EVI1-Positive AML Cells Show Distinct Features and High Dependency on ETS Transcription Factor ERG

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1 is indispensable for normal hematopoiesis. We have previously reported that EVI1 expression is restricted to hematopoietic stem cell fraction and EVI1-expressing cells show robust long-term reconstitution capacity using Evi1-IRES-GFP knock-in (EVI1-GFP) mice, which enable us to track Evi1 expression on a single cell basis. In this study, we tried to elucidate the functional implication of EVI1 expression in AML using these mice. We generated murine EVI1-GFP AML model by retrovirally transducing MLL-AF9 or -ENL fusion gene into Lineage- Sca-1+ c-kit+ (LSK) cells from EVI1-GFP mice followed by transplantation into lethally irradiated syngeneic mice. Clonogenic and leukemogenic potentials of AML cells, especially leukemic cells with a granulocyte-macrophage progenitor phenotype (L-GMPs) from these mice, were compared according to GFP expression. Remarkably, GFP-positive L-GMPs tended to show lower colony-forming activity in semi-solid media and lower leukemia-initiating potential than GFP-negative L-GMPs. GFP-positive L-GMPs, however, induced a more aggressive form of AML, characterized by shorter survival in the secondary transplantation model. When EVI1-GFP AML mice underwent cytotoxic chemotherapy with cytarabine, the GFP-positive fraction was enriched during myelosuppression, indicating the survival advantage of EVI1-positive cells. To investigate the downstream target of EVI1, we employed murine EVI1-AML model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we performed chromatin-immunoprecipitation coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody. To identify leukemia-specific targets of EVI1, the result was compared with the result of ChIP-seq obtained from 32D-cl3 murine hematopoietic progenitor cells with 3×FLAG tag inserted into 3'-end of the coding region of the EVI1 gene. Gene ontology analysis revealed that genes involved in immune processes are explicitly enriched in the leukemia samples. Among the list of EVI1-bound genes, we tried to refine functional downstream targets of EVI1, which are upregulated in murine EVI1-AML cells and of which expressions are positively correlated with EVI1. By combining the ChIP-seq data with murine transcriptome data that compare hematopoietic progenitor cells expressing empty-vector and EVI1+ AML cells, and public gene expression datasets of human AML (Valk et al. NEJM 2004), we picked out 18 genes as candidate EVI1 downstream genes. Functional screening using EVI1-AML cells and shRNAs against these genes revealed that silencing of ETS transcription factor ERG (ETS-related gene) markedly suppressed proliferation and colony-forming activity of EVI1-AML cells, as well as rendered them vulnerable to cytotoxic agents. Normal c-kit+ hematopoietic progenitor cells were less affected by shRNAs against ERG. By comparing MLL-ENL immortalized murine hematopoietic cells with high and low EVI1 expression, EVI1-high MLL-ENL cells showed higher ERG dependency than EVI1-low MLL-ENL cells. Pharmacological inhibition of ERG also led to marked inhibition of EVI1-AML cells and EVI1-high MLL-ENL cells. Finally, knockdown of ERG remarkably delayed AML development in bone marrow transplantation model of EVI1-AML and EVI1-expressing MLL-ENL AML. Our data suggest that EVI1-positive AML cells are characterized by an aggressive nature and resistance to cytotoxic agents, as well as low leukemia stem cell capacity. ERG would be a novel downstream target of EVI1, on which survival of EVI1-expressing AML cells depends. Disclosures Masamoto: Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; MSD K.K.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau. Kurokawa: Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau.

939 Autocrine/paracrine factors in cancer associated fibroblasts drive immunosuppression through impaired myeloid cell functions

BackgroundCancer associated fibroblasts (CAFs) promote tumorigenesis by secreting immunosuppressive cytokines, stimulating angiogenesis, and supporting the growth of tumor cells. Through their interactions with immune cells, CAFs are known to directly impact the functionality of T cells and macrophages. However, CAF interaction with dendritic cells (DCs) and DC progenitor cells and its impact on DC function is relatively understudied and was the main focus of this study.MethodsTwo types of coculture systems were used in this study. For the human system, fibroblasts from lung squamous cell carcinoma (LUSC) were cocultured with MUTZ3 cells (hematopoietic progenitor cells) in the presence of DC differentiation stimuli, sometimes followed by DC maturation stimuli. For the mouse coculture system, activated (YPSC-c) and inactivated (PSC-b) pancreatic stellate cells (PSCs) were isolated from the pancreas of C57BL/6 mice by the density gradient method and co-cultured in the presence of bone marrow cells in the presence of DC differentiation and maturation stimuli. For human tumor antigen processing and cross presentation assay MART1 peptide (10mer and 20mer) was used.ResultsCo-culture of human and murine hematopoietic progenitor cells with fibroblasts (human LUSC CAFs and murine PSC results in decrease in differentiation and maturation of DCs. DCs differentiated and matured in the presence of fibroblasts have impaired ability to process and present tumor antigen to T cells. In the presence of PSC fibroblasts DC differentiation from murine bone marrow cells is skewed more towards MDSC and macrophages. In contrast to inactivated PSC-b, activated PSC-c influence DC differentiation in a contact dependent manner. Furthermore, PSC-b and PSC-c show transcriptionally distinct signatures which translate to unique secretory profiles as measured by Luminex. Analysis of the conditioned media from the coculture demonstrated that PSC-c secrete (among others) CXCL1, IL6, and CCL5 chemo/cytokines. These and other factors may play an important role in mediating fibroblast induced suppression of DC differentiation from monocytes.ConclusionsOur study demonstrates that cancer associated fibroblasts, or their precursors directly impact DC differentiation and antigen presentation via cytokines that could be targeted therapeutically to improve DC expansion and activity in the tumor microenvironment.

Targeted Therapies and Druggable Genetic Anomalies in Acute Myeloid Leukemia: From Diagnostic Tools to Therapeutic Interventions

Acute myeloid leukemia (AML) is a clonal disorder resulting from acquired somatic mutations in hematopoietic progenitor cells that lead to the dysregulation of differentiation and the proliferation of hematopoietic cells [...]

The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.

Effects of Glucan Supplementation on Aged Hematopoietic Progenitor Cells

Age related changes in activity of hematopoietic progenitor cells led us to investigate the possibility that glucan supplementation might overcome this suppression. In our study, we focused on differences of these cells in young (8 weeks old) and old (18 months old) mice after supplementation of yeast-derived insoluble glucan. Our results showed significant differences in reconstitution ability of progenitor cells as well as changes in level of stromal cell-derived factor 1α between young and old mice exist and can be partly restored by glucan supplementation.

Nilai Prognostik FLT3 Pada Pasien Leukemia Mieloid Akut

Acute myeloid leukemia (AML) is a disease characterized by neoplastic transformation and abnormality of progenitor cells differentiation from myeloid cells. In AML there is a heterogeneous abnormality in hematopoietic progenitor cells, this abnormality has been identified and affects the balance between proliferation, survival and differentiation of body cells. Mutation of FMS-like tyrosin kinase 3 (FLT3) gene causes overexpression which is one of the most often found mechanism that has been identified could trigger genetic changes that can disturb intracellular signaling tissue in pathogenesis of leukemia. Besides being used as a diagnostic tool, FLT3 overexpression can also be used to assess the prognosis of AML.