1.The leukemia stem cell (LSC) model has received considerable attention in the past 20 years with the identification of rare cell populations expressing stem cell markers in leukemia cell populations. LSCs are thought to be responsible for leukemia initiation, recurrence, and drug resistance, thus being regarded as excellent targets for developing curative therapies. However, characters of LSCs that are resistant to specific types of therapies have not been fully understood. Using a mouse model for myeloid leukemia, we found that a p53-Mdm2 interaction inhibitor (DS-5272) showed the robust anti-leukemia effect against MLL-AF9 leukemia in vivo. Nevertheless, all mice eventually developed leukemia, indicating the existence of therapy-resistant LSCs. These LSCs were relatively enriched within the bone marrow (BM) endosteal region where osteoblasts reside, and expressed higher levels of Hif1 and PD-L1. The antileukemia effect of DS-5272 was markedly attenuated in immunodeficient mice, indicating the critical impact of systemic immune responses that drive p53-mediated leukemia suppression. Furthermore, inhibition of Hif1 and PD-L1 sensitized leukemia cells to DS-5272. Our study showed the potent activity of a p53-MDM2 interaction inhibitor against MLL-fusion leukemia, which is further augmented by antitumor immunity.

2.Myelodysplastic syndromes (MDS) arise in the elderly through stepwise acquisitions of many possible somatic mutations. The clinical diversity of this disease reflects heterogeneity of the resulting clonal architecture. Through analysis of 1,809 MDS patients (of which over 150 were studied serially), we demonstrate using specifically developed bio-statistical/analytic pipeline that the ancestral hits are mutually exclusive and defining of subsequent evolution not via a random, but rather preferential acquisition of specific secondary mutations. We have also objectively digitalized major morphologic features including the presence of various forms of dysplasia, cytopenia and myeloproliferative features (monocytosis, fibrosis increased megakaryocytes) obtained through a uniform unbiased and blinded pathologic evaluation. These features have been correlated with the presence of specific mutations and common mutational combinations. A number of important relationships between specific morphologic profiles and mutation combinations have been uncovered allowing to determine predictive value of morphologic configurations with regard to certain genotypes and vice versa. Similarly, our analysis revealed a convergence of molecular pathways steaming from distinct lesions towards common shared morphology. We also show that mutations in some genes exert different effects of the clinical features depending on their position in the clonal hierarchy. The latency between ancestral and secondary hits can be long, as evidenced by their presence in a prevalent condition known as clonal hematopoieisis of indeterminate potential (CHIP). Separating ancestral and secondary hits in MDS patients and comparing frequencies of mutations identified in meta-analysis of CHIP enabled us to speculate molecular and clinical characteristics of CHIP-related MDS and contrast them to MDS arising as de novo disease characterized by very penetrant mutations with a shorter latency to manifest disease. Patients with CHIP-related MDS had dominant TET2, DNMT3A and ASXL1 mutations, and overall higher mutational burden, were more elderly, exhibited less -7/del(7q) and complex karyotype, and showed better prognosis compared to those in CHIP-unrelated de novo MDS. In sum, we conclude that MDS patients can be broadly partitioned into pathogenically distinct CHIP-related vs. –unrelated MDS forms. Certain mutational configurations create morphologic configurations with subclonal hits further shaping the resultant morphology. Distinction of MDS by founder lesions and the presence of sweeping subclonal hits has wide-ranging therapeutic consequences.

1.Hematopoietic stem cells (HSCs) exhibit multilineage differentiation and self-renewal activities that maintain the entire hematopoietic system during an organism's lifetime. These abilities are sustained by intrinsic transcriptional programs and extrinsic cues from the microenvironment or niche. A particular gap is our understanding of the age-related alteration of HSC niche, which bridges environmental cues to HSC aging. Recent studies reveal that metabolic regulation plays an essential role in HSC maintenance. Metabolic pathways provide energy and building blocks for other factors required for HSC integrity at steady state and in stress. To approach aging-related alteration in HSC niche from a metabolic perspective, we recently took advantage of technical developments in metabolomics and in vivo imaging. In this mini-symposium, I will address the environmental regulation that tunes arrival of HSC to bone marrow after transplantation and its alteration during aging.

2.AML develops from the sequential acquisition of multiple mutations in a single lineage of cells. These mutations initially occur in HSCs, termed pre-leukemic HSCs, and are enriched in genes involved in regulation of the epigenome. The pre-leukemic cells acquire additional mutations, often in genes involved in proliferation, resulting in development of AML. Stratification of a cohort of AML patients into high or low pre-leukemic HSC groups demonstrated that the high group had much worse overall and relapse-free survival, indicating that the presence of pre-leukemic HSC may be critical for clinical outcomes. In bulk AML, sequencing studies demonstrated that most cases harbor multiple subclones with a complex evolutionary structure. These subclones potentially exhibit distinct features including leukemia stem cell properties, clonal dominance, and responses to chemotherapy and targeted agents. Isolating and investigating these subclones is essential to understanding their properties, and is facilitated by a novel humanized ossicle xenotransplantation model, single cell methods, and CRISPR/Cas9 engineering of normal and leukemic cells. Eventually, all subclones must be targeted in order to improve long-term outcomes and for potential cures in AML. Notably, mutation-specific targeted agents have the potential to improve outcomes in AML. Several approaches were employed to identify mutation-specific vulnerabilities including epigenomic profiling, computational approaches, and metabolomic profiling. Several targets were validated through genetic and/or pharmacologic modulation in primary AML cells and xenograft models, making them strong candidates for therapeutic development.

2003年に始まった「オーダーメイド医療の実現プログラム」では、個別化医療の実現を目指し、疾患や薬剤感受性に関連する遺伝因子の大規模全ゲノム関連解析（GWAS）を中心とした研究が進められてきた。本プログラムでは15年間を通じて51疾患、約27万人の生体試料および臨床情報の収集が行われ、世界最大規模のバイオバンク・ジャパン（BBJ）が構築されている。我々は本プログラムの第1期に登録された47疾患、20万人の方の臨床情報と追跡調査から得られた予後に関する情報について、疫学的手法を用いた分析を行い、研究参加者の全体像や疾患ごとの参加者の特性、生存期間等を明らかにした。さらにBBJが保有する臨床情報や生体試料の研究への利活用を促進すべく、この臨床情報解析に用いたデータベースを元にBBJ保有試料検索システムを構築し、BBJの試料やゲノム解析情報を誰もが任意の条件で検索することを可能にした。
一方、我々は2014年に骨軟部腫瘍ゲノムコンソーシアムを立ち上げ、全国の骨軟部腫瘍診療の基幹病院と研究機関との連携を進めながら、骨軟部腫瘍のゲノム解析を遂行している。本プロジェクトではまず脱分化型脂肪肉腫のゲノム解析より開始し、総計で100症例を超える腫瘍組織を用いてその遺伝子異常の特徴や予後関連因子、腫瘍原性や悪性転化に関わる遺伝子異常を明らかにしてきた。また、我々は軟骨肉腫のゲノム解析で高頻度に認められたIDH1/2遺伝子変異に着目し、その機能解析や薬剤スクリーニングを行うため軟骨肉腫マウスモデルの確立も進めている。
本セミナーでは、上記プロジェクトを通して実施した臨床情報解析やゲノム解析の研究成果、今後の展望について紹介する。

Chromosome structure and functions dramatically change during mitosis. Although the nucleosome is a fundamental structural and functional unit of eukaryotic chromatin, its regulatory roles in mitosis are poorly understood. Through comparative chromatin proteomics analysis to identify proteins whose chromatin binding is altered in mitosis, we discovered the novel nucleosome remodeling complex CHIRRC, composed of CDCA7 and HELLS (homolog of plant DDM1), both of which are mutated in the Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome. Lymphoblastoid cell lines from ICF patients exhibit hypomethylation and fragility at juxtacentromeric heterochromatin of chromosome 1 and 16. Our finding provides a mechanistic insight into how CDCA7 and HELLS contribute to DNA methylation. We also found that the cytoplasmic DNA sensor, cGAS, a critical component of the innate immune system, gains an access to mitotic chromosomes upon nuclear envelope break down, but its DNA-induced activation is suppressed by the nucleosome. However, when cells are arrested in mitosis by taxol, cGAS slowly becomes activated and promotes apoptosis. Implications of these findings in the ICF syndrome and cancers will be discussed.

がん細胞はそのhallmarkとして知られる染色体不安定性を背景として、しばしば過剰な中心体を持つ。過剰中心体はaneuploidyを引き起こし、外的ストレスへの耐性や細胞増殖促進などがん細胞の生存に有利な特性をもたらす一方で、細胞分裂時の染色体の分離異常は細胞に有害である側面も持つ。我々は肺癌細胞においてCDK1/CDK2阻害により、細胞分裂の際に過剰中心体の二極への収束が阻害され、細胞の多極分裂とそれに伴う染色体分離異常を経て娘細胞にアポトーシスが誘導されることを明らかにし、これをanaphase catastropheと名付けた。CDK1/CDK2阻害によるanaphase catastropheの誘導は、過剰中心体を持たない正常細胞では回避され、がんに比較的に特異性を持つ治療戦略として期待できる。その分子メカニズムとして、中心体タンパクの一つであるCP110のリン酸化阻害を介することがわかった。興味深いことにCP110タンパクは KRASの活性化でユビキチン機構により不安定化し、そのためanaphase catastropheは特にKRAS変異のある肺癌細胞で誘導されやすい。anaphase catastropheの誘導は、肺癌のみならず膵癌や大腸癌、リンパ腫などでも確認された。本セミナーではanaphase catastropheについてこれまでにわかっている知見を紹介し、がん治療におけるこの機構の可能性を考察したい。

視覚運動の方向や速度の検出は視覚系の重要な機能の一つであり、動物の生存にとって重要である。特定の方向の視覚運動に選択的に応答する神経細胞を方向選択性細胞と呼び、マウスやウサギを用いた研究から大脳視覚皮質のみならず 網膜にも方向選択性細胞が存在することが知られている。演者はこれまでマウス網膜をモデルとして方向選択性細胞の構造(文献１)や機能(文献２)、発達(文献３)を研究することにより、異なるタイプの神経細胞同士の相互作用がどのようにして感覚情報を処理するのかを明らかにしようとしてきた。最近では、網膜の方向選択性細胞の機能異常が先天性眼振という眼球運動の疾患に関連していることも明らかにした(文献４)。また、２０１５年からはデンマークで研究室を主宰し、２光子イメージング、電気生理、ウイルスベクターによる神経回路標識、分子生物学、マウス遺伝学などを組み合わせ、網膜で処理された視覚情報が下流の神経中枢においてさらにどのように処理されるのか(文献５、６)などを明らかにするべく研究を展開している。本セミナーでは、近年の我々の一連の取り組みを紹介し、また将来の展望を議論する。

肺癌は、分子標的治療薬や免疫チェックポイント阻害薬など新しい治療薬が開発されているにも関わらず、依然として日本における癌死亡原因の第1位を占める。全く異なる治療アプローチの開発が急がれる中、がん細胞で選択的に複製してがん細胞を破壊するがん治療用ウイルスを用いた「ウイルス療法」が注目されている。G47Δは、がん細胞特異的なウイルス複製能と特異的抗腫瘍免疫の惹起力がいずれも増強された第三世代の遺伝子組換え単純ヘルペスウイルスＩ型で、当研究所を中心に、世界に先駆けた臨床開発が展開している。動物モデルでは様々な癌腫に対し有効性が示されており、脳腫瘍から開始された臨床試験は、徐々に他の難治性がんに適応を拡大しつつある。肺癌に対しても、臨床応用を目指した基礎開発を進めている。G47Δの有効性の検証に加え、他の治療との併用実験、胸腔内および肺内投与の安全性試験など、これまでに蓄積した非臨床データを紹介する。

Carbohydrates (glycans) on the surface of cells present a complex code that can be recognized by a range of carbohydrate-binding proteins including antibodies and lectins. Blood group related glycans such as Lewis system antigens are often over-expressed by cancer cells and have been associated with tumour progression and invasiveness. On healthy and inflamed tissues similar Lewis glycans can become targets for bacterial adhesion. In this presentation, I will provide an overview of our structural studies using crystallography, automated docking and molecular dynamics that have probed recognition of Lewis system antigens by antibodies and lectins with implications for development of cancer-targeting antibodies and carbohydrate inspired anti-infectives. This work will be contrasted with recent crystal structures we have determined that define how a single antibody can bind a wide range of microbes through recognition of a common core oligosaccharide chain.

The functional interactions of the human gut microbiome with its host are largely driven by diet, yet the underlying mechanisms have remained poorly understood. By employing a gnotobiotic mouse model—containing a synthetic human gut microbiota composed of 14 commensal bacteria—we recently showed that a dietary fiber-deprived microbiota degrades the colonic mucus layer, which is the first line of defense against invading microorganisms. Furthermore, a fiber-deprived, mucus-eroding microbiota enhances susceptibility to the enteric pathogen Citrobacter rodentium (Desai et al. Cell (2016), 167, 1339–1353). Our follow-up experiments indicate that such deterioration of the colonic mucus barrier is associated with the immunological changes observed in inflammatory bowel disease. In the face of changing Western diets, our results suggest that dietary therapeutics, including next-generation prebiotics, could be utilized to modulate the the gut barrier alterations and to reduce the incidence of colitis.

近年、グローバリゼーションや人口構造の変化あるいは土地利活用の変化により、これまで認識されていなかった未知のウイルスによる感染症が加速的に台頭している。このような新興感染症の原因ウイルスの過半数は動物を宿主としているが、その多くのウイルスの感染リスクや感染経路は未だ予測できていない。この問題を鑑み、米国国際開発庁（USAID）グローバル保健局の科学者を含む世界中の専門家は、「グローバル・バイローム・プロジェクト」を発足させた。このプロジェクトは、地球上の動物からヒトに感染する可能性のある「未知のウイルス」を野生動物から発見、識別し、その特性を理解することにより、病原体がヒトに感染する前に、新興感染症の対策を打つことを目的としている。感染症のアウトブレイクを事前に予測し、その情報を感染症予防に直結させるという予防的アプローチに転換させることが期待されている。

今回の講演では、米国国際開発庁で長年感染症のプロジェクトに関わってきたデニス・キャロル博士をお招きし、今、全世界から注目を集めている感染症対策の大規模プロジェクト「グローバル・バイローム・プロジェクト」について、その科学的な根拠、ガバナンス、技術的な枠組みについてお話しいただきます。