Language：English   Publisher：Blood Advances  

Immunomodulatory drugs (IMiDs) are key drugs for treating multiple myeloma and myelodysplastic syndrome with chromosome 5q deletion. IMiDs exert their pleiotropic effects through the interaction between cell-specific substrates and cereblon, a substrate receptor of the E3 ubiquitin ligase complex. Thus, identification of cell-specific substrates is important for understanding the effects of IMiDs. IMiDs increase the risk of thromboembolism, which sometimes results in fatal clinical outcomes. In this study, we sought to clarify the molecular mechanisms underlying IMiDs-induced thrombosis. We investigated cereblon substrates in human megakaryocytes using liquid chromatography–mass spectrometry and found that thrombospondin-1 (THBS-1), which is an inhibitor of a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs 13, functions as an endogenous substrate in human megakaryocytes. IMiDs inhibited the proteasomal degradation of THBS-1 by impairing the recruitment of cereblon to THBS-1, leading to aberrant accumulation of THBS-1. We observed a significant increase in THBS-1 in peripheral blood mononuclear cells as well as larger von Willebrand factor multimers in the plasma of patients with myeloma, who were treated with IMiDs. These results collectively suggest that THBS-1 represents an endogenous substrate of cereblon. This pairing is disrupted by IMiDs, and the aberrant accumulation of THBS-1 plays an important role in the pathogenesis of IMiDs-induced thromboembolism.

DOI： 10.1182/bloodadvances.2023010080

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Language：English   Publisher：Cancer Science  

Metabolic alterations, especially in the mitochondria, play important roles in several kinds of cancers, including acute myeloid leukemia (AML). However, AML-specific molecular mechanisms that regulate mitochondrial dynamics remain elusive. Through the metabolite screening comparing CD34+ AML cells and healthy hematopoietic stem/progenitor cells, we identified enhanced lysophosphatidic acid (LPA) synthesis activity in AML. LPA is synthesized from glycerol-3-phosphate by glycerol-3-phosphate acyltransferases (GPATs), rate-limiting enzymes of the LPA synthesis pathway. Among the four isozymes of GPATs, glycerol-3-phosphate acyltransferases, mitochondrial (GPAM) was highly expressed in AML cells, and the inhibition of LPA synthesis by silencing GPAM or FSG67 (a GPAM-inhibitor) significantly impaired AML propagation through the induction of mitochondrial fission, resulting in the suppression of oxidative phosphorylation and the elevation of reactive oxygen species. Notably, inhibition of this metabolic synthesis pathway by FSG67 administration did not affect normal human hematopoiesis in vivo. Therefore, the GPAM-mediated LPA synthesis pathway from G3P represents a critical metabolic mechanism that specifically regulates mitochondrial dynamics in human AML, and GPAM is a promising potential therapeutic target.

DOI： 10.1111/cas.15835

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Language：English   Publisher：John Wiley & Sons Australia, Ltd  

急性骨髄性白血病(AML)に特異的なミトコンドリア機能の制御に関与する分子メカニズムを検討した。CD34陽性AML細胞と健常な造血幹/前駆細胞を比較した代謝物スクリーニングを通じて、AMLにおけるリゾホスファチジン酸(LPA)合成活性の亢進を同定した。LPAは、LPA合成経路の律速酵素であるグリセロール-3-リン酸アシルトランスフェラーゼ(GPAT)によってグリセロール-3-リン酸から合成される。GPATの4つのアイソザイムのうち、グリセロール-3-リン酸アシルトランスフェラーゼ、ミトコンドリア(GPAM)がAML細胞で高発現していた。GPAMサイレンシングまたはFSG67(GPAM阻害剤)でLPA合成を阻害すると、ミトコンドリア分裂が誘導され、その結果、酸化的リン酸化が抑制され、活性酸素種が増加することで、AMLの増殖が著しく阻害された。さらに、FSG67によるGPAM阻害は、in vivoでの正常な造血に影響を与えることなく、AMLに対して有効であることが示された。以上より、GPAMを介したグリセロール-3-リン酸からのLPA合成経路は、AMLにおけるミトコンドリア動態を特異的に制御する重要な代謝機構であり、GPAMは有望な治療標的であることが示唆された。

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   Publisher：Blood Advances  

The activation of β-catenin plays critical roles in normal stem cell function, and, when aberrantly activated, the maintenance and enhancement of cancer stemness in many solid cancers. Aberrant β-catenin activation is also observed in acute myeloid leukemia (AML), and crucially contributes to self-renewal and propagation of leukemic stem cells (LSCs) regardless of mutations in contrast with such solid tumors. In this study, we showed that the AML-specific autocrine loop comprised of T-cell immunoglobulin mucin-3 (TIM-3) and its ligand, galectin-9 (Gal-9), drives the canonical Wnt pathway to stimulate self-renewal and propagation of LSCs, independent of Wnt ligands. Gal-9 ligation activates the cytoplasmic Src homology 2 domain of TIM-3 to recruit hematopoietic cell kinase (HCK), a Src family kinase highly expressed in LSCs but not in HSCs, and HCK phosphorylates p120-catenin to promote formation of the LDL receptor–related protein 6 (LRP6) signalosome, hijacking the canonical Wnt pathway. This TIM-3/HCK/p120-catenin axis is principally active in immature LSCs compared with TIM-3–expressed differentiated AML blasts and exhausted T cells. These data suggest that human AML LSCs constitutively activates β-catenin via autocrine TIM-3/HCK/p120-catenin signaling, and that molecules related to this signaling axis should be critical targets for selective eradication of LSCs without impairing normal HSCs.

DOI： 10.1182/bloodadvances.2022008405

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Language：Japanese   Publisher：(一社)日本造血・免疫細胞療法学会  

急性骨髄性白血病における同種移植後の再発は極めて予後不良であり,大きな臨床的課題である。この再発を早期に予測するためには,測定可能/微小残存病変(measurable/minimal residual disease,MRD)の定量的評価が有用である。European Leukemia Net(ELN)は,マルチパラメーターフローサイトメトリ(MFC)によるMRD評価法を確立した手法と定義しているが,遺伝子変異で定義されるMRDとの関係性や,同種移植後MRDを用いた予後予測法の最適化など明らかにすべき課題は依然として存在する。本稿では,細胞表面抗原によるMRDと遺伝子変異によるMRDの関係性を明らかにするためのシングルセル解析を用いたアプローチ,及び,本邦におけるMFC-MRD評価法の確立に向けたTIM-3を用いた微小残存白血病幹細胞評価法の有用性の検討について報告する。(著者抄録)

Language：Japanese   Publisher：(一社)日本血液学会-東京事務局  

ヒト急性骨髄性白血病(acute myeloid leukemia,AML)においては,一部の白血病幹細胞(leukemic stem cells,LSCs)のみが,自己複製能と白血病細胞への限定的分化能を有し白血病細胞集団を維持している。我々は先行研究において,ヒト正常造血幹細胞には発現せずにLSCsにのみ特異的に発現する表面抗原としてT-cell immunoglobulin mucin-3(TIM-3)を同定した。本研究においてヒトAMLにおいては,リガンドのTIM-3分子への結合により,Src family kinaseの一つであるHCKがTIM-3細胞質内ドメインと結合し活性化すること,HCKは基質としてp120-cateninをリン酸化することでWntリガンド非依存的に恒常的なcanonical Wnt pathwayの活性化を誘導することを明らかにした。(著者抄録)