PHF6在正常和惡性造血中的作用

Abstract

人類PHF6基因位於X染色體上，和轉譯調控與染色質重塑相關。它在人類和小鼠 的中樞神經系統、B和T細胞中都有很高的表現量。遺傳性的PHF6突變會導致一 種罕見的X-染色體相關的萊曼綜合症(BFLS)。這個症狀的表現包括特殊的臉部特徵和智能低下。依目前文獻顯示BFLS可能是一個容易產生癌症的症狀。同時，體細胞的PHF6的突變也在成人急性T淋巴芽細胞白血病(T-ALL)中被發現，發生率大約有11%到39.5%。大部分的PHF6突變包含缺失、遺傳密碼位移突變、無譯或錯譯突變，因此PHF6突變被認為會造成基因失效。PHF6突變也在其他腫瘤中被發 現，例如急性骨髓性白血病、骨髓分化不良症侯群、肝細胞癌等，然而突變的比例 很少。目前PHF6在活體內的病理生理學角色仍尚待研究。 在一個藉由shRNA基因抑制的研究中發現，PHF6在T-ALL中是扮演抑癌基因的角色，而在急性B淋巴芽細胞白血病(B-ALL)中是致癌基因。在B-ALL細胞中剔除Phf6，會導致與正常B細胞發育和功能相關的基因表現下降，而與T細胞信息傳 導相關的基因表現上升。這些結果顯示Phf6的功能是情境依賴式的(context- dependent)。基於有很高比例的T-ALL病人有PHF6突變，所以本論文假設PHF6會影響T細胞的發育、分化與功能活化及可在血液腫瘤生成中扮演腫瘤抑制基因的角色。這個研究最大的限制是缺乏Phf6突變的小鼠模型。 為了解決這個問題，本論文以CRISPR/cas9 system研發了一個新創的條件式Phf6剔除的小鼠。本論文研究結果顯示，與野生型同窩小鼠相比，8週齡的Phf6剔除小鼠外週血中CD4+和CD8+ T細胞的數量減少。在8週至12週齡的Phf6剔除小鼠的骨髓中，骨髓單核細胞前驅細胞(granulocyte-monocytic progenitors)減少，但Lin-c-Kit+Sca-1+細胞增加。功能研究，包括競爭性再增殖單元和連續移植試驗，揭示了Phf6剔除的造血幹細胞(HSC)有增強重建和自我更新能力。18個月大的Phf6基因剔除小鼠表現出類似骨髓分化不良症候群，包括血小板計數減少、巨核細胞發育不良和與髓外造血相關的脾臟腫大。此外，本論文發現Phf6缺失至少部分通過增加白血病起始細胞，降低了NOTCH1誘導的白血病轉化的閾值。對骨髓造血幹細胞(hematopoietic stem cells) 亞群的轉錄組分析揭示了上調的細胞週期和致癌功能，以及這些途徑中關鍵基因表達的改變。綜上，本論文的研究表明Phf6在生理和惡性造血中的體內關鍵作用。 本論文研究結果顯示條件式Phf6剔除小鼠，在18個月大時，並不會產生白血病，因此假設需要第二個突變才會導致白血病。急性白血病的發病機制涉及遺傳改變之間的相互作用。IDH1/2和PHF6的突變在一些造血系統惡性腫瘤患者中很常見並且共存，但它們的協同作用仍未得到探索。本論文進一步繁殖出同時具有PHF6和IDH2R172K突變的小鼠來探討此問題。結果發現共同突變的Phf6KOIdh2R172K小鼠表現出偏向骨髓譜系的造血分化(biased hematopoietic differentiation toward myeloid lineages)，並減少了長期造血幹細胞(long-term hematopoietic stem cells)。與單突變和野生型小鼠相比，它們迅速發展出骨髓性和淋巴性相關的腫瘤，存活時間縮短許多。與攜帶Idh2R172K的小鼠相比，共同突變的小鼠其骨髓和脾細胞產生的2-羥基戊二酸量(2-hydroxyglutarate)顯著增加。單細胞RNA測序揭示了來自共同突變小鼠的造血幹/前驅細胞轉錄組的不同模式，包括代謝酶的異常表達、幾種癌基因的表達增加和DNA修復受損，本論文透過骨髓和脾細胞中增強的γH2AX表達證實DNA修復受損，且Idh2和Phf6突變在白血病發生中具有協同作用，至少通過 2-羥基戊二酸的過量產生和DNA修復受損。 總結來說，在我的博士班研究中，我發現了PHF6的許多功能，這些功能對其在血液惡性腫瘤中的作用很重要，包括它作為HSC和前驅細胞增殖的負調節劑的作用以及它作為腫瘤抑制物的作用。

Human PHF6 (plant homeodomain (PHD) finger 6), located in X chromosome, is involved in transcriptional regulation and chromatin remodeling. It is highly expressed in the central nervous system as well as B- and T-lymphoid cells in human and mice, involved in multiple physiological pathways through chromatin regulation by interaction with the nucleosome remodeling and deacetylation (NuRD) complex. Germline mutations of PHF6 lead to Borjeson-Forssman- Lehmann syndrome (BFLS), which is a rare X-linked disorder with distinctive facial features and mental retardation. Although less than 30 cases of BFLS with PHF6 mutations have been reported, two patients with BFLS developed T-cell acute lymphoblastic lymphoma (T-ALL) and Hodgkin lymphoma, respectively. These observations indicate that BFLS may be a cancer predisposition syndrome. Meanwhile, the somatic PHF6 mutations were found in adult T-ALL patients with an incidence from 11% to 39.5%. The mutations mainly consist of deletions, frameshifts, nonsense mutations, or missense mutations, indicating a loss of function. The PHF6 mutations were also reported in other neoplasms, such as acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and hepatocellular carcinoma, albeit with much lower incidences. However, the functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this problem, this study developed a novel conditional Phf6 knock out mouse model through CRISPR/cas9 system. This study knocked out Phf6 specifically in hematopoietic cells. This study found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, this study found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis. The pathogenesis of acute leukemia involves interaction among genetic alterations. Mutations of IDH1/2 and PHF6 are common and co-exist in some patients of hematopoietic malignancies, but their cooperative effects remain unexplored. As a result, this study addressed the question by characterizing the hematopoietic phenotypes of mice harboring neither, Phf6 knockout, Idh2 R172K, or combined mutations. This study found that the combined Phf6KOIdh2R172K mice showed biased hematopoietic differentiation toward myeloid lineages and reduced long-term hematopoietic stem cells. They rapidly developed neoplasms of myeloid and lymphoid lineages, with much shorter survival compared with single mutated and wild-type mice. The marrow and spleen cells of the combined mutated mice produced a drastically increased amount of 2-hydroxyglutarate compared with mice harboring Idh2 R172K. Single cell RNA sequencing revealed distinct patterns of transcriptome of the hematopoietic stem/progenitor cells from the combined mutated mice, including aberrant expression of metabolic enzymes, increased expression of several oncogenes, and impairment of DNA repairs, as confirmed by the enhanced γH2AX expression in the marrow and spleen cells. These results conclude that Idh2 and Phf6 mutations are synergistic in leukemogenesis, at least through overproduction of 2-hydroxyglutarate and impairment of DNA repairs. In conclusion, during my PhD projects I have discovered a number of functions of PHF6 that are important for its role in hematological malignancies, including its role as a negative regulator of HSC and progenitor proliferation and its function as a tumor suppressor.