DOI： 10.1182/blood-2023-182620

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DOI： 10.1182/blood-2023-188986

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Language：English   Publisher：Communications Biology  

Protection of telomeres 1a (POT1a) is a telomere binding protein. A decrease of POT1a is related to myeloid-skewed haematopoiesis with ageing, suggesting that protection of telomeres is essential to sustain multi-potency. Since mesenchymal stem cells (MSCs) are a constituent of the hematopoietic niche in bone marrow, their dysfunction is associated with haematopoietic failure. However, the importance of telomere protection in MSCs has yet to be elucidated. Here, we show that genetic deletion of POT1a in MSCs leads to intracellular accumulation of fatty acids and excessive ROS and DNA damage, resulting in impaired osteogenic-differentiation. Furthermore, MSC-specific POT1a deficient mice exhibited skeletal retardation due to reduction of IL-7 producing bone lining osteoblasts. Single-cell gene expression profiling of bone marrow from POT1a deficient mice revealed that B-lymphopoiesis was selectively impaired. These results demonstrate that bone marrow microenvironments composed of POT1a deficient MSCs fail to support B-lymphopoiesis, which may underpin age-related myeloid-bias in haematopoiesis.

DOI： 10.1038/s42003-023-05374-0

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00428-023-03562-1

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Language：English   Publisher：Blood Advances  

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia–specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.

DOI： 10.1182/bloodadvances.2022008242

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Nature  

Sex chromosome disorders severely compromise gametogenesis in both males and females. In oogenesis, the presence of an additional Y chromosome or the loss of an X chromosome disturbs the robust production of oocytes1–5. Here we efficiently converted the XY chromosome set to XX without an additional Y chromosome in mouse pluripotent stem (PS) cells. In addition, this chromosomal alteration successfully eradicated trisomy 16, a model of Down’s syndrome, in PS cells. Artificially produced euploid XX PS cells differentiated into mature oocytes in culture with similar efficiency to native XX PS cells. Using this method, we differentiated induced pluripotent stem cells from the tail of a sexually mature male mouse into fully potent oocytes, which gave rise to offspring after fertilization. This study provides insights that could ameliorate infertility caused by sex chromosome or autosomal disorders, and opens the possibility of bipaternal reproduction.

DOI： 10.1038/s41586-023-05834-x

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Language：English   Publisher：Leukemia  

To identify molecules/pathways governing Venetoclax (VEN) sensitivity, we performed genome-wide CRISPR/Cas9 screens using a mouse AML line insensitive to VEN-induced mitochondrial apoptosis. Levels of sgRNAs targeting March5, Ube2j2 or Ube2k significantly decreased upon VEN treatment, suggesting synthetic lethal interaction. Depletion of either Ube2j2 or Ube2k sensitized AML cells to VEN only in the presence of March5, suggesting coordinate function of the E2s Ube2j2 and Ube2k with the E3 ligase March5. We next performed CRISPR screens using March5 knockout cells and identified Noxa as a key March5 substrate. Mechanistically, Bax released from Bcl2 upon VEN treatment was entrapped by Mcl1 and Bcl-XL and failed to induce apoptosis in March5 intact AML cells. By contrast, in March5 knockout cells, liberated Bax did not bind to Mcl1, as Noxa likely occupied Mcl1 BH3-binding grooves and efficiently induced mitochondrial apoptosis. We reveal molecular mechanisms underlying AML cell-intrinsic VEN resistance and suggest a novel means to sensitize AML cells to VEN.

DOI： 10.1038/s41375-023-01879-z

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Pediatric Blood and Cancer  

DOI： 10.1002/pbc.29915

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Language：English   Publisher：International Journal of Hematology  

Donor-derived hematological malignancies have been recognized as rare but serious late complications in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Most cases in the literature were diagnosed as myelodysplastic syndrome or acute leukemia, with very few malignant lymphoma reported. We herein present another case of donor-derived Burkitt lymphoma that occurred 9 years after allo-HSCT under continued administration of immunosuppressants for chronic graft-versus-host disease (GVHD). The patient achieved a partial response after rituximab-combined intensive chemotherapy. To reduce the risk of relapse and to avoid organ toxicities due to repeated chemotherapies, we performed upfront high-dose chemotherapy followed by stem cell rescue using donor-derived CD34+ cells, called pseudo-autologous HSCT (pASCT), and adjusted immunosuppressants appropriately. The patient remained disease-free for 23 months after pASCT without exacerbation of cGVHD. Although the observation period has been relatively short and longer follow-up is needed, pASCT may be a feasible option for donor-derived lymphoma even in patients with active cGVHD.

DOI： 10.1007/s12185-022-03458-x

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Language：Japanese   Publisher：The Japanese Society of Hematology  

<p>A 27-year-old woman with pancytopenia was admitted to our hospital. Bone marrow aspiration revealed 52.2% myeloperoxidase-positive myeloblasts, leading to a diagnosis of acute myeloid leukemia. While a screening test for chimeric genes related to leukemia initially yielded negative results, including for the <i>CBFB::MYH11</i> fusion gene, G-banded karyotyping uncovered the presence of inv (16)(p13.1q22). Further investigation by fluorescence <i>in situ</i> hybridization (FISH) confirmed the split signals for <i>CBFB</i>. A second screening test for leukemia-related chimeric genes with different PCR primers revealed the elusive <i>CBFB::MYH11</i> fusion gene. Subsequently, the type I <i>CBFB::MYH11</i> fusion gene was identified through exhaustive exploration using RNA sequencing for fusion gene discovery. This exceptional case highlights the existence of a distinctive subtype of <i>CBFB::MYH11</i> that may yield false-negative results in conventional chimeric fusion screening, thus emphasizing the indispensable utility of PCR primer modification, FISH, and RNA sequencing in the investigative process.</p>

DOI： 10.11406/rinketsu.64.1503

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1182/blood-2022-164647

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DOI： 10.1182/blood-2022-168650

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Gastric Cancer  

Background: Loss of E-cadherin expression is frequently observed in signet ring carcinoma (SRCC). People with germline mutations in CDH1, which encodes E-cadherin, develop diffuse gastric cancer at a higher rate. Loss of E-cadherin expression is thus assumed to trigger oncogenic development. Methods: To investigate novel therapeutic targets for gastric SRCC, we engineered an E-cadherin-deficient SRCC model in vitro using a human gastric organoid (hGO) with CDH1 knockout (KO). Results: CDH1 KO hGO cells demonstrated distinctive morphological changes similar to SRCC and high cell motility. RNA-sequencing revealed up-regulation of matrix metalloproteinase (MMP) genes in CDH1 KO hGO cells compared to wild type. MMP inhibitors suppressed cell motility of CDH1 KO hGO cells and SRCC cell lines in vitro. Immunofluorescent analysis with 95 clinical gastric cancer tissues revealed that MMP-3 was specifically abundant in E-cadherin-aberrant SRCC. In addition, CXCR4 molecules translocated onto the cell membrane after CDH1 KO. Addition of CXCL12, a ligand of CXCR4, to the culture medium prolonged cell survival of CDH1 KO hGO cells and was abolished by the inhibitor, AMD3100. In clinical SRCC samples, CXCL12-secreting fibroblasts showed marked infiltration into the cancer area. Conclusions: E-cadherin deficient SRCCs might gain cell motility through upregulation of MMPs. CXCL12-positive cancer-associated fibroblasts could serve to maintain cancer-cell survival as a niche. MMPs and the CXCL12/CXCR4 axis represent promising candidates as novel therapeutic targets for E-cadherin-deficient SRCC.

DOI： 10.1007/s10120-022-01307-8

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Language：English   Publisher：シュプリンガー・ジャパン(株)  

E-カドヘリン欠損胃印環細胞癌(SRCC)における新規治療標的を同定することを目的とした。E-カドヘリンをコードするCDH1遺伝子をノックアウト(KO)したヒト胃オルガノイド(hGO)を用いて、in vitroのE-カドヘリン欠損胃癌モデルを作製し、新規治療標的を探索した。CDH1 KO hGO細胞は、SRCCに類似した特徴的な形態変化と高い細胞運動性を示した。RNA配列解析の結果、CDH1 KO hGO細胞では、野生型と比較して、マトリックスメタロプロテアーゼ(MMP)遺伝子の発現が増加していた。MMP阻害剤は、in vitroでCDH1 KO hGO細胞およびSRCC細胞株の細胞運動を抑制した。95例の臨床胃癌組織を用いた免疫蛍光分析により、MMP-3はE-カドヘリン異常のSRCCに特異的に多く存在することが示された。また、CDH1 KO後、CXCR4分子が細胞膜上に移行した。CXCR4のリガンドであるCXCL12を培養液に添加すると、CDH1 KO hGO細胞の細胞生存率が延長し、CXCR4アンタゴニストであるAMD3100によってその効果が消失した。SRCCの臨床サンプルでは、CXCL12を分泌する線維芽細胞が癌領域に著しく浸潤していることを確認した。以上より、MMPとCXCL12/CXCR4軸は、E-カドヘリン欠損SRCCの新規治療標的として有望な候補であると考えられた。

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Cancer Letters  

Cancer-associated fibroblasts (CAFs) play an important role in cancer progression. However, the origin of CAFs remains unclear. This study shows that macrophages in malignant ascites and pleural effusions (cavity fluid-associated macrophages: CAMs) transdifferentiate into fibroblast-like cells. CAMs obtained from gastrointestinal cancer patients were sorted by flow cytometry and cultured in vitro. CD45+CD14+ CAMs transdifferentiated into CD45−CD90+ fibroblast-like cells that exhibited spindle shapes. Then, cDNA microarray analysis showed that the CD45−CD90+ fibroblast-like cells (macrophage-derived CAFs: MDCAFs) had a fibroblast-specific gene expression signature and produced growth factors for epithelial cell proliferation. Human colon cancer cells transplanted into immunodeficient mice with MDCAFs formed larger tumors than cancer cells alone. Gene ontology analyses showed the involvement of TGFβ signaling and cell-matrix adhesion in MDCAFs, and transdifferentiation of CAMs into MDCAFs was canceled by inhibiting TGFβ and cell adhesion. Furthermore, the acquired genetic alterations in hematopoietic stem cells (HSCs) were shared in CAMs and MDCAFs. Taken together, CAMs could be a source of CAFs and might originate from HSCs. We propose the transdifferentiation process of CAMs into MDCAFs as a new therapeutic target for fibrosis associated with gastrointestinal cancer.

DOI： 10.1016/j.canlet.2022.215597

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Language：English   Publisher：Blood Advances  

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy, with varying prognosis after the gold standard rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Several prognostic models have been established by focusing primarily on characteristics of lymphoma cells themselves, including cell-of-origin (COO), genomic alterations, and gene/protein expressions. However, the prognostic impact of the lymphoma microenvironment and its association with characteristics of lymphoma cells are not fully understood. Using the nCounter-based gene expression profiling of untreated DLBCL tissues, we assess the clinical impact of lymphoma microenvironment on the clinical outcomes and pathophysiological, molecular signatures in DLBCL. The presence of normal germinal center (GC)-microenvironmental cells, including follicular T cells, macrophage/dendritic cells, and stromal cells in lymphoma tissue indicates a positive therapeutic response. Our prognostic model, based on quantitation of transcripts from distinct GC-microenvironmental cell markers, clearly identified patients with graded prognosis independently of existing prognostic models. We observed increased incidences of genomic alterations and aberrant gene expression associated with poor prognosis in DLBCL tissues lacking GC-microenvironmental cells relative to those containing these cells. These data suggest that the loss of GC-associated microenvironmental signature dictates clinical outcomes of DLBCL patients reflecting the accumulation of “unfavorable” molecular signatures.

DOI： 10.1182/bloodadvances.2021004618

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Clonal hematopoiesis (CH) refers to the disproportionate expansion of hematopoietic stem cell clones and their corresponding progeny following the acquisition of somatic mutations. CH is common at the time of diagnosis in patients with blood cancers, including multiple myeloma (MM) and lymphoma. The presence of CH mutations correlates with IL-6 mediated inflammation and may result in lymphoma or MM modulation through microenvironment effects or by manifestations of the mutations themselves within the founding tumor clone. As might be expected with a variety of mutations and multiple potential mechanisms, CH exerts context-dependent effects, being protective in some settings and harmful in others. Though CH is very common in patients with hematologic malignancies, how it intersects with therapy and the natural disease course of these cancers are active areas of investigation. In lymphomas and MM specifically, patients have high rates of CH at diagnosis and are subsequently exposed to therapies, such as cytotoxic chemotherapy, that can cause CH progression to overt hematologic malignancy. The expanding diversity of treatment modalities for these cancers also increases the opportunities for CH to impact clinical outcome and modulate clinical responses. Here we review the basic biology and known health effects of CH, and we focus on the clinical relevance of CH in lymphoma and MM.

DOI： 10.1016/j.jaccao.2022.01.098

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Language：English   Publisher：Journal of Immunology  

T follicular regulatory (Tfr) cells are a subset of CD4+ T cells that express CXCR5 and migrate into germinal centers (GCs). They regulate GC reactions by communicating with T follicular helper (Tfh) and B cells. TNF inhibitors are used in inflammatory diseases; however, the generation of autoantibodies or anti-drug Abs sometimes causes problems. Because TNFR2 signaling is important for suppressive functions of regulatory T cells, we investigated the role of TNFR2 on human Tfr cells. Tfr cells stimulated with MR2-1 (an anti-TNFR2 agonistic Ab) were analyzed for cell proliferation, Foxp3 expression, and surface molecules. Tfh/B cell proliferation, IgM production, and differentiation in cocultures with MR2-1-stimulated Tfr cells were examined. Tfr cells express a high level of TNFR2. MR2-1 stimulation altered the gene expression profile of Tfr cells. Cell proliferation and Foxp3 expression of Tfr cells were enhanced by MR2-1. MR2-1-stimulated Tfr cells expressed ICOS and Programmed cell death protein 1 and significantly suppressed Tfh/B cell proliferation, IgM production, and B cell differentiation. TNFR2-stimulated Tfr cells retained the migration function according to the CXCL13 gradient. In conclusion, we showed that TNFR2-stiumulated Tfr cells can regulate Tfh and B cells. Aberrant antibody production during TNF inhibitor treatment might be, at least in part, associated with TNFR2 signaling inhibition in Tfr cells. In addition, expansion and maturation of Tfr cells via TNFR2 stimulation in vitro may be useful for a cell-based therapy in inflammatory and autoimmune diseases to control GC reactions.

DOI： 10.4049/jimmunol.2100323

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Blood  

Acute lymphoblastic leukemia (ALL) harboring the IgH-CRLF2 rearrangement (IgH-CRLF2-r) exhibits poor clinical outcomes and is the most common subtype of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). While multiple chemotherapeutic regimens, including ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. To identify molecules/pathways relevant for IgH-CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of ruxolitinib using 2 IgH-CRLF2-r ALL lines that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled, for the first time, highly efficient transduction of human B cells. While single-guide RNAs (sgRNAs) targeting CRLF2, IL7RA, or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B, or STAT3 did not, suggesting that STAT signaling is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and ruxolitinib sensitivity and that CRKL depletion enhances ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that blocks CRKL phosphorylation, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with ruxolitinib. We further show that combining gilteritinib with trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status. Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes.

DOI： 10.1182/blood.2021012976

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Leukemia  

Acute myeloid leukemia (AML) is a devastating disease, and clinical outcomes are still far from satisfactory. Here, to identify novel targets for AML therapy, we performed a genome-wide CRISPR/Cas9 screen using AML cell lines, followed by a second screen in vivo. We show that PAICS, an enzyme involved in de novo purine biosynthesis, is a potential target for AML therapy. AML cells expressing shRNA-PAICS exhibited a proliferative disadvantage, indicating a toxic effect of shRNA-PAICS. Treatment of human AML cells with a PAICS inhibitor suppressed their proliferation by inhibiting DNA synthesis and promoting apoptosis and had anti-leukemic effects in AML PDX models. Furthermore, CRISPR/Cas9 screens using AML cells in the presence of the inhibitor revealed genes mediating resistance or synthetic lethal to PAICS inhibition. Our findings identify PAICS as a novel therapeutic target for AML and further define components of de novo purine synthesis pathway and its downstream effectors essential for AML cell survival.

DOI： 10.1038/s41375-021-01369-0

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