Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of the Philadelphia (Ph) chromosome, formally termed as der(22)t(9;22)(q34;q11), in which the ABL gene at 9q34 is juxtaposed to the 5.8-kb limited region of the BCR gene at 22q11, resulting in the generation of 210 kD BCR-ABL oncoprotein, p210. In rare cases of CML, two other breakpoint regions of the BCR gene are fused to the ABL gene and produce 190 and 230 kD chimeric proteins, respectively. This active tyrosine kinase is responsible for the clonal amplification of leukemia cells by inhibition of apoptosis and stimulation of cell cycling.CML is believed to originate from a hematopoietic stem cell carrying the BCR-ABL fusion gene. The concept that leukemic cells in CML originate from a deregulated stem cell compartment was at first indicated by the demonstration of the Ph chromosome in erythroid and myeloid cells as well as megakaryocytes and later also in B-lymphoblastoid cells. Furthermore, evidence that some of the BCR-ABL+ cells can also differentiate into endothelial cells was reported, suggesting the hemangioblast origin of the leukemic clone. In spite of the putative stem cell origin and the competence for differentiation toward mature B cells, there is a longstanding consensus that CML never involves the T cell lineage at least in chronic phase. One possible explanation is that the BCR-ABL oncoprotein may be harmful to differentiating T cell progenitors, as has been suggested to be the case in fibroblasts

To gain insight into the reason why the BCR-ABL+ leukemic clone has a very limited ability to differentiate toward mature T cells, I applied the conditionally-active BCR-ABL kinase to the in vitro model system of T cell differentiation from murine iPSCs and HSCs. We used induced pluripotent stem cell (iPSCs) in our in vitro study because the concept leukemic clones have been originated from hemangioblast, beyond the hematopoietic stem cell level.

C57BL/6 MEFs were reprogrammed using a polycistronic lenti viral Tet-On vector encoding human Oct4, Sox2 and Klf4, which were tandemly linked via porcine teschovirus-1 2A peptides, together with another lentiviral vector expressing rtTA driven by the EF-1α promoter. Almost all the vector sequences including the transgenes were deleted by adenovirus-mediated transduction of Cre-recombinase after derivation of iPSCs, and only remnant 600-bp LTRs containing a single loxP site remained in the genome. A clone of MEF-iPSCs were retrovirally transduced with p190ΔccER, a ligand-controllable p190-estrogen receptor fusion protein, whose tyrosine kinase activity absolutely depends on 4-hydroxytamoxyfen (4-HT).For T cell lineage differentiation, p190ΔccER-MEF-iPSCs were recovered from a feeder-free culture supplemented with LIF and plated onto a sub-confluent OP9-DL1 monolayer in the presence of 5ng/ml Flt3 ligand and 1ng/ml IL7 with or without 0.5 μM 4-HT.After 3 weeks of culture, iPSC-derived blood cells were collected and subjected to FACS analysis for their lineage confirmation. About 70% of lymphocyte-like cells from the 4-HT (-) culture expressed CD3, but only 20% of counterparts from the 4-HT(+) culture expressed CD3, suggesting impaired T cell development by BCR-ABL.

To define the stages of impairment in T cell development more specifically we did the same study by using murine hematopoietic stem cell. Next C57BL/6 mouse bone marrow was collected after treatment intravenous of 5FU injection and bone marrow cells were sorted by lin-c-kit+Sca1+ mouse antibody KSL cells were transduced with same p190ΔccER retroviral vector or CS-CDF- tetO-BCR-ABL and CS-EF-rtTA2AEGFP lenti viral vector and induced them into T cells differentiation on sub-confluent OP9-DL1 monolayer in the presence of human Flt3 ligand and mouse IL7 with or without 0.5 μM 4-HT or doxycycline 4μg/ml. After 2 weeks of culture FACS analysis was done and it revealed that 95% 4-HT (-) cells were expressed CD3+TCRβ double positive but only 30% cells were double positive in case of 4HT (+) cultured cells. Next to specify the stages of T cells developmental arrest by BCR-ABL 4HT (+) and 4-HT (-) cultured cells were labeled by CD117, CD25 and CD44 anti mouse antibody and analyzed by FACS. Approximately 90% of 4-HT (-) cultured cells were in DN2 (CD44+CD25+CD117+) stage where in 4HT (+) cultured cells were only 50% in DN2 (CD44+CD25+CD117+) stage rest of 50% cells in DN1 (CD44+CD25-CD117+) stage. BCR-ABL positive cells constituted the two populations according to the expression level of EGFP, and the EGFPlow population arrested at the DN1 (CD44+CD25-CD117+) stage, while EGFPhigh population. BCR-ABL impairs T cell development possibly through interfering of IL-7Receptor alpha down regulation.

It appears that BCR-ABL disturbs transition from the DN1 (c-Kit+CD44+CD25-) to the DN2 (c-Kit+CD44+CD25+) stage of T cell differentiation from iPSCs and HSCs. Intriguingly, BCR-ABL positive cells constituted the two populations according to the expression level of EGFP, and the EGFPlow population arrested at the DN1 stage, while EGFPhigh population proceeded to the DN2 stage. Assuming that the EGFP intensity reflects the expression level of BCR-ABL directly or inversely, absolute BCR-ABL kinase activity may determine the T cell fate. Although the precise mechanism of impaired T cell development by BCR-ABL is to be elucidated, it is likely that BCR-ABL down-regulates the Notch and/or IL7 signal, considering that the majority of BCR-ABL+ progenitor cells arrested at the DN1 stage. Especially, one possible speculation is that BCR-ABL may perturb the IL7 signaling pathway at any point, since IL7 signal is also transmitted through tyrosine kinase cascades including the Jak-Stats. Furthermore, it should be noted that DN1 thymocytes are still capable of transdifferentiation toward myeloid cells in murine hematopoietic system. Direct reprogramming of somatic cells to pluripotent stem cells is markedly valuable for pathophysiological investigation as well as therapeutic application in the near future. Widely used reprogramming vectors were retrovirus- or lentivirus-based vectors, which integrated into the host genome in multiple copies, resulting in the potential risk of insertional mutagenesis of endogenous genes. For the above investigation I developed and tested a single, polycistronic lentiviral vector that can be deleted after reprogramming to pluripotency. Additionally, I applied the Tet-on system to expression of reprogramming factors as another safe-guard, instead of constitutive promoters. Unexpected low efficiency of reprogramming is probably due to the low titer of viral vectors produced in small scale and unpurified.

本研究は、新たに構築したレンチウイルスベクター・システムを用いて樹立した外来初期化因子フリーのiPS細胞と骨髄造血幹細胞をそれぞれT細胞に分化誘導する過程で、BCR-ABLがん遺伝子を発現させたときに分化が阻害されるかどうか、される場合にどの分化段階で起こるかの解析を試みたものであり、下記の結果を得ている。

1.3つの初期化因子(Oct4, Klf4, Sox2)を豚テシオウイルス由来2Aペプチドでつないだポリシストロンをドキシサイクリン(Dox)応答性のプロモーター/エンハンサーの下流に挿入した自己不活化型レンチウイルス発現ベクターを構築した。なお、予め一部配列を欠損させた3'LTR内にCreリコンビナーゼが認識するloxP配列を挿入した。

2.上記ベクターとrtTA発現レンチウイルスベクターをマウス胎仔線維芽細胞(MEF)に同時感染させてiPS細胞株iPS-C6を樹立した。さらに、この細胞株にCreリコンビナーゼ発現アデノウイルスベクターを感染させ、iPS細胞ゲノムに挿入されたプロウイルスのうちloxPに挟まれたポリシストロンを含む外来配列の大部分を除去することに成功した。ゲノムにはエンハンサー機能を喪失した3'LTRのみが残った。

3.上記iPS-C6細胞に4-HT応答性にキナーゼ活性が誘導されるp190Bcr-Abl誘導体(p190ΔccER)を導入した。このiPS細胞をOP9細胞との共培養で血球系へ分化誘導後、Flt3LとIL7を含む培地中でOP9-DL1細胞と共培養することによりCD3陽性T細胞へ分化誘導させた。この過程で4-HT添加によりBcr-Ablを活性化させると、添加群では非添加群と比較してCD3陽性細胞の出現が約1/3に低下した。

4.C57BL/6マウス骨髄より幹細胞を含むc-Kit陽性Sca1陽性Lineageマーカー陰性(KSL)細胞を純化して、Dox誘導性野生型p190を導入した。OP9-DL1との共培養によるT細胞分化誘導系においてDox添加によりp190を誘導すると、添加群では非添加群と比較してCD3/TCR β陽性T細胞の出現が1/10に低下した。

5.同じくKSL細胞にp190ΔccERを導入し、OP9-DL1との共培養によるT細胞分化誘導系において4-HT添加によりp190を活性化すると、添加群では非添加群と比較してCD3/TCR β陽性T細胞の出現が1/3に低下した。さらに、表面抗原の発現で識別可能なT細胞分化段階を解析すると、非添加群は90%以上がCD25陽性CD44陽性のDN2まで分化していたが、添加群では約半分がCD25陰性のDN1にとどまっていた。

6.p190ΔccER導入時の標識マーカーであるGFPの発現レベルはBcr-Ablの発現と逆相関しており、GFP高(Bcr-Abl低)発現細胞はDN2へ分化する一方、GFP低(Bcr-Abl高)発現細胞はDN1にとどまることから、Bcr-AblはDN1からDN2への分化を阻害すると考えられた。

以上、本論文はマウスiPS細胞および骨髄造血幹細胞を用いて、Ph陽性白血病の病因遺伝子産物であるBcr-AblがT細胞への分化を阻害している証拠を提供した。幹細胞レベルで発症するPh陽性白血病がなぜT細胞系譜は回避するのか数十年にわたって合理的な説明がされていないが、本研究はこの疑問を解決し、Ph陽性白血病の病態解明に重要な貢献をなすと考えられ、学位の授与に値すると考えられる。