Cell-context dependent functions of mTOR complexes in Leukemia

International Symposium on Tumor Biology in Kanazawa & Symposium on Drug Discoverry in Academics 2014 [DATE]: January 23(Thu)-24(Fri),2014, [Place]:Kanazawa Excel Hotel Tpkyu, Kanazawa, Japan, [Organizers]:Kanazawa Association of Tumor Biologists / Cancer Research Institute, Kanazawa Universit

Mechanistic / mammalian target protein of rapamycin signaling in hematopoietic stem cells and leukemia

がん進展制御研究所Mechanistic/mammalian target protein of rapamycin (mTOR) is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants such as nutrient availability, energy sufficiency, stress, and growth factor concentration. mTOR participates in two complexes, designated mTOR complex 1 (mTORC1) and 2 (mTORC2), both of which phosphorylate multiple substrates. Recent studies have revealed that the fine-tuning activity of mTOR complexes contributes to both maintenance of hematopoietic stem cells (HSCs) and suppression of leukemogenesis. Dysregulation of mTORC1 activity results in impaired HSC homeostasis. Abnormalities of mTOR signaling are observed in many patients with leukemia and genetic studies clearly show that the leukemogenesis associated with Pten deficiency involves both mTORC1 and mTORC2. Although the several mTOR inhibitors have been developed for cancer therapy, effectiveness of the inhibitors for eradication of leukemia stem cells (LSCs) is unknown. Advances in understanding of how mTOR signaling is involved in mechanisms of normal HSC and LSC homeostasis may lead to novel therapeutic approaches that can successfully eradicate leukemia. © 2013 Japanese Cancer Association

Novel therapeutic approach to eradicate tyrosine kinase inhibitor resistant chronic myeloid leukemia stem cells

がん進展制御研究所Although discovery of the tyrosine kinase inhibitor (TKI) imatinib mesylate has significantly improved the prognosis of chronic myeloid leukemia (CML) patients, a rare population of CML stem cells is known to be resistant to TKI therapy, causing recurrence of CML. However, recent progress in CML stem cell biology may present a novel therapeutic avenue for CML patients. In this review, we focus on mechanisms used by CML stem cells to maintain TKI-resistance. Comprehensive approaches including mouse genetics, prospective identification of CML stem cells, and syngenic transplantation techniques have identified several key molecules or signaling pathways, including hedgehog (Hh)/Smo, promyelocytic leukemia (PML), 5-lipoxygenase (5-LO), and forkhead box class O (FOXO), that function in CML stem cell maintenance. Inhibiting some of these factors in combination with TKI administration successfully antagonized resistance of CML stem cells to TKI therapy, resulting in efficient eradication of leukemia cells in vivo. Thus, development of methods that sensitize CML stem cells to TKI therapy may lead to novel therapies to treat CML patients. © 2010 Japanese Cancer Association

Molecular mechanisms of cell fate determination in the cells that undergo stepwise differentiation to multiple pathways

金沢大学がん進展制御研究所mTORC1による生体内での骨髄球系細胞分化における役割を明らかにするため、骨髄球分化の促進が認められるKrasの恒常的な活性化モデルと、mTORC1活性を欠損するRaptor欠損マウスを組み合わせて解析を行った。前年度までの解析で、Raptor欠損は顆粒球分化が阻害されることを見出していたが、Kras活性化状態においても顆粒球分化は著しく阻害されており、骨髄球細胞の終末分化に必須であると考えられた。一方で、骨髄球前駆細胞は顕著に増加していることを見出した。mTORC1はRasシグナルの下流分子であると考えられているが、本研究の結果から、mTORC1抑制による分化抑制効果とKras活性化による骨髄球増殖促進効果がそれぞれ独立して働き、骨髄球前駆細胞を増加させうることが示唆された。　またmTORC1抑制によって細胞に生じる変化を同定するため、mTORC1欠損下で生存する急性骨髄性白血病細胞集団を用いて、表面抗原の網羅的な解析を行った。その結果、mTORC1抑制後には、コントロールの白血病細胞では発現低く、リンパ球系細胞や幹細胞で発現が高いことが報告されている分子が多数発現増加していることを見出した。このことはmTORC1抑制が積極的に細胞分化に影響していることを示唆している。そのいくつかはmRNAレベルで顕著に発現変動しており、mTORC1の変動を介して、翻訳後制御と転写制御の両者で遺伝子発現が変化していると考えられる。今後このような分子の細胞分化における機能とmTORC1との相関を明らかにすることによって、mTORC1による細胞分化制御のメカニズムが明らかになると期待された。研究課題/領域番号:23118511, 研究期間(年度):2011-04-01 – 2013-03-3

TGF-Β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia

金沢大学がん研究所がん幹細胞研究センターChronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL, a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO), supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy, imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here, using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model, we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a+/+ and Foxo3a-/- mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore, we find that TGF-Β is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-Β inhibition, Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore, the treatment of human CML LICs with a TGF-Β inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-Β-FOXO pathway in the maintenance of LICs, and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo. © 2010 Macmillan Publishers Limited. All rights reserved

mTOR複合体1による正常造血幹細胞・白血病幹細胞制御機構の解明

金沢大学がん進展制御研究所mTORC1の異常な活性化は白血病化を促進するが、白血病幹細胞の維持における役割は明らかではない。本研究では白血病マウスモデルにおいてmTORC1活性を特異的に欠損させることにより、分化した白血病細胞は細胞死の誘導により速やかに消失するが、未分化型白血病細胞は長期的に生存可能であることを見出した。mTORC1の再活性化により、白血病が再発したことから、mTORC1抑制下でも、白血病幹細胞は自己複製を行うことが明らかとなった。Although dysregulation of mTOR complex 1(mTORC1) promotes leukemogenesis, how mTORC1 affects established leukemia is unclear. We investigated the role of mTORC1 in mouse model of acute myeloid leukemia. Raptor deficiency significantly suppressed leukemia progression by causing apoptosis of differentiated, but not undifferentiated, leukemia cells. Strikingly, a subset of AML cells with undifferentiated phenotypes survived long-term in the absence of mTORC1 activity. Thus, AML cells lacking mTORC1 activity can self-renew as AML stem cells.研究課題/領域番号:22790906, 研究期間(年度):2010-2011出典：研究課題「mTOR複合体1による正常造血幹細胞・白血病幹細胞制御機構の解明」課題番号22790906（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-22790906/22790906seika/）を加工して作

mTOR複合体1による白血病幹細胞自己複製維持メカニズムの解明

金沢大学がん進展制御研究所mTORは、二つの複合体（mTORC1とmTORC2）を形成して機能するキナーゼである。これまでの成果から、mTORC1欠損下で分化AML細胞の産出と白血病発症は抑制されるが、AML幹細胞は自己複製し、生体内で維持されることを明らかにしていた。本研究では、mTORC1欠損AML細胞を用いて、自己複製を司る分子の同定を主題として研究を展開し、新規のmTORC1下流候補分子やAML幹細胞関連分子を同定した。また、mTORC2-FOXO経路の生体内AML幹細胞における役割を解明する研究基盤を構築した。今後の研究によって白血病幹細胞の新たな治療標的となることが期待される。Mammalian target of rapamycin (mTOR) is identified as a target protein of immunosuppressive agent rapamycin, and this protein kinase is known to form two different complexes, named mTORC1 and mTORC2. In past years, we reported that mTORC1 inactivation, by Raptor deficiency, apparently suppressed the acute myeloid leukemia (AML) cell propagation and differentiation, but some AML cells with stem cell properties survived and proliferated in vivo. To identify the molecular mechanisms to maintain the stem cell like population after mTORC1 inactivation, we performed a quantitative phosphoproteomics study and cell surface proteomics study. We identified mTORC1 downstream candidate genes from phosphoproteomics study, and AML-stem cell associated cell surface molecules from cell surface protemics study. The findings will provide the new therapeutic targets for drug-resistant leukemia therapy.研究課題/領域番号:24790967, 研究期間(年度):2012-04-01 – 2014-03-3