line HL-60, did not show significant correlation with FAB subtypes. However, the bone marrow cells from AML patients with high myeloblast counts (>80%) generally showed brighter IGF-IR expressions, which indicated the IGF-IR pathway might play an important role for AML blast proliferation and survival. Indeed, blocking the IGF-1R pathway by neutralizing monoclonal antibodies could reduce the proliferation of HL-60 cells by 38.28% at 48 hr. This inhibitory effect on blast growth was observed in 4 of 5 AML samples. In the same IGF-1R blocking treatment, the apoptosis of HL-60 cells was significantly induced, resulting in apoptosis of 57% of the cell population with the measurement of Annexin V vs PI staining by flow cytometry. The control contained only 20% apoptotic cells. We also demonstrated that the blockade of the IGF-1R pathway inhibited the phophorylation of the PI3K pathway component Akt in HL-60 cells when cultured in a serum free system with a supplement of 50ng/ml exogenous IGF. Since PI3K pathway activation greatly contributes to the proliferation, survival and drug resistance of AML, it is of interest to study whether blockading IGF-IR could also inhibit the PI3K pathway in primary AML blasts and synergize other anti-leukemia agents to improve the therapeutic effectiveness. Conclusions: IGF-IR may play an important role in the proliferation and survival of the AML blast population; Blocking the IGF-IR pathway could significantly inhibit the growth of AML blasts and considerably induce the apoptosis of AML blasts; IGF-IR could become a critical molecular target in anti-leukemia drug discovery.

Achieving Hematologic Remission with Combination Chemotherapy and Donor Leukocyte Infusions for Relapse of Acute Myeloid Leukemia Six Years After Human Leukocyte Antigen-Mismatched Transplantation: A Case Report

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4476-4476
Author(s):  
Jingyan Xu ◽  
Jian Ouyang ◽  
Rong-Fu Zhou
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Combination Chemotherapy ◽  
Myeloid Leukemia ◽  
Low Dose ◽  
Mononuclear Cells ◽  
Bone Marrow Examination ◽  
Intensive Chemotherapy ◽  
Bone Marrow Blast ◽  
Acute Myeloid

Abstract Abstract 4476 Hematopoietic Stem Cell Transplantation (HSCT) from partially HLA-matched (haploidentical) family donors represents a promising therapy for high-risk acute myeloid leukemia (AML). However, for patients with AML relapsed after HSCT from an HLA-mismatched familial donor, there is no standard therapy. They may receive conventional chemotherapy, cyclosporine withdrawal, second HSCT, and donor leukocyte infusion (DLI) with or without prior mobilization. Recently, combination chemotherapy and DLI showed achieving hematologic remission. We report a case of successful combination chemotherapy and donor leukocyte infusions from original donor in a patient with AML relapsing 6 years after HSCT from an HLA-Mismatched Familial Donor. A 37-year-old male presented with fever in June 2003.Bone marrow aspirate confirmed the diagnosis of AML(M5 subtype according to FAB classification). The patient initially received intensive chemotherapy. However, the patient with AML that was refractory to conventional therapy. He received HSCT in first CR from his mother 1-loci HLA-mismatched (HLA-A) using BuCY- Conditioning regimen on June 11, 2004. He showed a medullary relapse 6 years after HSCT. His bone marrow blast counts exceeded 80% with 8.25% of donor karyotypes (46 XX FISH). We decided to try to use his mother as the donor for DLI. Cytoreductive chemotherapy was commenced prior to DLI. He was treated twice with DLI on August 02, 2010 and September 23, 2011. He was treated chemotherapy before in first DLI, chemotherapy regimens; FLAG-ida [fludarabine 30 mg/m2/d from day-6 to-2 of cell infusion, cytosine arabinoside 2 g/m2/d from day-6 to-2 of cell infusion, idarubicine 20 mg/d day-1 and G-CSF 300μ g/day from day-7 to +30]. The donors received G-CSF 10μ g/kg subcutaneously daily starting day-3 of cell infusion for 5 days. Donor peripheral blood mononuclear cells were collected by CS-3000 Plus cell separator (Baxter Corp.) on the fifth days of G-CSF administration and infused through a central venous catheter into the patients on the same day. 8.33×107/kg mononuclear cells, 6×107/kg CD3+ cells were reinfused without manipulation. Cyclosporine at the dose of 3 mg/kg were administered for the prevention of GVHD. On days 36 Bone marrow blast counts exceeded 45% with 44% of donor karyotypes (46 XX FISH) after first Chemo-DLI. He received cyclosporine withdrawal. He was treated chemotherapy by low-dose Ara-C and aclarubicin with concomitant use of G-CSF before in second DLI.,chemotherapy regimens;CAG[ Low-dose Ara-C was given subcutaneously at a dosage of 10 mg/m2 every 12 hours on days-14 to-1. Aclarubicin was administered intravenously at a dosage of 7 mg/m2 on days-14 to-7. Recombinant G-CSF was given subcutaneously at a dosage of 200μ g/m2 per day on days-14 to-1]. On day 0,1.4×108/kg mononuclear cells,1×108/kg CD3+ cells were reinfused. On days 25 bone marrow examination showed CR with 89% of donor karyotypes (46 XX FISH). He was treated consolidation chemotherapy by regimens; CAG.On days 62 bone marrow examination showed CR with 100% of donor karyotypes (46 XX FISH). He developed chronic GVHD with limited disease at day 123 of DLI. In the patient whose cGVHD resolved with the use of steroid, cyclosporine plus methotrexate. The patient died from pneumonia without evidence of recurrent leukemia on day +230. From the cases reported, combination chemotherapy and subsequent mobilized DLI produced a CR with AML in relapse six years after HLA-Mismatched transplantation. We demonstrate that of the patient who relapsed after 6 years, treatment with chemotherapy followed by intensive chemotherapy followed by DLI, can effectively salvage a patient with attainment of durable remissions. Although limited by the small number of one patient, AML in relapse six years after HLA-Mismatched transplantation requires particular attention in future studies, as well as in designing future treatment programs. Clearly a large number of patients is required to confirm the real efficacy of this treatment. Disclosures: No relevant conflicts of interest to declare.

Identifying Small Molecules That Overcome HoxA9-Mediated Differentiation Arrest in Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3513-3513
Author(s):  
David B. Sykes ◽  
Mark K Haynes ◽  
Nicola Tolliday ◽  
Anna Waller ◽  
Julien M Cobert ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Small Molecules ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Myeloid Differentiation ◽  
Prostacyclin Receptor ◽  
Acute Myeloid

Abstract Abstract 3513 AML in adults is a devastating disease with a 5-year survival rate of 25%. We lack new treatments for AML, and the chemotherapy standard of care remains unchanged in thirty years. One success story in the treatment of AML has been the discovery of drugs that trigger the differentiation of leukemic blasts in the small subset of patients with acute promyelocytic leukemia. However, differentiation therapy is unfortunately not available for the remaining 90% of non-APL acute myeloid leukemia patients. Understanding and targeting the mechanism of differentiation arrest in AML has been under investigation for more than four decades. There is growing evidence to support the role of the homeobox transcription factors in normal hematopoietic differentiation as well as malignant hematopoiesis. The persistent, and inappropriate, expression of the homeobox gene HoxA9 has been described in the majority of acute myeloid leukemias. This implicates HoxA9 dysregulation as a common pathway of differentiation arrest in myeloid leukemias and suggests that by understanding and targeting this pathway, one might be able to overcome differentiation arrest. In cultures of primary murine bone marrow, constitutive expression of HoxA9 blocks myeloid differentiation and results in the outgrowth of immature myeloid cell lines. The mechanism by which HoxA9 causes differentiation arrest is not known and no compounds exist that inhibit HoxA9. We developed a murine cell line model in which the cells were blocked in differentiation by a conditional version of HoxA9. In this system, an estrogen-dependent ER-HoxA9 protein was generated by fusion with the estrogen receptor hormone-binding domain. When expressed in cultures of primary murine bone marrow, immortalized myeloblast cell lines can grow indefinitely in the presence of stem cell factor and beta-estradiol. Upon removal of beta-estradiol, and inactivation of HoxA9, these cell lines undergo synchronous and terminal myeloid differentiation. We took advantage of an available transgenic mouse model in which GFP was expressed downstream of the lysozyme promoter, a promoter expressed only in mature neutrophils and macrophages. Cell lines derived from the bone marrow of this lysozyme-GFP mouse were GFP-negative at baseline and brightly GFP-positive upon differentiation. In this manner, we generated a cell line with a built-in reporter of differentiation. These cells formed the basis of a high-throughput screen in which cells were incubated with small molecules for a period of four days in 384-well plate format. The cells were assayed by multi-parameter flow cytometry to assess for toxicity and differentiation. Compounds that triggered green fluorescence were scored as “HITS” and their pro-differentiation effects confirmed by analysis of morphology and cell surface markers. Given the availability of cells and the simple and reliable assay, we performed both a pilot screen of small molecules at The Broad Institute as well as an extensive screen of the NIH Molecular Libraries Small Molecule Repository. The screen of more than 350,000 small molecules was carried out in collaboration with the University of New Mexico Center for Molecular Discovery. We have identified one lead class of compounds - prostacyclin agonists – capable of promoting myeloid differentiation in this cell line model of AML. Using a parallel cell line derived from a prostacyclin receptor knock-out mouse, we confirmed that activity was due to signaling through the prostacyclin receptor. The role of prostacyclin signaling in myeloid differentiation has not been previously described. Analysis of gene expression demonstrated that the expression of the prostacyclin receptor is seen in ∼60% of in primary human AML samples. This is a potentially exciting finding as prostacyclin agonists (e.g. treprostinil) are clinically relevant as well as FDA-approved. Their potential role in the treatment of acute myeloid leukemia is unknown. Here we present the details of our high-throughput flow cytometry system and preliminary identification of pro-differentiation agents in AML. If successful, we anticipate that one of these small molecules may offer insight into a mechanism for overcoming differentiation arrest, and may also translate into a novel, clinically relevant treatment for acute myeloid leukemia. Disclosures: Sklar: IntelliCyt: Founder of IntelliCyt, the company that sells the HyperCyt high-throughput flow cytometry system. Other. Zon:Fate Therapeutics: Founder Other.

C/EBPα and MiR-182 Generate a Negative Feedback Loop Which Is Dysregulated in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 776-776
Author(s):  
Alexander Arthur Wurm ◽  
Dennis Gerloff ◽  
Daniela Braeuer-Hartmann ◽  
Christiane Katzerke ◽  
Jens-Uwe Hartmann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Feedback Loop ◽  
Mononuclear Cells ◽  
Myeloid Differentiation ◽  
Negative Feedback Loop ◽  
U937 Cells ◽  
Leukemia Development ◽  
Acute Myeloid

Abstract The transcription factor CCAAT enhancer binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is silenced in approximately 50% of all acute myeloid leukemia (AML) cases. There are several mechanisms known how C/EBPα is inactivated in AML, including promoter hypermethylation, posttranslational modifications and mutations in the ORF of the CEBPA gene. MicroRNAs, a class of small non-coding RNAs, were identified as important regulators of normal hematopoiesis and leukemia development. We have already shown that microRNAs, such as miR-223, miR-34a and miR-30c, are essential elements in C/EBPα triggered granulocytic differentiation. But to our knowledge nothing is known about inactivation of C/EBPα by microRNAs in acute myeloid leukemia. In this study, we identified a novel network between C/EBPα and miR-182. In a next generation sequencing approach based on inducible K562-C/EBPα-ER cell line, we found miR-182 strongly downregulated by wildtype C/EBPα. We could further demonstrate an inverse correlation between C/EBPα protein amount and miR-182 expression level in several in vitro systems, including leukemic cell lines and G-CSF treated primary human CD34+progenitor cells. Additionally, C/EBPα and miR-182 showed reciprocal expression in sorted murine bone marrow subpopulations in vivo. To discover the mechanism how miR-182 is blocked by C/EBPα, we analyzed the minimal promoter region of miR-182 and performed chromatin immunoprecipitation (ChIP). Here, we could demonstrate a strong binding of C/EBPα to the miR-182 promoter, particularly to a conserved E2F binding site. Because E2F is a well known inhibitor of C/EBPα function, we tested whether E2F also effects miR-182 expression. An overexpression of E2F1 in U937 cells leads to an elevated miR-182 expression level. In addition, we measured the expression of miR-182 in bone marrow from AML patients regarding to their CEBPA mutation status. We could show that only patients with mutations in the C-terminal region of C/EBPα showed elevated miR-182 expression, while patients with N-terminal CEBPA mutations revealed no abnormal miR-182 expression compared to healthy donors or AML patients with no CEBPA mutation. The C-terminal domain of C/EBPα is necessary for E2F inhibition. These findings illustrate the importance of C/EBPα-E2F interaction during miR-182 regulation. Next, we found a highly conserved binding site of miR-182 in the 3’UTR of CEBPA itself, suggesting a possible negative feedback loop. To test this, we performed overexpression of miR-182 in U937 cells, umbilical cord blood mononuclear cells (UCB-MNCs) and primary blasts from AML patients. Here, we observed a strong reduction of C/EBPα protein after miR-182 overexpression in all cell types. Furthermore, we could demonstrate a direct binding of miR-182 to the 3’UTR of CEBPA via luciferase activity assay. Finally, we were interested in the functional impact of miR-182 in myeloid differentiation and leukemia development. We showed that enforced expression of miR-182 in U937 cells reduced the percentage of Mac-1 positive myeloid cells after treatment with all-trans retinoid acid (ATRA). Additionally, lentiviral overexpression of miR-182 induces a block of differentiation and hyperproliferation in G-CSF treated 32D cells and an enhanced replating capacity of primary mouse bone marrow mononuclear cells. Taken together, we identified miR-182 as novel oncogenic microRNA that directly blocks C/EBPα during myeloid differentiation and leukemia development. Thus, our data display a potential new strategy for therapeutics in C/EBPα dysregulated AML. Disclosures No relevant conflicts of interest to declare.

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