Reverting to Quiescence Preserves Hematopoietic Stem Cells Following Genome Editing

Abstract Hematopoietic stem cells (HSCs) in steady-state are quiescent in cell cycle. CRISPR-Cas9 genome editing has revolutionized the HSC research and therapeutic application of HSCs for hematological diseases. Although these methods and clinical results are promising, keeping HSC function after highly efficient genome editing is still challenging because HSCs gradually lose their repopulation capacity following cell cycle activation. Preserving the function of HSCs after genome editing is an urgent issue. In this study, we interrogated the culture method following genome editing to reverse the cell cycle status of HSCs into a quiescent state to test how cell cycle status affects the genome editing efficiency and the HSC potential. To assess the relationship between genome editing efficiency and cell cycle status, we compared genome editing efficiency among the freshly-isolated or cultured HSCs and progenitors (HSPCs) at various time points. Ribonucleoprotein (RNP) complex was delivered into cells by electroporation. Genome-editing efficiency was evaluated by CD45 knockout rate comparing sgRNA for Rosa and CD45. All the HSPC fractions cultured displayed higher genome-editing efficiency following over-night preculture. Genome-editing efficiency of fresh HSC was lower than 20% while fresh granulocyte/monocyte progenitor (GMP) showed higher than 40%. Cell cycle analysis using EdU or Ki67 and Hoechst 33342 staining confirmed that genome-editing efficiency positively correlated with cell cycle activation. We further tested the effect of pre- and post-culture conditions for genome editing. While preculture with high cytokine concentration for a long period of time (> 16 hours) is required for the efficient genome editing, post-culture condition did not compromise the genome-editing efficiency. Given the quiescent nature of HSCs, we hypothesized that reverting activated HSCs back to quiescent state may improve the function of HSCs following genome editing. To test this, genome-edited HSCs were cultured in the quiescence-maintaining condition (SCF 1.5 ng/mL and TPO 1.0 ng/mL) under 1% O 2 atmosphere. After 7-day culture, more than 30% of cells kept the surface marker phenotype of CD150 + CD48 - LSK, and over 60% of cells were successfully underwent genome editing. Less than 10% of HSCs were EdU +, suggesting that HSCs reverted to cell cycle quiescence after genome editing. By contrast, HSCs cultured in a conventional high cytokine condition (proliferative condition) lost the surface marker phenotypes and highly incorporated EdU. To assess the long-term reconstitution potential of edited HSCs, the Evi1 expression level was evaluated using Evi1-IRES-GFP mice. The expression level of Evi1 was significantly higher in quiescent HSCs than proliferating HSCs after editing. These results suggest that, as post-electroporation culture, quiescence-maintaining condition reverts precultured HSCs back to a quiescent state in cell cycle. This protocol maintains phenotypic HSCs without compromising the genome-editing efficiency. To further determine the function of genome-edited HSCs, single cell colony assay was performed. Clonally sorted CD45 knockout HSCs cultured in the quiescence-maintaining condition after gene editing fully maintained colony-forming capacity, but HSCs cultured in the proliferating condition lost their capacity. We then performed transplantation assays using Ubc-GFP mice. GFP + HSCs were genome-edited for Rosa and transplanted into lethally-irradiated recipient mice with competitor cells. The donor-derived chimerism of edited HSCs with quiescence-maintaining condition in peripheral blood and bone marrow was generally superior to that of edited HSCs with proliferative condition. These data demonstrates that edited HSCs cultured in quiescence-maintaining condition maintain stem cell potential in vitro and in vivo. Altogether, we established an HSC-optimized, highly efficient genome-editing protocol. This study demonstrated that effectiveness of keeping HSC in a quiescent state even in the setting of genome editing. Our protocol is suitable for unveiling the function of genes distinguishing cycling and quiescent HSCs. Disclosures Kataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties .

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The Outcomes of Systemic Chronic Active EBV Infection Treatment By Allogeneic Hematopoietic Stem Cell Transplantation: An Analysis of Japanese Registry Data

Blood ◽

10.1182/blood-2021-147046 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3965-3965

Author(s):

Ayako Arai ◽

Masahide Yamamoto ◽

Maho Sato ◽

Yasushi Onishi ◽

Yoji Sasahara ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Research Funding ◽

Skin Lesions ◽

Hematopoietic Stem ◽

Chugai Pharmaceutical ◽

Ebv Infection ◽

Otsuka Pharmaceutical ◽

Definition Of

Abstract Background and aims Systemic chronic active Epstein-Barr virus infection (sCAEBV) is classified as T- or NK-cell neoplasms in the WHO classification revised in 2017. Although allogeneic stem cell transplantation (allo-HSCT) is efficacious for sCAEBV, the effects are yet to be analyzed in a large number of cases due to the disease rarity. To investigate the outcomes and the prognostic factors of allo-HSCT in sCAEBV under the definition of the WHO 2017 classification, we analyzed retrospectively using the database of Japanese Society for Transplantation and Cellular Therapy (JSTCT). Methods Data collection We used the clinical data of hematopoietic stem cell transplantation (HSCT) recipients of the Transplant Registry Unified Management Program (TRUMP) sponsored by JSTCT and Japanese Data Center for Hematopoietic Cell Transplantation (JDCHCT). Patients who underwent HSCT to cure EBV-associated diseases, secondary hemophagocytic lymphohistiocytosis (HLH), and virus-associated hemophagocytic syndrome between January 1993 and December 2016 were selected in TRUMP database, and on our behalf, JDCHCT sent out survey questions to the institutions of these patients to collect additional data to check if the diagnosis of sCAEBV matches our criteria, to analyze disease status at the time of HSCT, and to evaluate the efficacy of different treatment methods. The diagnosis of sCAEBV sCAEBV was diagnosed according to criteria suggested in 2016 by the Research group of Measures against Intractable Diseases by Ministry of Health, Labour and Welfare of Japan: (1) elevated EBV DNA load in peripheral blood (PB) (> 10 2.5 copies/μg DNA), (2) detection of EBV infection in T or NK cells from the affected tissues or PB, (3) systemic inflammatory symptoms such as fever, lymphadenopathy, liver dysfunction, progressive skin lesions, vasculitis, and uveitis persisting for > 3 months, and (4) exclusion of other possible diseases, such as primary EBV infection, autoimmune disease, immunodeficiencies, and lymphomas. Patients who fulfilled all (1) to (4) were diagnosed as sCAEBV. These criteria are compatible with the definition of sCAEBV described in the WHO definition of 2017. The definitions of disease activities and responses The disease activities are defined in previous reports (Blood. 2012;119, p673 and BMT. 2016;51, p879) as follows: positive of fever, ALT level elevation, vasculitis, progressive skin lesions, or uveitis. We defined the complete resolution of disease activity as complete response (CR) and CR with a significant decrease in PB EBV-DNA load (< 10 2.5 copies/μg DNA) as virological CR (vCR). Results 81 patients who met the diagnostic criteria of sCAEBV were analyzed. The median age at HSCT was 24 years old, and the three-year overall survival rate (3-year OS) was 74.0%. Of 74 patients whose viral load after HSCT evaluated, 49 (66.2%) achieved vCR. The multivariate cox proportional hazard model revealed that advanced age, adolescent and young adult (AYA) (age, 15-39; n = 48) and adult (age, > 40; n = 13), was a risk factor of poor OS. The hazard ratios (HR) of AYA and adult groups were 10.14 and 4.63 respectively. It also showed that the presence of HLH at HSCT (HR 4.55), high sIL-2R (≥ median, 691 U/mL) at HSCT (HR 5.27), and conditioning without total body irradiation (HR 3.23) were independently associated with poor survival. Moreover, the median survival time of patients with active disease and extremely high sIL-2R level (≥ 3 × median, 2073 U/mL) was 0.9 months, whereas the other groups did not reach the median. Conclusion Although HSCT is the only curative treatment for sCAEBV, the strategies need improvements in high-risk cases, especially of high sIL-2R. Disclosures Arai: ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Kyowa Kirin CO., LTD.: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Elsai Co Ltd: Research Funding; Abbott Japan LLC: Honoraria; Nippon Shinyaku Co. Ltd: Honoraria, Research Funding; Otsuka Pharmaceuticals Co. Ltd: Research Funding; Novartis Pharma KK: Honoraria; Takeda Pharmaceuticals Co Ltd: Honoraria, Research Funding; Shionogi & Co Ltd: Research Funding; Asahi Kasri Pharma Corporation: Research Funding; Sanofi: Honoraria; Pfizer japan: Honoraria; Astellas Pharma Inc.: Honoraria. Yamamoto: Bristol-Myers Squibb Company: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; NIPPON SINYAKU CO., LTD: Honoraria; Novartis Pharma: Honoraria; ONO PHARMACEUTICAL CO.: Honoraria; Otsuka Pharmaceutical: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria. Nakamae: Astellas Pharma Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Simon-Kucher & Partners: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria; Novartis: Honoraria, Research Funding; Pfizer Japan Inc.: Honoraria; Bristol-Myers Squibb　Company: Honoraria, Research Funding; Alexion: Research Funding; PPD-SNBL K.K: Research Funding; CMIC HOLDINGS Co., Ltd: Research Funding. Ichinohe: Repertoire Genesis Inc.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria; Celgene: Honoraria; Zenyaku Kogyo Co.: Research Funding; Takara Bio Inc.: Research Funding; Taiho Pharmaceutical Co.: Research Funding; Sumitomo Dainippon Pharma Co.: Honoraria, Research Funding; Otsuka Pharmaceutical Co.: Research Funding; Nippon Shinyaku Co: Research Funding; Ono Pharmaceutical Co.: Honoraria, Research Funding; Kyowa Kirin Co.: Honoraria, Research Funding; FUJIFILM Wako Chemicals.: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Eisai Co.: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Chugai Pharmaceutical: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co.: Honoraria; AbbVie Pharma: Research Funding; Astellas Pharma: Honoraria, Research Funding. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria; Meiji Seika Pharma Co, Ltd.: Honoraria.

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Functional Roles of DDX41 Mutations in the Development of Myeloid Malignancies

Blood ◽

10.1182/blood-2021-152269 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 150-150

Author(s):

Ayana Kon ◽

Masahiro Marshall Nakagawa ◽

Keisuke Kataoka ◽

Hideki Makishima ◽

Manabu Nakayama ◽

...

Keyword(s):

Stem Cells ◽

Innate Immunity ◽

Research Funding ◽

Pharmaceutical Research ◽

Mutant Mice ◽

Loss Of Function ◽

Chugai Pharmaceutical ◽

Myeloid Malignancies ◽

Myeloid Neoplasms ◽

Otsuka Pharmaceutical

Abstract DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, little is known about the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. BM-specific biallelic Ddx41 deletion using Vav1-Cre resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoietic development. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), homozygous Ddx41 KO (Ddx41-/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with BM from Ddx41-/- or Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF). None of the mice transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice developed myeloid neoplasms. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41-/- or Ddx41R525H/- mice-derived cells in PB was markedly reduced compared to that of cells derived from Ddx41+/+ mice. In contrast, Ddx41+/- or Ddx41R525H/+ mice-derived cells showed no significant changes in competitive bone marrow reconstitution compared to Ddx41+/+ mice-derived cells. Notably, about half of the recipient mice died due to BMF when Ddx41R525H/--derived BM cells were co-transplanted with Ddx41+/--derived BM cells but not with wild-type BM cells, suggesting some non-cell autonomous effect of Ddx41R525H/- cells on Ddx41+/- cells. Transcriptome analysis of stem cells (Kit +Sca-1 -Lin low cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in stem cells from all the mutant mice, with larger changes for Ddx41R525H/- than Ddx41+/- or Ddx41 R525H/+ cells. Notably, Ddx41R525H/- -derived stem cells exhibited a significant upregulation of genes involved in innate immunity, including an upregulation of cGAS-STING innate immunity pathways, as well as an enhanced Trp53 pathway, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis. Proteomics analysis confirmed the significant downregulation of ribosomal proteins in hematopoietic cells derived from Ddx41R525H/- mice. In summary, our results revealed an essential role of Ddx41 in normal hematopoiesis. While both heterozygous Ddx41 KO and heterozygous R525H knock-in did not develop myeloid neoplasm, compound biallelic loss-of function and R525 alleles led to a compromised function of hematopoietic stem cells, which was evident from reduced competitive repopulation capacity and impaired hematopoietic differentiation, where activated innate immunity and impaired ribosome functions may play important roles. Their roles in myeloid neoplasms need further evaluation. Disclosures Nakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Kataoka: Celgene: Honoraria; Eisai: Honoraria; Astellas Pharma: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Sumitomo Dainippon Pharma: Honoraria; Kyowa Kirin: Honoraria; Janssen Pharmaceutical: Honoraria; MSD: Honoraria; Takeda Pharmaceutical: Honoraria; Otsuka Pharmaceutical: Honoraria; Asahi Genomics: Current equity holder in publicly-traded company; Otsuka Pharmaceutical: Research Funding; Chordia Therapeutics: Research Funding; Chugai Pharmaceutical: Research Funding; Takeda Pharmaceutical: Research Funding; Bristol-Myers Squibb: Research Funding; Eisai: Other: Scholarship; Otsuka Pharmaceutical: Other: Scholarship; Ono Pharmaceutical: Other: Scholarship; Kyowa Kirin: Other: Scholarship; Shionogi: Other: Scholarship; Takeda Pharmaceutical: Other: Scholarship; Summitomo Dainippon Pharma: Other: Scholarship; Chugai Pharmaceutical: Other: Scholarship; Teijn Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Genetic Alterations: Patents & Royalties: PD-L1 abnormalties . Ogawa: Ashahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding.

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Prediction for the Risk of Gvhd after Allogeneic HSCT in Patients with ATL Treated By Mogamulizumab

Blood ◽

10.1182/blood-2019-126912 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3290-3290

Author(s):

Yuhei Kamada ◽

Ayako Kuroki ◽

Naosuke Arima ◽

Maiko Hayashida ◽

Daisuke Nakamura ◽

...

Keyword(s):

Serum Concentration ◽

Research Funding ◽

Mononuclear Cells ◽

Significant Risk ◽

Peripheral Blood Mononuclear ◽

Hematopoietic Stem ◽

Chugai Pharmaceutical ◽

Number Of Patients ◽

Otsuka Pharmaceutical ◽

Grade Ii

Introduction: Mogamulizumab (Mog) is an anti-CCR4 (C-C motif chemokine receptor 4) antibody, which induce antibody dependent cellular cytotoxicity against cells expressing CCR4. Administration of Mog before allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with adult T-cell leukemia/lymphoma (ATL) is considered to raise the risk of graft versus host disease (GVHD). Since not only ATL cells but regulatory T cells (Tregs) express CCR4, both are targeted by Mog. Tregs play a crucial role in maintaining and suppressing excessive immune responses especially in GVHD, and the depletion of Tregs by Mog is considered to result in the progression of GVHD. The aim of this study is to elucidate the characteristics of GVHD observed in patients treated by Mog, and further tried to predict the risk of GVHD by incorporating the information of serum concentration of Mog, the number of Tregs, and the alteration in Treg fractions (naïve/resting Treg; nTreg, effector Treg; eTreg). Patients and Methods: In this study, we analyzed 25 ATL patients who received allo-HSCT. The serum concentration of Mog was measured by SRL Medisearch (Tokyo, Japan). The fraction of Tregs in peripheral blood mononuclear cells (PBMNCs) were analyzed by flow cytometry (BD FACSCalibur, BD biosciences). Statistical analysis (the cumulative incidence of Grade II-IV GVHD and the number of Tregs) were performed with EZR (Easy R) software (Jichi Medical University, Saitama, Japan). Results: In comparison to allo-HSCT without Mog (Mog-, n=14), the risk of Grade II-IV GVHD was increased in the allo-HSCT with Mog (Mog+, n=11). Stage 3 or more skin GVHD was observed in 4 patients of Mog+ (36 %) and 1 patient of Mog- (7 %), respectively. Notably, stage 3 or more gut GVHD was observed in 6 patients of Mog+ (54 %), and 4 patients died with GVHD or infectious complications, whereas 3 Mog- (21 %) experienced non-fatal gut GVHD. Moreover, cytomegalovirus enteritis was observed only in the Mog+ cases. Mog was detectable in serum for approximately 6 months after the last administration (cut off level; 5.0 ng/ml), and the number of Tregs, especially eTreg (Foxp3 high, CD45RA low) was continuously suppressed during this period. In the Mog+ case, the number of eTregs recovered 180 days after allo-HSCT in most cases. Among 7 patients who received Mog before allo-HSCT, 5 patients who developed gut GVHD were performed allo-HSCT on average 52 (19-87) days after the last administration of Mog. The serum level of Mog was 100-1000 ng/ml in the period, and this concentration of Mog was considered to be critical for the onset of severe GVHD. Conclusion: Use of mogamulizumab before allo-HSCT for ATL patient is a significant risk for severe GVHD, especially gut GVHD. Although the number of patients in this study is limited, our results suggest that the serum concentration of mogamulizumab (≥10 ng/ml) and the number of eTregs in PB at the time of allo-HSCT may enable to predict the risk of developing GVHD. Disclosures Yoshimitsu: Sanofi: Speakers Bureau; Chugai: Speakers Bureau; Takeda: Speakers Bureau; Bristol-Myer-Squibb,: Speakers Bureau; Shire: Speakers Bureau; Novartis: Speakers Bureau. Ishitsuka:Daiichi Sankyo: Consultancy, Honoraria; Alexion: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Otsuka Pharmaceutical: Honoraria; Eisai: Honoraria, Research Funding; MSD: Research Funding; Asahi kasei: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; mundiharma: Honoraria; MSD: Research Funding; Celgene: Honoraria; Shire: Honoraria; Mochida: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Eli Lilly: Research Funding; Genzyme: Honoraria; sanofi: Honoraria; Bristol-Myers Squibb: Honoraria; sanofi: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Genzyme: Honoraria; Alexion: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; mundiharma: Honoraria; Eisai: Honoraria, Research Funding; Taiho Pharmaceutical: Honoraria, Research Funding; Mochida: Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria; Shire: Honoraria; Otsuka Pharmaceutical: Honoraria; Novartis: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Teijin Pharma: Research Funding; Pfizer: Honoraria; Yakult: Research Funding; Astellas Pharma: Honoraria, Research Funding; Asahi kasei: Research Funding; Eli Lilly: Research Funding; Janssen Pharmaceutical: Honoraria; Celgene: Honoraria; Teijin Pharma: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Yakult: Research Funding.

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Results of the Phase 1b Dose Escalation Study of OPB-111077, Decitabine, and Venetoclax for the Treatment of Newly Diagnosed or Relapsed/Refractory AML

Blood ◽

10.1182/blood-2020-140729 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 10-10

Author(s):

Lindsay Wilde ◽

Ubaldo Martinez-Outschoorn ◽

Neil Palmisiano ◽

Gina Keiffer ◽

Margaret Kasner

Keyword(s):

Dose Escalation ◽

Research Funding ◽

Acid Metabolism ◽

Pharmaceutical Research ◽

Refractory Disease ◽

Newly Diagnosed ◽

Dose Escalation Study ◽

Phase Ib ◽

Otsuka Pharmaceutical ◽

Refractory Aml

Background: The combination of a hypomethylating agent (HMA) and the Bcl-2 inhibitor venetoclax (VEN) has revolutionized the treatment of elderly patients with newly diagnosed acute myeloid leukemia (AML). However, patients treated with this regimen inevitably relapse and subsequent treatment options are limited. Furthermore, the success of this combination in the relapsed/refractory setting has been less impressive. Mechanisms of resistance to HMA/VEN are of great interest, and alterations in leukemic cell metabolism have been implicated. It is hypothesized that drugs that target oxidative phosphorylation (OXPHOS), fatty acid metabolism, and /or amino acid metabolism may be successful in overcoming HMA/VEN resistance. OPB-111077 is a novel, oral, low-molecular-weight compound that was shown in preclinical models to inhibit mitochondrial electron transport and have an inhibitory effect on the growth of AML cells. When given in combination with decitabine, OPB-111077 showed a more potent antitumor effect in a KG-1 tumor-bearing mouse model, thus providing support for conducting clinical trials of this combination. AML cells treated with OPB-111077 and venetoclax have also been shown to have decreased proliferation and increased apoptosis. The effects on proliferation and apoptosis of the combination of OPB-111077 and venetoclax were more pronounced in AML cells that were genetically engineered to increase OXPHOS. These data formed the basis for the development of a clinical trial utilizing the triplet of OPB-111077, decitabine, and venetoclax for the treatment of newly diagnosed or relapsed/refractory AML. Herein, we report results from the Phase Ib dose escalation study. Methods: In this phase Ib single center study (NCT03063944), OPB-111077 is administered daily starting on day 1 and continuing throughout the treatment cycle. Decitabine 20mg/m2 is given for 5 days starting on day 4 of cycle 1. Venetoclax 70mg daily (if receiving posaconazole prophylaxis) or 100mg daily (if receiving voriconazole) is started on day 4 and given continuously until day 28. If a response is seen within 2 cycles, treatment continues until toxicity, disease progression, or availability of an alternative therapy. Patients were enrolled in cohorts of escalating dose levels of OPB-111077 using a traditional 3+3 design. The primary objectives were to determine preliminary safety and tolerability as well as maximum tolerated dose (MTD) of OPB-111077. The secondary objective was to measure preliminary efficacy. Correlative studies including metabolomics, ATP measurement, apoptosis, and proliferative assays were performed on samples of blasts and non-cancerous cells that were collected from blood or marrow. Results: As of July 15, 2020, 2 patients with newly diagnosed AML and 7 patients with relapsed/refractory AML were treated with the triplet. Patients received OPB-111077 at 150mg (n=3), 200mg (n=3), and 250mg (n=3). Median age was 73 years (range, 23-79) and 7 patients (78%) were male. The median number of prior systemic anticancer regimens for patients with relapsed/refractory disease was 2 (range, 1-5); no patients had undergone prior stem cell transplant. The median treatment duration has not yet been reached; 3 patients are receiving ongoing treatment. No patients experienced a dose limiting toxicity. The most common Grade ≥3 adverse event (AE) was febrile neutropenia (56%). No patients discontinued due to AEs. No pts experienced tumor lysis syndrome. Three patients died after completing their study treatment, all due to progressive AML. The best overall response to therapy was a complete remission in 2 (22%) patients (1 with newly diagnosed AML and 1 with relapsed/refractory disease). Metabolomics using liquid chromatography/mass spectrometry was performed on paired pre- and post-treatment samples and intracellular metabolic changes in glycolysis and the tricarboxylic acid (TCA) cycle were observed consistent with on-target effects. Conclusion: The triplet of OPB-111077, decitabine, and venetoclax for the treatment of newly diagnosed or relapsed/refractory AML was safe and well tolerated, and showed preliminary anti-leukemic efficacy. A planned expansion phase is underway utilizing the 250mg daily dose of OPB-111077. Disclosures Martinez-Outschoorn: Otsuka Pharmaceutical: Research Funding. Palmisiano:AbbVie: Research Funding; Genentech: Research Funding. Kasner:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka Pharmaceutical: Research Funding.

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Hematopoiesis in the Elderly: Age-Associated Effects in Frequency, Function, and Gene Expression of Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.1505.1505 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1505-1505

Author(s):

Wendy W. Pang ◽

Elizabeth A. Price ◽

Irving L. Weissman ◽

Stanley L. Schrier

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Expression Profiles ◽

Frequency Function ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Cycle Status

Abstract Abstract 1505 Poster Board I-528 Aging of the human hematopoietic system is associated with an increase in the development of anemia, myeloid malignancies, and decreased adaptive immune function. While the hematopoietic stem cell (HSC) population in mouse has been shown to change both quantitatively as well as functionally with age, age-associated alterations in the human HSC and progenitor cell populations have not been characterized. In order to elucidate the properties of an aged human hematopoietic system that may predispose to age-associated hematopoietic dysfunction, we evaluated and compared HSC and other hematopoietic progenitor populations prospectively isolated via fluorescence activated cell sorting (FACS) from 10 healthy young (20-35 years of age) and 8 healthy elderly (65+ years of age) human bone marrow samples. Bone marrow was obtained from hematologically normal young and old volunteers, under a protocol approved by the Stanford Institutional Review Board. We determined by flow cytometry the distribution frequencies and cell cycle status of HSC and progenitor populations. We also analyzed the in vitro function and generated gene expression profiles of the sorted HSC and progenitor populations. We found that bone marrow samples obtained from normal elderly adults contain ∼2-3 times the frequency of immunophenotypic HSC (Lin-CD34+CD38-CD90+) compared to bone marrow obtained from normal young adults (p < 0.02). Furthermore, upon evaluation of cell cycle status using RNA (Pyronin-Y) and DNA (Hoechst 33342) dyes, we observed that a greater percentage of HSC from young bone marrow are in the quiescent G0- phase of the cell cycle compared to elderly HSC, of which there is a greater percentage in G1-, S-, G2-, or M-phases of the cell cycle (2.5-fold difference; p < 0.03). In contrast to the increase in HSC frequency, we did not detect any significant differences in the frequency of the earliest immunophenotypic common myeloid progenitors (CMP; Lin-CD34+CD38+CD123+CD45RA-), granulocyte-macrophage progenitors (GMP; Lin-CD34+CD38+CD123+CD45RA+), and megakaryocytic-erythroid progenitors (MEP; Lin-CD34+CD38+CD123-CD45RA-) from young and elderly bone marrow. We next analyzed the ability of young and elderly HSC to differentiate into myeloid and lymphoid lineages in vitro. We found that elderly HSC exhibit diminished capacity to differentiate into lymphoid B-lineage cells in the AC6.21 culture environment. We did not, however, observe significant differences in the ability of young and elderly HSC to form myeloid and erythroid colonies in methylcellulose culture, indicating that myelo-erythroid differentiation capacity is preserved in elderly HSC. Correspondingly, gene expression profiling of young and elderly human HSC indicate that elderly HSC have up-regulation of genes that specify myelo-erythroid fate and function and down-regulation of genes associated with lymphopoiesis. Additionally, elderly HSC exhibit increased levels of transcripts associated with transcription, active cell-cycle, cell growth and proliferation, and cell death. These data suggest that hematopoietic aging is associated with intrinsic changes in the gene expression of human HSC that reflect the quantitative and functional alterations of HSC seen in elderly bone marrow. In aged individuals, HSC are more numerous and, as a population, are more myeloid biased than young HSC, which are more balanced in lymphoid and myeloid potential. We are currently investigating the causes of and mechanisms behind these highly specific age-associated changes in human HSC. Disclosures: Weissman: Amgen: Equity Ownership; Cellerant Inc.: ; Stem Cells Inc.: ; U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties.

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Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells

The Journal of Cell Biology ◽

10.1083/jcb.122.4.897 ◽

1993 ◽

Vol 122 (4) ◽

pp. 897-902 ◽

Cited By ~ 196

Author(s):

WH Fleming ◽

EJ Alpern ◽

N Uchida ◽

K Ikuta ◽

GJ Spangrude ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Fetal Liver ◽

Significant Proportion ◽

Cell Activity ◽

Rhodamine 123 ◽

Hematopoietic Stem ◽

Cell Cycle Status

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

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Intrapatient Complex Transcriptional Responses to Conventional Chemotherapy in Relapsed Acute Myeloid Leukemia Revealed By Single Cell RNA-Seq Analysis

Blood ◽

10.1182/blood-2021-153848 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1179-1179

Author(s):

Hideaki Mizuno ◽

Akira Honda ◽

Mineo Kurokawa

Keyword(s):

Single Cell ◽

Signaling Pathway ◽

Myeloid Leukemia ◽

Research Funding ◽

Chemotherapy Resistance ◽

Rna Seq ◽

Conventional Chemotherapy ◽

Chugai Pharmaceutical ◽

Dna Mutation ◽

Otsuka Pharmaceutical

Abstract Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

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Genotype-Phenotype Relationships and Therapeutic Targets in Acute Erythroid Leukemia

Blood ◽

10.1182/blood-2020-141750 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 17-18

Author(s):

June Takeda ◽

Kenichi Yoshida ◽

Akinori Yoda ◽

Lee-Yung Shih ◽

Yasuhito Nannya ◽

...

Keyword(s):

Cell Line ◽

Board Of Directors ◽

Research Funding ◽

Pharmaceutical Research ◽

Private Company ◽

Advisory Committees ◽

Otsuka Pharmaceutical ◽

Group A ◽

Genetic Profiles ◽

Stat Pathway

Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although gene mutations in AEL have been described in several reports, genotype phenotype correlations are not fully understood with little knowledge about the feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 105 AEL cases with the median age of 60 (23-86), using targeted-capture sequencing of commonly mutated genes in myeloid neoplasms, together with 1,279 SNPs for copy number measurements. Among these 105 cases, 13 were also analyzed by RNA sequencing. Genetic profiles of these 105 AEL cases were compared to those of 775 cases with non-erythroid AML (NEL) including 561 cases from The Cancer Genome Atlas and Beat AML study. An immature erythroid cell line (TF1) and three patient-derived xenografts (PDX) established from AEL with JAK2 and/or EPOR amplification. Cell line and samples from patients were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 subgroups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype and showed very poor prognosis. Remarkably, all PEL cases were categorized into Group A. Conspicuously, 80% of PEL cases had amplifications of JAK2 (6/10; 60%), EPOR (7/10;70%), and ERG (6/10;60%) loci on chromosomes 9p, 19q, and 21q, respectively, frequently in combination, although they were rarely seen in NEL cases. All cases in Group B (n=19, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. To further characterize this subgroup, we compared genetic profiles of STAG2-mutated AEL and NEL. Prominently, 70% (14/20) of STAG2-mutated cases in AEL had KMT2A-PTD, whereas it was found only in 8.8% (3/34) of NEL. CEBPA mutations were also more common in AEL (6/21; 29%) than NEL (4/34; 12%). While Group C was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in NEL, NPM1-mutated patents in this subgroup lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in NEL (25/209; 12%). The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, U2AF1 and KMT2C. Recurrent loss-of-function mutations in USP9X were unique to this subtype, although USP9X mutations have been reported in ALL with upregulation of JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only those cases with JAK2 or EPOR amplification, but also those without, suggesting that aberrantly upregulated STAT5 activation might represent a common defect in AEL. Based on this finding, we evaluated the effect of a JAK inhibitior, ruxolitinib, on an AEL-derived cell line and three PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest, ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with TF1 and 2 PDX models with STAT5 downregulation, although the other model was resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2, EPOR and/or ERG. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a role of JAK inhibition was suggested. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Miyazaki:Astellas Pharma Inc.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Takaori-Kondo:Celgene: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; MSD: Honoraria. Kataoka:Asahi Genomics: Current equity holder in private company; Otsuka Pharmaceutical: Research Funding; Takeda Pharmaceutical Company: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding. Usuki:Alexion: Research Funding, Speakers Bureau; Apellis: Research Funding; Novartis: Research Funding, Speakers Bureau; Chugai: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Ganser:Novartis: Consultancy; Celgene: Consultancy. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Ruxolitinib is used for drug efficacy test using patient-derived xenografts established from acute erythroid leukemia.

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Age-Dependent Decrease in Requirement for Glycolysis in Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2018-99-111616 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 328-328

Author(s):

Watanuki Shintaro ◽

Hiroshi Kobayashi ◽

Daiki Karigane ◽

Yukako Ootomo ◽

Ayumi Okinaga ◽

...

Keyword(s):

Cell Cycle ◽

Research Funding ◽

Energy Production ◽

Consumption Rate ◽

Proliferating Cells ◽

Hematopoietic Stem ◽

Atp Production ◽

Atp Concentration ◽

Age Dependent

Abstract Metabolic process governs cell cycle and differentiation status of hematopoietic stem cells (HSCs) through regulating the balance between oxidative phosphorylation and glycolysis. Cell cycle and self-renewal capacity dynamically changes during developmental and aging process; in early stage of life, HSCs are highly cycling and self-renew, whereas adult HSCs exhibit quiescent in cell-cycle, and gradually decline in its function as they age. How functional alteration of HSCs is coupled with metabolic program is an important issue for understanding the lifecourse of HSCs. Phosphoglycerate mutase 1 (Pgam1) is an enzyme catalyzing conversion of 3-phosphoglycerate into 2-phosphoglycerate. By deleting Pgam1, ATP production through glycolysis was abrogated while pentose-phosphate pathway (PPP) was spared, allowing us to examine dependence on glycolytic energy production without affecting nucleotide and NADPH production through PPP. Following inducible Pgam1 deletion in mice at various ages, the number of HSCs was measured. At an early life stage (5-week old), Pgam1 deletion resulted in decreased number of HSCs, suggesting an essential role of glycolysis for HSC maintenance. Notably, the number of HSCs in Pgam1-deleted mice restored in an age-dependent manner, with 2-year old mice exhibiting almost comparable number of HSCs with wild type mice. By contrast, upon HSC transplantation, the repopulation capacity of Pgam1-deficient HSCs from any stages of life was uniformly abrogated, indicating that glycolytic energy production is essential for stressed condition in which HSCs are forced to proliferate. Reverse transplantation of wild type HSCs into Pgam1-floxed recipients followed by conditional deletion in non-hematopoietic cells showed no phenotypes in hematopoietic lineages, suggesting a cell-autonomous requirement of Pgam1 for blood cells. To further determine the relevance of glycolysis with maintenance/proliferation balance, we performed in vitro culture utilizing newly-developed quiescence-maintaining condition with or without glucose. The number of HSCs under the quiescence-maintaining condition was irrelevant to the presence or absence of glucose, while cell number under the proliferating condition was markedly decreased in glucose-deprived condition. Furthermore, lymphoid-primed multipotential progenitors (LMPP) with marker phenotype of Flt3+ linage markers- Sca-1+ c-Kit+ cells, which show high cycling status in vivo, were highly dependent on glucose for survival in vitro. Collectively, proliferating cells require glucose irrespective to its cell type, while quiescent HSCs are less dependent on glycolytic energy production in line with age-dependent decrease of requirement for glycolysis in vivo. ATP concentration directly reflects ATP production/consumption rate inside cells. We utilized ATP-biosensor (GO-ATeam2)-transgenic mice to monitor ATP concentration in live HSCs under various metabolic environments in vitro. HSCs cultured in the proliferating condition exhibited gradual decrease in ATP concentration during 7-day culture, whereas in the quiescence-maintaining condition ATP was kept high. In the steady state, cycling multipotent progenitors (MPPs) exhibited lower ATP concentration than quiescent HSCs. These results suggest that quiescent state is characterized by lower ATP production/consumption rate than the proliferating cells. We then compared ATP concentration in young and old HSCs using the same system. The decrease of ATP under the proliferating condition was less prominent in old HSCs, while ATP concentration was comparable in the quiescence-maintaining condition. The result indicates the lower ATP production/consumption rate in old HSCs, presumably explaining lower dependence on glycolytic energy production in the steady state hematopoiesis. Altogether, we concluded that the dependence on glycolysis-driven ATP production derived from glucose decreased with age, underlying mechanism of which is explained by lower ATP consumption rate in old HSCs. Disclosures Okamoto: Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co.,Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.:: Research Funding; Nippon Shinyaku Co., Ltd: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Asahi Kasei Pharma Corp.:: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

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Hydroxyurea Can Be Used to Increase Mouse c-kit+Thy-1.1loLin−/loSca-1+ Hematopoietic Cell Number and Frequency in Cell Cycle In Vivo

Blood ◽

10.1182/blood.v90.11.4354 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4354-4362 ◽

Cited By ~ 25

Author(s):

Nobuko Uchida ◽

Annabelle M. Friera ◽

Dongping He ◽

Michael J. Reitsma ◽

Ann S. Tsukamoto ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Cell Number ◽

Short Term ◽

Synthesis Inhibitor ◽

Hematopoietic Stem ◽

Cell Cycle Status

Abstract The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

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