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Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. Significance: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten–eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type–specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis. Significance: We show that 5-hydroxymethylation profiles are cell type–specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes. This article is highlighted in the In This Issue feature, p. 265

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics. Significance: Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten–eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type–specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis.Significance:We show that 5-hydroxymethylation profiles are cell type–specific and associated with transcriptional abundance and chromatin accessibility across human hematopoiesis. TET2 loss caused aberrant growth and differentiation phenotypes and disrupted 5hmC and transcriptional landscapes. Treatment of TET2 KO HSPCs with ascorbate or azacitidine reverted 5hmC profiles and restored aberrant phenotypes.This article is highlighted in the In This Issue feature, p. 265

Supplementary Figure from The Cell Type–Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia

Supplementary Figure file showing Figures S1-S7

Supplementary Table from The Cell Type–Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia

Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax.Significance:Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions.See related commentary by Robinson and Levine, p. 266.This article is highlighted in the In This Issue feature, p. 247

Supplementary Table from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Supplementary Figure from Reengineering Ponatinib to Minimize Cardiovascular Toxicity

Small molecule tyrosine kinase inhibitors (TKI) have revolutionized cancer treatment and greatly improved patient survival. However, life-threatening cardiotoxicity of many TKIs has become a major concern. Ponatinib (ICLUSIG) was developed as an inhibitor of the BCR-ABL oncogene and is among the most cardiotoxic of TKIs. Consequently, use of ponatinib is restricted to the treatment of tumors carrying T315I-mutated BCR-ABL, which occurs in chronic myeloid leukemia (CML) and confers resistance to first- and second-generation inhibitors such as imatinib and nilotinib. Through parallel screening of cardiovascular toxicity and antitumor efficacy assays, we engineered safer analogs of ponatinib that retained potency against T315I BCR-ABL kinase activity and suppressed T315I mutant CML tumor growth. The new compounds were substantially less toxic in human cardiac vasculogenesis and cardiomyocyte contractility assays in vitro. The compounds showed a larger therapeutic window in vivo, leading to regression of human T315I mutant CML xenografts without cardiotoxicity. Comparison of the kinase inhibition profiles of ponatinib and the new compounds suggested that ponatinib cardiotoxicity is mediated by a few kinases, some of which were previously unassociated with cardiovascular disease. Overall, the study develops an approach using complex phenotypic assays to reduce the high risk of cardiovascular toxicity that is prevalent among small molecule oncology therapeutics.Significance:Newly developed ponatinib analogs retain antitumor efficacy but elicit significantly decreased cardiotoxicity, representing a therapeutic opportunity for safer CML treatment.

Without a protective atmosphere, space-exposed surfaces of airless Solar System bodies gradually experience an alteration in composition, structure and optical properties through a collective process called space weathering. The return of samples from near-Earth asteroid (162173) Ryugu by Hayabusa2 provides the first opportunity for laboratory study of space-weathering signatures on the most abundant type of inner solar system body: a C-type asteroid, composed of materials largely unchanged since the formation of the Solar System. Weathered Ryugu grains show areas of surface amorphization and partial melting of phyllosilicates, in which reduction from Fe3+ to Fe2+ and dehydration developed. Space weathering probably contributed to dehydration by dehydroxylation of Ryugu surface phyllosilicates that had already lost interlayer water molecules and to weakening of the 2.7 µm hydroxyl (–OH) band in reflectance spectra. For C-type asteroids in general, this indicates that a weak 2.7 µm band can signify space-weathering-induced surface dehydration, rather than bulk volatile loss.

Introduction: Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects. Hypothesis: Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity. Methods: Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.

Cancer-related anemia is present in more than 60% of newly diagnosed cancer patients and is associated with substantial morbidity and high medical costs. Drugs that enhance erythropoiesis are urgently required to decrease transfusion rates and improve quality of life. Clinical studies have observed an unexpected improvement in hemoglobin and RBC transfusion-independence in patients with acute myeloid leukemia (AML) treated with the isocitrate dehydrogenase 2 (IDH2) mutant-specific inhibitor enasidenib, leading to improved quality of life without a reduction in AML disease burden. Here, we demonstrate that enasidenib enhanced human erythroid differentiation of hematopoietic progenitors. The phenomenon was not observed with other IDH1/2 inhibitors and occurred in IDH2-deficient CRISPR-engineered progenitors independently of D-2-hydroxyglutarate. The effect of enasidenib on hematopoietic progenitors was mediated by protoporphyrin accumulation, driving heme production and erythroid differentiation in committed CD71(+) progenitors rather than hematopoietic stem cells. Our results position enasidenib as a promising therapeutic agent for improvement of anemia and provide the basis for a clinical trial using enasidenib to decrease transfusion dependence in a wide array of clinical contexts.

Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

著者人数: 66名 (所属. 宇宙航空研究開発機構宇宙科学研究所 (JAXA)(ISAS): 岩田, 隆浩; 安部, 正真; 大竹, 真紀子; 仲内, 悠佑; 松岡, 萌; L. Riu; 中藤, 亜衣子; 横田, 康弘; 坂谷, 尚哉; 山本, 幸生; 岡田, 達明; 澤田, 弘崇, 早川, 雅彦; 白井, 慶; 嶌生, 有理; 尾川, 順子; 照井, 冬人; 竹井, 洋; 佐伯, 孝尚; 中澤, 暁; 田中, 智; 吉川, 真; 渡邊, 誠一郎; 津田, 雄一), Accepted: 2019-03-07, 資料番号: SA1180385000

Analyses of meteorites and theoretical models indicate that some carbonaceous near-Earth asteroids may have been thermally altered due to radiative heating during close approaches to the Sun1–3. However, the lack of direct measurements on the subsurface doesn’t allow us to distinguish thermal alteration due to radiative heating from parent-body processes. In April 2019, the Hayabusa2 mission successfully completed an artificial impact experiment on the carbonaceous near-Earth asteroid (162173) Ryugu4,5, which provided an opportunity to investigate exposed subsurface material and test potential effects of radiative heating. Here we report observations of Ryugu’s subsurface material by the Near-Infrared Spectrometer (NIRS3) on the Hayabusa2 spacecraft. Reflectance spectra of excavated material exhibit a hydroxyl (OH) absorption feature that is slightly stronger and peak-shifted compared with that observed for the surface, indicating that space weathering and/or radiative heating have caused subtle spectral changes in the uppermost surface. The strength and shape of the OH feature suggests that the subsurface material experienced heating above 300 °C, similar to the surface. In contrast, thermophysical modelling indicates that radiative heating cannot increase the temperature above 200 °C at the estimated excavation depth of 1 m, even at the smallest heliocentric distance possible for Ryugu. This supports the hypothesis that primary thermal alteration occurred on Ryugu’s parent body. Hayabusa2 created an artificial crater on Ryugu to analyse the subsurficial material of the asteroid. Results show that the subsurface is more hydrated than the surface. It experienced alteration processes that can be traced back to Ryugu’s parent body.

Introduction: Loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) are commonly found in both germline and somatic hematopoietic malignancies and confer particularly poor prognosis in AML. However, it remains unclear how RUNX1 functions during hematopoietic and leukemic development, particularly because RUNX1 mutations alone are not sufficient to cause myeloid malignancy and some models show that RUNX1 mutations confer hematopoietic stem cell defects. Recently, mouse models have shown that RUNX1-deficient neutrophils upregulate NFκB activity, and hematopoietic stem and progenitor cells (HSPCs) with overactive inflammatory pathways gain competitive advantage under chronic inflammation. Thus, we hypothesized that while RUNX1 mutations impair normal HSPC function, inflammation may select for or rescue RUNX1 mutant HSPCs. Methods: To interrogate the effect of RUNX1 loss in human CD34+ HSPCs, we disrupted the RUNX1 locus using CRISPR/Cas9 and AAV6-mediated homology directed repair. Importantly, by using an AAV6 vector that carries arms of homology flanking a fluorescent reporter expression cassette, we are able to track and isolate cells edited at the RUNX1 locus for in vitro and in vivo functional analyses and for molecular characterization using RNA-seq and ATAC-seq. Results: First, we used this system to evaluate the functional consequences of RUNX1 knockout (KO) in human CD34+ HSPCs. Loss of RUNX1 caused early erythroid-megakaryocytic differentiation arrest and skewing toward monocytic differentiation. RUNX1 KO cells demonstrated decreased proliferation, cell cycle arrest, and reduction in serial replating potential in vitro. In competitive transplantation experiments in NSG mice, RUNX1 KO engraftment decreased over time in both primary and secondary transplant, revealing a competitive disadvantage. Second, ATAC-seq peak motif analysis showed that PU.1 and NFκB motifs are more accessible upon RUNX1 KO whereas GATA, TAL1, and RUNX motifs were less accessible. Similarly, gene set enrichment analysis of transcriptional data confirmed the broad upregulation of NFκB-mediated inflammatory programs; downregulation of GATA1-dependent heme metabolism and platelet development pathways; and downregulation of MYC- and E2F-dependent cell cycle programs. These observations imply that RUNX1 directs cell fate decisions by recruiting and activating lineage-specific hematopoietic transcription factors and augmenting stem cell proliferation programs. We next sought to determine which cytokines are sufficient to drive RUNX1 KO cell expansion. RUNX1 KO cells not only expanded preferentially in NSG mice expressing human SCF, GM-CSF, and IL-3 (NSGS mice), but also were no longer defective in competitive transplants in these mice. Further, treatment with IL-3 was sufficient to significantly expand RUNX1 KO cells in vitro. Flow cytometry revealed that the IL-3 receptor CD123 is upregulated in RUNX1 KO cells compared to control. Similarly, RUNX1-mutant AML patient samples express higher levels of CD123 than RUNX1-wildtype AML patient samples. Finally, evaluation of publicly available RUNX1 ChIP-seq of bone marrow CD34+ HSPCs revealed that RUNX1 directly binds the promoter of CD123. Ongoing efforts are aimed at determining whether targeting CD123 and IL-3 signaling may be a viable therapeutic approach for the prevention or treatment of RUNX1-mutant myeloid malignancies. Conclusion: In summary, we established a RUNX1-deficient human HSPC model not only to evaluate the role of RUNX1 in hematopoiesis, but also to characterize intrinsic and extrinsic factors involved in RUNX1-deficient clonal expansion and leukemic transformation. We show that RUNX1 KO causes monocytic skew at the expense of erythro-megakaryocytic potential and severely limits HSC engraftment and expansion in vivo. Molecular profiling reveals that these effects are associated with dysregulation of both transcription factor activity and cytokine signaling. However, exposure to IL-3 rescues RUNX1-deficient cell proliferative defects in vitro and competitive engraftment defects in vivo. This hypersensitivity to IL-3 signaling is mediated in part by increased expression of the IL-3 receptor CD123. These findings reveal how RUNX1 mutations may initially behave in a deleterious manner but can ultimately confer an advantage to HSPCs under certain environmental conditions. Disclosures Majeti: CD47 Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Kodikaz Therapeutic Solutions Inc: Membership on an entity's Board of Directors or advisory committees.

Although loss-of-function RUNX1 mutations are commonly found in hematopoietic malignancies, how RUNX1 functions during hematopoietic and leukemic development is unclear. Using a CRISPR/AAV6 system to target the RUNX1 locus in human CD34+ hematopoietic stem and progenitor cells (HSPCs), we show that RUNX1 deficiency causes monocytic skew at the expense of erythro-megakaryocytic potential and stem cell activity, including a severe in vivo stem cell competitive defect. RNA-seq and ATAC-seq review that these effects are mediated by broad upregulation of PU.1 and NFKB transcriptional programs; downregulation of GATA1- and TAL1-dependent erythro-megakaryocytic differentiation; and downregulation of cell cycle programs mediated by MYC and E2F. Treatment with IL-3 rescues RUNX1-deficient cell proliferative and stem cell defects. Together, these results show that RUNX1 controls transcription factor activity and cytokine signaling, and loss of RUNX1 causes monocytic skewing and hypersensitivity to IL-3-dependent expansion. We are currently studying how the IL-3 selects for inflammation-sensitive RUNX1 deficient cells and whether targeting IL-3 signaling may be a viable therapeutic in the prevention or treatment of RUNX1 mutant malignancies.

Acute Myeloid Leukemia (AML) remains one of the most difficult cancers to treat, with a 30% 2-year survival rate. High-throughput sequencing of AML patients has identified mutations, including FLT3, IDH1, and IDH2, for which targeted therapies have been developed. Enasidenib is an FDA-approved, first-in-class agent that preferentially inhibits IDH2-mutant activity and reduces levels of the oncometabolite 2-HG, inducing differentiation of IDH2-mutated blasts. Interestingly, greater than 50% of enasidenib-treated patients who had no objective clinical response still demonstrated improvement in their peripheral blood counts and reached RBC transfusion independence. The mechanism underlying this phenomenon is unknown but is of great clinical relevance given the high transfusion dependence and anemia-associated complications universally associated with AML. Thus, we sought to investigate how enasidenib drives normal hematopoiesis to improve quality of life and reduce morbidity in AML patients. In this study, we demonstrate that enasidenib enhances erythropoiesis from normal CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from cord blood (CB) and bone marrow. Enasidenib doubled the proportion and total number of mature CD71+/GPA+ erythroblasts after 8 days of culture with EPO, SCF, and IL-3. In the presence of EPO, enasidenib induced a gene signature characteristic of maturing erythrocytes, with increased expression of GATA1 (1.3 fold), EPOR (2 fold), and KLF1 (1.4 fold), and decreased PU.1 (0.5 fold) and GATA2 (0.7 fold). Enasidenib-treated progenitor cells further demonstrated increased hemoglobin production (1.9 fold) and morphologic characteristics of increased erythroid maturation. Next, we sought to determine if enasidenib augments erythroid differentiation through IDH2 and IDH2-dependent pathways. First, we found that other IDH inhibitors (AG-120, AGI-6780, and AG-881) did not increase erythropoiesis at doses ranging from 1-10μM. As expected for normal HSPCs, 2-HG was not present at detectable levels in either the DMSO or enasidenib-treated conditions, and addition of 2-HG (50, 200μM) did not affect the ability of enasidenib to increase the proportion of CD71+GPA+ cells. Because it is possible that enasidenib acts through inhibition of wild-type IDH2, we generated CRISPR-Cas9 engineered IDH2 knockout (KO) CD34+ cells and treated them with enasidenib. Similar to wildtype cells, IDH2 KO CB CD34+ cells demonstrated a 3.4-fold increase in %CD71+GPA+ erythroid cells. Thus, enasidenib augments erythropoiesis independently of both mutant and wildtype IDH2 pathways. We then investigated the progenitor population that enasidenib acts on to drive erythroid maturation. Enasidenib did not increase the number of BFU-E or CFU-E colonies or the proportion of BFU-E (IL3R-CD34+CD36-) and CFU-E (IL3R-CD34-CD36+) progenitors in colony forming or liquid culture assays, respectively, leading us to conclude that enasidenib acts on more mature erythroid progenitors. Indeed, treating sorted mature CD71+ erythroid progenitors with enasidenib increased %CD71+GPA+ cells compared to DMSO control, whereas enasidenib treatment of CD71- early erythroid progenitors showed no effect. These observations provide evidence that enasidenib acts on CD71+ erythroid progenitors to increase late-stage erythroid differentiation. Given that CD71 allows for iron uptake into erythroid progenitors, we hypothesized that enasidenib modulates the heme biosynthesis pathway. Enasidenib inhibited the ABCG2 transporter, which effluxes protoporphyrin IX (PPIX), the direct precursor to heme, from the mitochondria and cytosol. Inhibition of ABCG2 by enasidenib could lead to PPIX accumulation within the cell, driving increased heme synthesis. To investigate this hypothesis, we treated cells with 20μM Ko143, a potent ABCG2 inhibitor, and observed a similar increase in %CD71+GPA+ cells as seen with enasidenib. Measurement of PPIX autofluorescence by flow cytometry and microscopy revealed an increase of PPIX in enasidenib-treated cells by 1.2-fold. Together, our data suggests that enasidenib drives maturation of CD71+ erythroid precursors independently of wildtype or mutant IDH2. Our results position enasidenib as a promising therapy to stimulate erythropoiesis and provide the basis for a clinical trial using enasidenib to improve anemia in a wide array of clinical contexts. Disclosures Majeti: Forty Seven Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy.

Introduction: Mutations in additional sex combs like 1 (ASXL1) are recurrently found in myeloid malignancies including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), but can also be found in pre-malignant states such as clonal hematopoiesis of indeterminate potential (CHIP). However, the mechanisms by which these mutations contribute to disease initiation and progression remain unresolved. Initial observations using murine models suggested a loss of function of the ASXL1 protein as relevant to disease. However more recently, several studies have suggested that recurrent mutations of ASXL1 result in a change-of function caused by a truncated ASXL1 protein. Most of these studies have been performed in a murine system and have used over-expression of truncated ASXL1. Methods: To interrogate the functional relevance of recurrent mutations in ASXL1 in human HSPCs, we generated a novel CRISPR/Cas9-mediated model that allows for the introduction of truncations or complete knockout of ASXL1 into CD34+ cord blood cells. Importantly, this is the first model which efficiently introduces these mutations into the native ASXL1 locus in human cord-blood and bone-marrow derived HSPCs while simultaneously introducing a fluorescent marker corresponding to the genotype, so individual cells can be tracked in vitro and in vivo. Results: Using this system, we demonstrate that truncated, but not deleted, ASXL1 shows a proliferative advantage, decreased ability to differentiate along the megakaryocyte and erythroid lineages, as well as increased serial replating in vitro. Interestingly, truncation of ASXL1 alone did not result in a myeloid differentiation block in vitro, in line with observations of mutant HSPCs in CHIP. In vivo, CD34+ cells harboring truncation of ASXL1 exhibited myeloid skewing and a proliferative advantage in competitive engraftment studies using a xenograft NSGS mouse model (both p Conclusion: In summary, our work demonstrates the utilization of a novel CRISPR/Cas9 model of human ASXL1-mutant HSPCs that closely resembles the phenotype seen in individuals with ASXL1 mutant CHIP and patients with myeloid malignancies carrying mutant ASXL1. Additionally, our model allows us to directly compare the deletion of ASXL1 with the truncation, adding to the evidence that truncated ASXL1 phenocopies pre-malignant and malignant hematopoiesis with recurrent ASXL1 mutations. Finally, we provide the first model for the spontaneous progression of pre-malignant human CHIP to myeloid malignancy, which should enable further studies of leukemogenesis and potentially disease prevention. Disclosures Majeti: FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy.

To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

Introduction: Mutations in IDH1 and IDH2 are recurrent in AML and several other cancers, resulting in the aberrant production of the onco-metabolite, R-2-hydroxyglutarate (2-HG), as well as an inability of mutant IDH1 to convert cytoplasmic alpha-ketoglutarate to isocitrate via reductive carboxylation. Currently, inhibitors of the neomorphic enzymes that abrogate the production of 2-HG, such as AG-120, are FDA-approved, but are not curative. Using a novel computational method (MiSL), we identified acetyl CoA carboxylase (ACACA) as a potential druggable target specifically in IDH1-mutated AML. ACACA regulates the de novo synthesis of lipid precursors by converting acetyl CoA to malonyl CoA building blocks. We hypothesize that IDH1 mutant AML exhibits a defect in reductive carboxylation and de novo fatty acid synthesis conferring preferential susceptibility to ACACA inhibition. Here, we investigate this hypothesis by comprehensively quantifying the metabolic landscape, including non-polar lipid metabolites, conferred by IDH1 R132H mutation compared to IDH2 mutation in isogenic cell lines and primary samples. Moreover, we investigate the in vitro and in vivo effects of targeting de novo lipid synthesis on IDH1 and IDH2 mutant AML. Methods: Comprehensive metabolomic profiling of primary FACS-purified AML blasts was performed using an in-house protocol optimised for extraction of non-polar lipid metabolites from less than 1 million primary cells. CD33+CD45+ leukemic blasts were profiled from 17 patient samples with IDH1 mutation (n=6), IDH2 mutation (n=5), or IDH1/2 wildtype (n=6) after culturing in serum-free media. 6 independent cord-blood CD34+ cells were profiled as a negative control. For validation of IDH1-specific effects, isogenic THP-1 cells transduced with doxycycline-inducible wildtype and R132H mutant IDH1 or R140Q mutant and wildtype IDH2 were profiled. Molecules were identified according to their molecular weight and retention time using Mass Hunter software (Agilent) and the Human Metabolome Database. For in vivo studies, primary AML samples were engrafted in NSG mice that were subjected to dietary modification with low lipid diet and/or treatment with selective inhibitors of ACACA and mutant IDH1. Results: Principle component analysis of metabolite abundance of 1400 unique compounds revealed striking differences between IDH1 and IDH2 mutant AML. Both IDH1 and IDH2 mutant samples produced high levels of 2-HG compared to wildtype AML and CD34+ cells (50 fold increase, P=4.5E-05). A major perturbation in multiple phospholipid fatty acid species was conferred by IDH1 R132H, but not by IDH2 mutation. The same pattern was observed in cell lines with 49 lipid species decreased in the presence of mutant IDH1 compared to only 2 perturbed with mutant IDH2. Direct comparison of IDH1 vs IDH2 mutant primary samples revealed 54 lipid metabolites significantly down-regulated in IDH1 mutant blasts (adjusted P value To investigate the effects of targeting de novo lipid synthesis on IDH1 mutant AML in vivo, we engrafted primary IDH1 mutant AML and tested growth with lipid-free compared to normal diet. At 12 weeks, IDH1 mutant AML showed reduced growth in the bone marrow of mice on lipid-free diet (SU389 11% vs 40%, n=10 mice, P=0.03 and SU372 21% vs 34%, n=10, P=0.02 Mann-Whitney U). IDH1 mutant AML was susceptible to ACACA inhibition with shRNA, CRISPR targeting, or selective nanomolar inhibitors. Knockdown of ACACA with independent shRNAs caused a defect in cell growth in the presence of IDH1 R132H, but not in its absence or with scrambled shRNA (p=0.009, shRNA #1 vs. scrambled; p=0.01, shRNA #2 vs. scrambled) in vitro and in xenografts. Primary IDH1 R132 mutated AML blasts were selectively sensitive to ACACA inhibitor treatment compared to IDH1 wildtype normal karyotype blasts (IC50 0.6 uM vs 6 uM, p=0.009). Notably, IDH1-mutant AML blasts pre-treated with 10mM AG-120 remained susceptible to ACACA inhibition, identifying a 2-HG independent vulnerability. Similar findings were observed in a solid tumor IDH1 mutant sarcoma model in vivo. Conclusion : These results support our hypothesis that IDH1 mutant AML exhibits a defect in de novo fatty acid synthesis conferring preferential susceptibility to ACACA inhibition, and suggests that pharmacologic inhibitors of ACACA may complement IDH1 mutation-specific inhibitors in the clinic. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine. To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in "Hit & Run," we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis. First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease. Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14). Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. Disclosures No relevant conflicts of interest to declare.

Summary Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs., Graphical Abstract, Highlights • Single-cell transplantation reveals dramatic age-related changes in HSC composition • MyRPs/MySCs increase with age as a frequency of whole BM cells and the HSC compartment • Latent-HSCs were identified exclusively in the aged bone marrow • Latent-HSCs have restricted potential in primary, but not secondary, transplants, Yamamoto et al. explore age-related changes to HSC function through large-scale clonal analysis using single-cell transplantation. They find large increases in myeloid-restricted repopulating progenitors (MyRPs) as well as a population of MyRPs that display a broader differentiation capacity only in secondary transplants, suggesting additional mechanisms contributing to hematopoietic aging.

著者人数: 13名, Accepted: 2017-8-18, 資料番号: SA1170090000

Graft-versus-host disease (GVHD), mediated by donor-derived alloreactive T cells, is a major cause of nonrelapse mortality in allogeneic hematopoietic stem cell transplantation. Its therapy is not well-defined. We established allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAbs) that specifically target HLA molecules, with steady death of target-expressing cells. One such ASHmAb, against HLA-A*02:01 (A2-kASHmAb), was examined in a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγ(null) mice were injected with healthy donor human peripheral blood mononuclear cells, some expressing HLA-A*02:01, some not. Administration of A2-kASHmAb promoted the survival of mice injected with HLA-A*02:01-expressing peripheral blood mononuclear cells (p0.0001) and, in humanized NOD/Shi-scid/IL-2Rγ(null) mice, immediately cleared HLA-A*02:01-expressing human blood cells from mouse peripheral blood. Human peripheral blood mononuclear cells were again detectable in mouse blood 2 to 4 weeks after A2-kASHmAb administration, suggesting that kASHmAb may be safely administered to GVHD patients without permanently ablating the graft. This approach, different from those in existing GVHD pharmacotherapy, may open a new door for treatment of GVHD in HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) using cord blood or haploidentical donor is a promising alternative option for patients who can not find an HLA-matched donor. However, HLA-mismatch allo-HSCT may be complicated by graft-versus-host disease (GVHD), a major cause of non-relapse mortality mediated by alloreactive T cells. Infusion of anti-thymocyte globulin (ATG) is used both as a treatment for and as a prophylactic against GVHD but ATG, like other immunosuppressive regimens, reacts against cells without distinguishing between donor and recipient cells. ATG is polyclonal and causes many side effects such as allergic reaction and increasing susceptibility to infection due to repression of T cells. Our ultimate goal is to develop a novel therapeutic approach to GVHD with anti-HLA monoclonal antibody specifically targeting the mismatched HLA molecule of donor origin to transiently ablate alloreactive T cells. To generate allele-specific anti-HLA antibody (ASHmAb), we established a rapid and efficient strategy using two different HLA-transgenic mouse strains, HLA-A24 and HLA-A2 and immunized them with HLA-A* 02:01 and HLA-A* 24:02 tetramers loaded with cytomegalovirus PP65 peptide, respectively. Antibodies were screened for HLA allele-specific complement-dependent cytotoxicity in vitro. We successfully established three killing ASHmAbs (kASHmAbs) against HLA-A* 02:01, -A* 02:01/–A* 03:01, and -A* 23:01/-A* 24:02. In vivo cytotoxicity and specificity of a kASHmAb against HLA-A* 02:01 (A* 02:01-kASHmAb) was examined in detail using a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγnull (NOG) mice were injected with either HLA-A2 or HLA-A26 positive human peripheral blood mononuclear cells (PBMCs) from healthy donors (i.e. HLA-A2 mice or HLA-A26 mice, respectively). Administration of A* 02:01-kASHmAb promoted the survival of HLA-A2 mice to 100%, with a mean survival of more than 6 months. In contrast, administration of A* 02:01-kASHmAb to nonreactive HLA was not effective; all HLA-A26 mice died within 2 months (p Next, we examined the cytotoxicity of the kASHmAb on the human graft in bone marrow using humanized NOG mice. Sublethally irradiated NOG mice were reconstituted with CD34 positive hematopoietic stem/progenitor cells from HLA-A2 positive cord blood. These humanized mice showed about 70-90% human T, B and myeloid cells in the peripheral blood. When these mice were administrated with A* 02:01-kASHmAb for five consecutive days (60 mg/day), mature human blood cells disappeared immediately from the mouse peripheral blood. Interestingly, human PBMCs were detectable again in the mouse blood in 2-4 weeks after the last kASHmAb administration, suggesting kASHmAb may have preferential cytotoxic effect on mature PBMCs sparing hematopoietic stem progenitor cells. The mechanism of this preferential killing is not clear, but an optimal dose of kASHmAb may be safely administered to GVHD patients without permanently ablating the graft and necessitating repeated transplantation. This study is the first reported instance of using the cytotoxic effect of HLA antibodies against GVHD. Unlike ATG, kASHmAb specifically killed donor cells and contributed to the survival of the recipients. While this novel therapeutic approach requires a panel of kASHmAbs to different HLA alleles, this may open a new door for treatment of severe GVHD. Furthermore, optimization of the kASHmAb administration protocol may contribute to tolerance induction. Disclosures: No relevant conflicts of interest to declare.

Abstract 4544 [Introduction] Hematopoietic cell transplantation (HCT) is a promising strategy for the treatment of both hematologic malignancies and non-malignant hematologic dyscrasias. However, HCT is sometimes complicated by infectious diseases, especially those caused by virus. Although the most important defense mechanism to control viral replication is inducing virus-specific cytotoxic T-lymphocytes (CTLs), the details of CTL development are not fully elucidated in human transplantation. In 1978, Zinkernagel, et al., clarified in murine hematopoietic stem cell transplantation models that MHC-restricted T cells are not governed by donor HLA expressed in bone marrow stem and progenitor cells. Instead, MHC restriction is acquired by interaction with recipient thymic epithelial cells. For this reason, virus-specific T cells are not induced by the mismatched HLA. Due to the increasing number of cord blood and haplo-identical transplantations, many transplantations are being performed under HLA-mismatched conditions. However, the full repercussions of this HLA-mismatch on viral immunity are still unknown. [Clinical case / Methods] For 4 years we followed a 35-year-old patient (HLA-A*24:02, A*33:03) with acute myeloid leukaemia of subtype t(8;21)(q22;q22);RUNX1(AML1)-RUNX1T1(ETO) of the 2008 WHO classification who underwent cord blood (HLA-A*02:01, A*24:02) transplantation following myeloablative conditioning regimen in first complete molecular remission. We analyzed the cytomegalovirus-specific CD8(+) T cells (CMV-CTLs) through HLA-A*02:01 tetramer (HLA-mismatched) and HLA-A*24:02 tetramer (HLA-matched) by flow cytometer. T cell receptor repertoire of these tetramer-positive CMV-CTLs were analyzed by CTL cloning and CDR3 sequencing. [Results / Discussion] The patient had a short period of CMV antigenemia but not CMV disease which was well-controlled by drip infusion of the anti-viral reagent ganciclovir after transplantation. On day 41 after transplantation, only A24-restricted (HLA-matched) tetramer-positive CMV-CTLs were present. By day 195, however, A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs also appeared. We considered that the donor-derived dendritic cells (DCs) induced A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs from naïve T cells, which are originally contained in cord blood inoculum. These mismatched donor HLA restricted memory T cells existed for a long time with a wide-ranging repertoire after transplantation, however, they were considered to have a limited functional role in infection immunity because lungs and intestinal tracts epithelial cells do not express HLA-A2. Usually, cord blood with 0, 1, or 2 loci mismatches is applicable for transplantation. Since HLA-A and B loci are checked only on a serological level, there are some possibilities in which every HLA-A and B loci are mismatched in the genetic level. In such a case, induction of virus-specific CTLs from donor naïve T cells might be impaired and result in uncontrollable viral infections. The findings of this case will be helpful to understand the impact of HLA-mismatch on viral immunity. Disclosures: No relevant conflicts of interest to declare.

Summary The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy., Graphical Abstract, Highlights • iPSC-derived rejuvenated CTLs are effective against EBV-induced tumors in vivo • Rejuvenated CTLs are implemented with an inducible caspase-9 (iC9)-based suicide system • Upon induction, the iC9 system efficiently leads to apoptosis in rejuvenated CTLs • The iC9-based system provides a safeguard for future iPSC-mediated cell therapy, In this article, Nakauchi and colleagues show that iPSC-derived rejuvenated CTLs (rejCTLs) implemented with an inducible caspase-9 (iC9)-based suicide system are effective against EBV-induced tumors in vivo. Upon induction, the iC9-based system efficiently and safely leads to apoptosis in these rejCTLs in vitro and in vivo and provides a safeguard for future iPSC-mediated cell therapy.