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

Abstract 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.

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GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Blood ◽

10.1182/blood-2020-134071 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 33-33

Author(s):

Akira Chiba ◽

Yosuke Masamoto ◽

Hideaki Mizuno ◽

Mineo Kurokawa

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Hematopoietic Progenitor Cells ◽

Rna Seq ◽

Hematopoietic Progenitor ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Knock Out

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

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Optimal Cycles of Bendamustine-Plus Rituximab Therapy for Indolent B Cell Lymphoma

Blood ◽

10.1182/blood-2019-124597 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5242-5242

Author(s):

Kazuhiro Toyama ◽

Toshiaki Takezaki ◽

Akira Honda ◽

Yasunori Kogure ◽

Akira Chiba ◽

...

Keyword(s):

B Cell ◽

Pharmaceutical Company ◽

Research Funding ◽

B Cell Lymphoma ◽

Study Cohort ◽

Indolent Lymphoma ◽

Chugai Pharmaceutical ◽

Company Limited ◽

Significant Difference ◽

Otsuka Pharmaceutical

Backgrounds Although bendamustine-plus rituximab therapy (BR) is considered as one of the standard therapy for several indolent B cell lymphomas, the optimal cycles of BR is not still uncovered. To elucidate the optimal cycles of BR, we performed a single center retrospective study of patients with indolent lymphoma treated with BR. Methods All patients with follicular lymphoma (n=40), lymphoplasmacytic lymphoma (n=11) and mucosa associated lymphoid tissue lymphoma (n=6) who underwent BR in our institute in the period between April 2011 and September 2017 were included in this study. The clinical information including the number of repeated cycles, overall survival (OS), progression free survival (PFS), laboratory findings, backgrounds, and the response to BR were analyzed retrospectively. Rituximab was administered at day 1 and bendamustine was administered at day 1 and 2, or 2 and 3, in each 28 days cycle. In the study cohort, the number of repeated cycles was allowed up to 6. The cessation of BR was allowed after 4 cycles in the patients with response to BR, according to the discretion of attending physicians. The dosage of bendamustine was reduced to 67% and 50% in 70 to 79 years and not less than 80 years, respectively. All patients in this study were prescribed trimethoprim-sulfamethoxazole combination or pentamidine for the prevention of pneumocystis pneumonia, and acyclovir for the prevention of herpes zoster. Results In total 57 patients, the median age was 65 years (range, 37 to 83). Thirty four were male, and 23 were female. Three patients were newly diagnosed and 54 were relapsed or refractory patients. The median observation period was 51.7 months (5.1 to 83.6). The overall response rate was 86.0% (CR 54.4% and PR 31.6%). The median number of repeated cycles of BR was 4 (1 to 6). There was no significant correlation between patient characteristics and the number of repeated cycles of BR. All patients were stratified by their number of repeated cycles of BR. The early cessation group (n=17) was identified that the number was from 1 to 3, and the late cessation group (n=40) was identified that the number was from 4 to 6. The 5-year OS rates in early and late cessation groups were 56.1% and 87.0%, respectively. The 5-year PFS rates in early and late cessation groups were 31.4% and 50.6%, respectively. Both 5-year OS and PFS rates in late cessation group were significantly longer than that in early cessation group (p=0.011 and p<0.01, respectively). In late cessation group, the number of the patient who underwent 4 cycles of BR (4 cycles group) was 21, and the number of the patient who underwent 5 or 6 cycles of BR (over 4 cycles group) was 19. The 5-year OS rates in 4 cycles group and over 4 cycles group was 85.7% and 85.2%, respectively. There was no significant difference between these groups in the 5-year OS rates (p=0.58). The 5-year PFS rates in 4 cycles group and over 4 cycles group was 71.8% and 31.0%, respectively. The 5-year PFS rates in 4 cycles group were significantly longer than that in over 4 cycles group (p<0.01). The most common reason of the cessation of BR was adverse event (n=15). BR were stopped in 9 patients who achieved response after 4 or 5 cycles, and in 8 patients who became relapsed or refractory. Conclusions Our study indicated that the outcome of the patients with indolent lymphoma who stopped BR after 4 cycles was not inferior to that of the patients who stopped BR after 5 or 6 cycles. The results suggest that the cessation of BR after 4 cycles may be permissible in the patients with response to BR. Disclosures Toyama: Celgene K.K.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau. Nakamura:Astellas Pharma Inc.: Speakers Bureau. Kurokawa:Shionogi & Co., Ltd: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Research Funding; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Pfizer Japan Inc.: Research Funding; Teijin Limited: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Shire Japan K.K.: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Celgene K.K.: Consultancy, Speakers Bureau; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Bioverativ Japan ltd.: Consultancy.

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Prognostic Factors of Idiopathic Hypereosinophilic Syndrome: A Nationwide Survey in Japan

Blood ◽

10.1182/blood-2021-147609 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4192-4192

Author(s):

Akira Honda ◽

Kazuhiro Toyama ◽

Yu Oyama ◽

Kensuke Matsuda ◽

Hideaki Mizuno ◽

...

Keyword(s):

Pharmaceutical Company ◽

Clinical Features ◽

Research Funding ◽

Hypereosinophilic Syndrome ◽

Clinical Information ◽

Nationwide Survey ◽

Line Treatment ◽

Chugai Pharmaceutical ◽

Company Limited ◽

Otsuka Pharmaceutical

Abstract Background Idiopathic hypereosinophilic syndrome (iHES) is a rare disease characterized by prolonged hypereosinophilia and organ damage without any known cause of eosinophilia. Since iHES was first proposed, the concept of the disease has continued to change, and its clinical features, optimal treatment, and prognosis have not yet been elucidated. Therefore, to clarify the clinical features and prognostic factors of iHES, we conducted a nationwide survey and collected detailed clinical information of iHES in Japan. Methods We conducted a nationwide postal questionnaire survey of iHES. A first simple questionnaire was sent to departments of hematology across the country to determine presence or absence of iHES in their department. Subsequently, a detailed questionnaire was sent to the department that responded that they had experience with iHES. After collecting the questionnaires, the validity of the diagnosis was determined. Only those that corresponded to iHES were used for subsequent analysis. In this research, iHES was defined as follows: absolute eosinophil counts more than 1,500/µL, presence of organ damage due to eosinophilia, with no known cause of eosinophilia. Allergy, collagen diseases, infection, asthma, drugs, vasculitis, and other diseases that can cause eosinophilia were ruled out. The absence of hematopoietic malignancies was confirmed by bone marrow examination, fluorescence in situ hybridization for FIP1L1-PDGFRA fusion gene, and chromosomal analysis by G-banding. Results The 1st questionnaire was sent to 492 departments of hematology, and we identified 152 patients with iHES in Japan. Among those patients, a detailed clinical information was collected from 68 patients. Of the 68 patients, 23 did not meet the criteria for iHES, and the remaining were subjected to subsequent analysis. Of the 45 patients with iHES, 27 (60%) were male, and 18 (40%) were female. The median age of diagnosis was 54 (range: 16-95) years, and the median number of involved organs per patient was 2 (range: 1-7). Symptoms caused by hypereosinophilia were consisted of systemic symptom (22, 49%), hematopoietic disorder (17, 38%), skin (16, 36%), digestive (15, 33%), respiratory (14, 31%), cardiovascular (14, 31%), and kidney (4, 9%). The median white blood cell and absolute eosinophil count were 18,800/µL (5,300-73,000/µL) and 9587/µL (2,067-63,370/µL), respectively. The median hemoglobin level was 13.3 g/dL (6.6-16 g/dL), and the median platelet count was 255 × 10 9/L (4.7-54 × 10 9/L). The median levels of lactate dehydrogenase and C-reactive protein were 299 U/L (123-972 U/L) and 0.96 mg/dL (0.02-13.9 mg/dL), respectively. Of the 45 cases, 37 (82%) required treatment, and 35 (78%) received corticosteroid as 1st line treatment. Although 28 (80%) patients responded to corticosteroid, 12 (34%) patients required subsequent 2nd line treatment, and 2 (6%) patients died. In addition, 6 patients required 3rd line treatment. Six of 45 patients died from any cause during the follow-up, and the median follow-up period for censored cases was 3.1 years (0.2-23 years). The median survival from diagnosis for all cases was 2.5 years (0.1- 23 years). In univariate analysis, hemoglobin less than 10 g/dL (P=0.02), the presence of renal symptoms (P<0.001), and the presence of respiratory symptoms (P<0.01) were statistically significant factors for overall survival. In multivariate analysis, hemoglobin less than 10 g/dL was a statistically significant factor for overall survival (HR, 17.2; 95% CI, 1.51-197; P=0.02). Conclusion In this nationwide survey, we clarified the clinical characteristics of iHES in Japan. In addition to clinical features at the time of diagnosis, the response rate to corticosteroid and long-term prognosis were also clarified. Furthermore, the presence of anemia was found to be a poor prognostic factor for iHES. Further accumulation of cases is necessary to establish the optimal treatment strategy for iHES. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee. Toyama: Celgene Corporation: Other: Lecture fee; Otsuka Pharmaceutical Co., Ltd.: Other: Lecture fee; NIHON PHARMACEUTICAL CO., LTD.: Other: Lecture fee; ONO PHARMACEUTICAL CO., LTD.: Other: Lecture fee; DAIICHI SANKYO COMPANY, LIMITED: Other: Lecture fee; CHUGAI PHARMACEUTICAL CO., LTD.: Other: Lecture fee; Takeda Pharmaceutical Company Limited: Other: Lecture fee. Matsuda: Ono Pharmaceutical: Other: Lecture fee; Kyowa Kirin: Other: Lecture fee. Komatsu: Fujifilm Wako Pure Chemical Corporation: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Japan Tobacco Inc.: Consultancy; Otsuka Pharmaceutical Co. Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharma KK: Consultancy, Research Funding, Speakers Bureau; Shire Japan KK: Consultancy, Research Funding, Speakers Bureau; PharmaEssentia Japan KK: Consultancy, Current Employment, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding. Kurokawa: Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

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Extracellular Vesicles Play an Important Role in Intercellular Communication Between Bone Marrow Stroma and Hematopoietic Progenitor Cells in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v128.22.1957.1957 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1957-1957

Author(s):

Teresa L. Ramos ◽

Luis Ignacio Sánchez-Abarca ◽

Beatriz Rosón ◽

Alba Redondo ◽

Concepción Rodríguez ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Board Of Directors ◽

Intercellular Communication ◽

Research Funding ◽

Hematopoietic Progenitor Cells ◽

Rt Pcr ◽

Hematopoietic Progenitor ◽

Advisory Committees ◽

Cd34 Cells

Abstract The complex interplay between bone marrow-derived mesenchymal stromal cells (BM-MSC) and neoplastic hematopoietic cells is involved in the progression of myeloproliferative neoplastic (MPN) diseases. Extracellular vesicles (EV) have emerged as a complex cell-to-cell communication system within the neoplastic microenvironment. EV are able to reprogram recipient cells by transferring proteins, mRNA and microRNA from their cell of origin. We aimed to analyze the microRNA content of EV obtained from MPN BM-MSC, as well as the changes induced when these EV are incorporated into hematopoietic progenitor cells (HPC). EV were isolated from BM-MSC of MPN patients (n=22) and healthy donors (HD) (n=19) by ultracentrifugation. Characterization of EV by transmission electron microscopy (TEM), immunoblot, multiparametric flow cytometry (MFC) and NanoSight analysis revealed vesicles with a typical bilayer-membrane characteristic morphology with a size inferior to 500 nm, which were positive for various EV markers as CD63 and CD81, and for MSC markers as CD73, CD90 and CD44 (Figure 1). MicroRNA profiling by 384-well microfluidic cards (TaqMan® MicroRNA Array A) showed an overall increase in the microRNA expression in the MPN-MSC-derived EV, when compared to EV from donor MSC. Using RT-PCR, we observed that miR-155 was selectively enriched in EV released by MPN-MSC. An overexpression of this microRNA was observed in EV (p=0.032), while a downregulation was observed in BM-MSC (p=0.0078) (Figure 2). EV incorporation was demonstrated by fluorescence microscopy and MFC, where HPC (CD34+ cells obtained by immunomagnetic selection) were co-cultured with Vybrant Dil-labeled EV. For functional studies apoptosis and clonogenic assays (CFU-GM) were performed. We observed an increase in CD34+ cell viability after incorporating EV from BM-MSC (HD and MPN). Moreover, an increase (p=0.04) in miR-155 expression was observed when HD HPC incorporated EV from MPN-MSC. When neoplastic CD34+ cells incorporated the EV derived from MPN-MSC an increase of CFU-GM number was also observed. We suggest that EV released from MPN-MSC represent a mechanism of intercellular communication between malignant stromal and hematopoietic cells, through the transfer of genetic information that may be relevant in the pathophysiology of these diseases. Funding: GRS 1034/A/14 (C. Sanidad, JCYL) and FCT (SFRH/BD/86451/2012) Figure 1 EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 1. EV characterization by TEM (A), Immunobloting - CD63 (B) and MFC (C). Scale bar: 200 and 500 nm. Figure 2 Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Figure 2. Expression of miR-155. RT-PCR from EV released from HD and MPN-MSC (A), and the expression of miR-155 in BM-MSC (B). Disclosures Sánchez-Guijo: Bristol-Myers-Squib: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Del Cañizo:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Arry: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Guided Polarization of iPSC-Derived CD4SP Helper T Cells By CRISPR/Cas9-Based Genome-Editing

Blood ◽

10.1182/blood-2019-122193 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1937-1937

Author(s):

Hisashi Yano ◽

Tokuyuki Shinohara ◽

Keiko Koga ◽

Shoichi Iriguchi ◽

Yasuyuki Miyake ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Pharmaceutical Company ◽

Progenitor Cells ◽

Research Funding ◽

Master Regulator ◽

Th Cells ◽

Company Limited ◽

Th Cell ◽

Ifn Γ

BACKGROUND: Large number of rejuvenated antigen-specific T cells generated from iPS cells (iPSCs) may have a large impact on the T-cell immunotherapy field. We previously reported the generation of functional CD8 single positive (SP) cytotoxic lymphocytes (CTLs) from iPSCs (Minagawa etal. CellStemCell, 23: 850-858. (2018)), and a regenerative CTL-based immunotherapy is about to begin in clinical trials. However, these two-dimensional differentiation protocols using the OP9DL1 murine feeder cell line or DLL4 recombinant proteins could differentiate iPSCs into CD8SP CTLs but not into robust CD4SP helper T (Th) cells. It is clear that, we could better control immune reactions if we could produce each Th cell fraction. For example, we could enhance antitumor immunity if we could specifically induce Th1 cells to command cellular immunity. The drastic therapeutic effect of CD19 CAR modified T cells including both CD8SP CTLs and CD4SP Th cells is clearly based on the role of CD4SP Th cells in helping CD8SP CTLs prolong the therapeutic effect against B-cell malignancy. This led us to hypothesize that Th cell induction from iPSCs is essential for efficient immunotherapy. In this situation, a three-dimensional (3D) method called artificial thymic organoid (ATO) was reported to support robust differentiation of both CD4SP and CD8SP TCRαβ cells from primary hematopoietic stem and progenitor cells (Seet etal. NatureMethods, 14: 521-530. (2017)) and hematopoietic progenitor cells derived from ES cells and iPSCs (Montel-Hagen etal. CellStemCell, 24: 376-389. (2019)). Here, we evaluated the advantages and unsolved tasks of the 3D method to induce antigen-specific and functional CD4SP Th cell subsets from iPSCs. METHODS and RESULTS: By applying the ATO methods, we cultured mixed pellets of iPSC-derived hematopoietic progenitor cells (HPCs) and Notch ligand-expressing MS5 feeder cells on cell culture inserts for up to 9 to 12 weeks. Next, we analyzed differentiated T cells in the ATOs. We used iPSCs derived from antigen-specific Th cells containing HLA class II restricted TCR genes corresponding to an original Th cell clone. Most importantly, the ATO method supported robust differentiation of CD4SP T cells as expected even from these iPSCs. These CD4SP T cells showed high expression of Th-POK as a master regulator of Th cells, and high secretion ability of several key cytokines produced by Th cells-IL-2, IFN-γ, and IL-4. However, the majority of iPSC-derived CD4SP T cells showed simultaneous secretion of both IFN-γ and IL-4 unlike normal peripheral CD4SP Th cells. We tried to make the regenerated CD4SP T cells separately produce the Th1 (IFN-γ) and Th2 (IL-4) cytokines by optimizing culture conditions, but we failed to achieve separated Th1/2 differentiation. To understand the reason for the bipolarized cytokine production profile of regenerated CD4SP T cells, we checked the master regulator expression profile. We found that a majority of the CD4SP T cell population highly expressed T-bet, the master regulator of Th1. Contrary to our expectation, GATA3's expression levels were not high in most CD4SP T cells. And, GATA3 is master regulator not only of Th2 but of T cell differentiation itself, so we guessed knock out (KO) of GATA3 led to failure of differentiation into T cell. To "polarize" the CD4SP T cells to have Th1 or Th2 functions, we knocked out TBX21 (coding T-bet) or the Th2 "master cytokine" IL4 of undifferentiated iPSCs using CRISPR-Cas9 to obtain TBX21KO/KO iPSCs or IL4KO/KO iPSCs, respectively. Those iPSCs were successfully differentiated into HPCs, and ATOs were then prepared using these cells. After 9 to 12 weeks, mature CD4SP T cells and CD8SP T cells were observed in both ATOs with the same surface marker profile as T cells from wild type iPSCs, and TBX21KO/KO CD4SP T cells or IL4KO/KO CD4SP T cells from iPSCs with selective production of IL-4 or IFN-γ, respectively (Figure1). These results also suggested the potential utility of ATO-based invitro T cell differentiation from genome-edited iPSC for understanding of human developmental immunology. CONCLUSIONS: "Polarized" CD4SP Th cells were successfully obtained from master regulator or cytokine gene-knockout iPSCs in ATO-based invitro differentiation. We are now investigating the actual helper function of these "polarized" iPS-Th cells that could be induced by the target peptide on HLA Class II molecules of antigen presenting cells. Disclosures Shinohara: Takeda Pharmaceutical Company Limited: Employment. Koga:Takeda Pharmaceutical Company Limited: Employment. Kassai:Takeda Pharmaceutical Company Limited: Employment. Kiyoi:Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Perseus Proteomics Inc.: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding. Kaneko:KIRIN holdings Co.,Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Other: Scientific adviser, Research Funding; TERUMO Co., Ltd.: Research Funding; TOSOH Co., Ltd.: Research Funding; Thyas Co., Ltd.: Other: Founder, Shareholder, Chief Science Officer, Research Funding.

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eIF4B Is a Novel Target of CAMK2G and Promotes Proliferation of Malignant Cells in Myelofibrosis

Blood ◽

10.1182/blood-2021-150349 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3586-3586

Author(s):

Ken Sasaki ◽

Hideaki Mizuno ◽

Tadayuki Ogawa ◽

Yosuke Masamoto ◽

Mineo Kurokawa

Keyword(s):

Mass Spectrometry ◽

Pharmaceutical Company ◽

Research Funding ◽

Kinase Activity ◽

Protein Complexes ◽

Direct Interaction ◽

Chugai Pharmaceutical ◽

Company Limited ◽

Otsuka Pharmaceutical ◽

Jak2 Inhibitors

Abstract Background Myelofibrosis (MF) is a myeloproliferative neoplasm which is associated with megakaryocytic atypia, fibrosis in the bone marrow (BM), and extramedullary hematopoiesis, followed by progressive hematopoietic failure and leukemic transformation. JAK1/JAK2 inhibitors are currently available and reduce spleen volume, improve symptoms related to MF and prolong the overall survival (OS). Although the benefits associated with JAK1/JAK2 inhibitors are well established, not all patients respond to these inhibitors, and long-term exposure to these inhibitors results in the emergence of resistant clones based on the reactivation of the JAK-STAT pathway. Therefore, new therapeutic strategies targeting other molecules can provide additional benefits to patients with MF. In the previous study, we identified Calcium/Calmodulin Dependent Protein Kinase II Gamma (CAMK2G) as a new therapeutic target for MF by performing compound screening. Furthermore, CAMK2G inhibition can overcome drug-resistance against JAK2 inhibitors. Therefore, it is important to investigate the mechanisms underlying the therapeutic effect of CAMK2G inhibition. In this study, to explore the mechanism underlying the therapeutic effect of CAMK2G inhibition, we performed the immunoprecipitation mass spectrometry to find out the protein that has the direct interaction with CAMK2G. Methods Quantitative Proteomic Analysis of the Target Proteins of CAMK2G The protein complexes with FLAG-tagged CAMK2G (FLAG-CAMK2G) were trapped by anti-FLAG antibody. The eluted assay mixtures were reduced, alkylated and digested into peptides. Each peptide solution and control mixture were labeled with differential stable isotope tags. The samples were quantitatively analyzed using a Q Exactive mass spectrometer (Thermo Fisher Scientific). The spectra were searched against the SWISS-PROT databases using SEQUEST on Proteome Discoverer software 2.2 (Thermo Scientific). Results To reveal the , we conducted the immunoprecipitation assays by FLAG-CAMK2G or FLAG-tag alone, overexpressed in MF model cells. Because CAMK2G has kinase activity, we attempted to reveal the kinase-activity-dependent targets using unhydrolyzable ATP analog, AMP-PNP（5'-adenylyl-imidodiphosphate）that can maintain the strong-binding state of kinase with its target. The protein complexes with FLAG-CAMK2G were trapped by anti-FLAG antibody. After samples were digested into peptides and the candidate proteins were identified and quantitatively analyzed by mass spectrometry. As a result, we identified eukaryotic translation initiation factor 4B (eIF4B) as a protein that has a direct interaction with CAMK2G. eIF4B is a part of the complex involved in the initiation of translation. It has been reported that eIF4B plays a role in the translation of factors involved in anti-apoptosis (Bcl2, Bclxl, Mcl1), cell cycle (Cdc25c), and cell proliferation (c-Myc). We then checked whether knockdown of eIF4B decrease proliferation of MF model cell line. shRNA-mediated silencing of eIF4B decreased cell growth of these cells. Furthermore, the phosphorylation of eIF4B was increased by the ectopic expression of MPL W515L, one of the common mutations found in MF. Also, the phosphorylation of eIF4B was increased by the overexpression of CAMK2G. We then explored the proteins regulated by eIF4B in MF and identified that knockdown of eIF4B decreased the amount of Bcl2 protein. Conclusion In our study, we performed immunoprecipitation mass spectrometry and identified eIF4B as a partner protein of CAMK2G. Since CAMK2G inhibition was shown to be effective against MF in vitro and in vivo, we focused on eIF4B as a potential effector in MF. We also showed that overexpression of MPL W515L and CAMK2G phosphorylates eIF4B and that knockdown of eIF4B inhibited proliferation of MF cells. Furthermore, Bcl2 can be one of the target proteins regulated by eIF4B. Based on these, eIF4B plays an important role in MF. We further perform ribosome profiling to comprehensively understand the regulation of translation by eIF4B. Our research not only elucidate the pathogenesis of MF but also identify new therapeutic targets for MF. Disclosures Masamoto: MSD K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Kurokawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: 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.

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A Nationwide Survey of Lower Risk Myelodysplastic Syndromes in Japan

Blood ◽

10.1182/blood-2021-146093 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4662-4662

Author(s):

Kumi Nakazaki ◽

Toshiaki Takezaki ◽

Yosuke Masamoto ◽

Yasushi Miyazaki ◽

Kinuko Mitani ◽

...

Keyword(s):

Pharmaceutical Company ◽

Lower Risk ◽

Research Funding ◽

Anabolic Steroids ◽

Risk Groups ◽

Free Survival ◽

Chugai Pharmaceutical ◽

Company Limited ◽

Otsuka Pharmaceutical ◽

Research Facilities

Abstract Introduction: There are some treatment options for lower risk myelodysplastic syndromes (MDS) such as erythropoiesis-stimulating agents (ESAs), anabolic steroids, hypomethylating agents, and immunosuppressants. The object of this multicenter retrospective study was to survey the current situation about treatment selection and the prognosis of the lower risk MDS cases in Japan. We also evaluated the prognosis of the cases with paroxysmal nocturnal hemoglobinuria (PNH) type cells and therapeutic effects of cyclosporine. Methods: We investigated the clinical information in the form of a questionnaire for joint research facilities as to each case of newly diagnosed MDS between 2013 and 2018 corresponding to the lower risk of International Prognostic Scoring System (IPSS) or revised IPSS (IPSS-R). The diagnosis of MDS was based on WHO 2008 or WHO 2016 classification. Survival analysis was conducted using Kaplan-Meier method and log-rank test. Multivariate analysis was performed using Cox proportional hazard regression model. This study was approved by the institutional review board of the University of Tokyo and other research facilities. This work was supported by the Research Program of Intractable Disease (the Japanese National Research Group on Idiopathic Bone Marrow Failure Syndromes) provided by the Ministry of Health, Labor, and Welfare of Japan. Results: 1,304 cases at thirty facilities nationwide were enrolled. Median age was 76 years [IQR, 68 - 83], and male and female ratio was 61.3% and 38.7%. At diagnosis, 19.0% and 4.4% of the cases were dependent on red blood cells and platelets transfusion, respectively. The risk classification of enrolled cases was as follows: very low, 217 (16.6%); low, 652 (50.0%); intermediate, 360 (27.6%); high, 56 (4.3%); very high, 4 (0.3%); not determined, 14 (1.1%). 1,230 cases of the very low, low and intermediate risk groups were included in subsequent analyzes. Serum erythropoietin levels were measured in 466 cases (37.9%) with a median of 61.8 IU/l, and 74.2% and 85.7% cases showed less than 200 IU/l and 500 IU/l, respectively. PNH type cells in the peripheral blood were evaluated in 231 cases and were positive in 33 cases (14.3%). Median follow-up period was 22 months. As an initial therapy, 26.4% and 11.6% of transfusion-dependent and independent cases started to receive ESAs, respectively. 16.6% and 11.5% took oral anabolic hormones, and azacytidine were administered to 17.0% and 7.2% of each group. 55.4% of transfusion-independent cases were just followed up at first. Median overall and acute myeloid leukemia (AML)-free survival was 70.0 months [95% CI, 61.0 - not reached] and 62.0 months [95% CI, 54.0 - 74.0], respectively. Log-rank analysis revealed significant differences among IPSS-R risk groups about overall and AML-free survival (p<0.01 and p<0.01, respectively). Multivariate analysis confirmed that initiating azacytidine at the time of diagnosis conferred an independent significant poor prognostic factor with respect to overall survival (hazard ratio for death, 1.74; p<0.01) and AML-free survival (hazard ratio for death or onset of AML, 1.86; p<0.01) in addition to sex, age, IPSS-R classification, and transfusion-dependency. Comparing thirty-three positive cases of PNH type cells with 198 negative cases, overall and AML-free survival was significantly better in the former group (p<0.01 and p<0.01, respectively). Median overall and AML-free survival were not reached in the positive group and 51.0 months [95% CI, 47.0 - 79.0] and 49.1 months [95% CI, 40.0 - 57.1] in the negative group, respectively. Interestingly, focused on the positive cases, 14 cases receiving cyclosporine revealed better AML-free survival than the others (p=0.033). Overall survival was tended to be better (p=0.052). On the other hand, cyclosporine did not improve the prognosis of the PNH type cells negative cases. It is thought to be consistent with the effectiveness of immunosuppressive therapy in aplastic anemia with PNH type cells. Conclusion: ESAs, anabolic steroids, and azacytidine were frequently selected as an initial treatment for lower risk MDS cases in Japan, however, when to start azacytidine is an issue for consideration. Good response to cyclosporine may be obtained in cases with PNH type cells. Disclosures Masamoto: Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; MSD K.K.: Speakers Bureau. Miyazaki: Kyowa-Kirin: Honoraria; Sumitomo-Dainippon: Honoraria, Research Funding; Abbvie: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Astellas: Honoraria; Janssen: Honoraria; Eisai: Honoraria; Daiichi-Sankyo: Honoraria; Chugai: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Mitani: Nippon Shinyaku Co.: Research Funding, Speakers Bureau; MSD Pharma.: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Pfizer Inc.: Speakers Bureau; Celgene Co.: Speakers Bureau; Takeda Pharma.: Research Funding, Speakers Bureau; Kyowa Kirin,: Research Funding, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Shire plc: Speakers Bureau; BML Inc: Speakers Bureau; Mochida Parma.: Speakers Bureau; Alexion Pharma.: Speakers Bureau; AbbVie Inc.: Speakers Bureau; Ono Pharma.: Speakers Bureau; Chugai Pharma.: Research Funding; Teijin Pharma.: Research Funding; Sumitomo Dainippon Pharma: Research Funding; Taiho Phama.: Research Funding; Otsuka Pharma.: Research Funding. Kurokawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau.

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