探討基因選擇性剪接對骨髓性血癌的臨床與生物學效應

Abstract

幾乎所有人類的基因都會有選擇性剪接(alternative splicing)，一旦這些原本自然現象的選擇性剪接產生變異(aberrant alternative splicing)，就有可能改變生物體原本正常的生理功能，甚至癌化。根據近幾年的研究，選擇性剪接產生變異可能是致癌的一個重要機轉。 本研究計畫首先利用基因轉錄體微陣列(transcriptome array)的方式，分析了176個骨髓造血不良症候群(myelodysplastic syndrome; MDS)病患的骨髓細胞，以20個正常捐髓者的骨髓造血細胞檢體當對照組，發現MDS患者相對於正常人，在骨髓細胞整個基因體平均約有25-30%的基因發生了變異選擇性剪接，若整個轉錄體變異選擇性剪接的程度越厲害，疾病的預後就越差。接著，利用RNA定序的方式(RNA sequencing)分析了341個急性骨髓性白血病(acute myeloid leukemia; AML)患者的骨髓檢體，進一步發現這些血癌細胞整個轉錄體整體選擇性剪接程度的改變會影響化療的療效與病患預後。因此，變異選擇性剪接可能在血癌致病機制中具有重要作用。 更進一步分析這些血癌患者發生變異選擇性剪接的基因，發現了一個候選基因Nuclear Transcription Factor Y Subunit Alpha (NFYA)可能與血癌的致病機制及預後有關聯。NFYA為NFY轉錄因子的最重要成分，它與基因上游的啟動子(promoter)結合後啟動其標的基因的表達。另外，NFYA基因主要有兩個因選擇性剪接產生的轉錄異構物(isoforms)：NFYA-L[長形式，包含所有10個外顯子(exons)]和NFYA-S[短形式，略過外顯子3 (skipping exon 3)]。研究結果顯示，從臍帶血樣本(cord blood sample)中獲取的CD34+正常造血幹細胞主要表達NFYA-S，而AML癌細胞中可能同時表達NFYA-S和NFYA-L。且較高的NFYA-L和較低的NFYA-S表達(NFYA-L高表達模式)會與不好的臨床特徵與較差的存活率有關。相反，有較高的NFYA-S和較低的NFYA-L表達(NFYA-S高表達模式)與化療後更高的緩解率和較好的存活率相關。這些血癌病患的轉錄體資料分析顯示，NFYA-S的高表達模式與細胞週期相關基因的高度表達相關，而NFYA-L的高表達模式會與KMT2基因重組的血癌細胞表現類似。這一發現與細胞實驗結果相符合，即OCI-AML3細胞中NFYA-S過度表達會增強細胞增殖能力，並增加對化療藥物Ara-C敏感性，但NFYA-L的過度表達卻會造成血癌細胞對化療藥物的抗藥性。這些研究結果突顯了NFYA基因選擇性剪接對AML的臨床與生物學重要性。

Almost all human genes undergo alternative splicing (AS). When this natural phenomenon results in aberrant AS, it can alter an organism's normal physiological functions and even lead to cancer. Recent research indicates that aberrant AS might be a crucial mechanism in carcinogenesis. In this research project, we first employed Affymetrix Human Transcriptome Array (HTA) 2.0 to identify global AS differences in 176 myelodysplastic syndrome (MDS) patients and 20 normal marrow donors. We found that, compared to normal individuals, MDS patients had approximately 25-30% of their genes undergoing aberrant AS in their bone marrow cells. The extent of global aberrant AS in MDS patients correlated with shorter leukemia-free survival, highlighting its involvement in the progression to acute leukemia. Subsequently, a comprehensive analysis of global AS in 341 de novo non-M3 acute myeloid leukemia (AML) patients using the RNA-seq platform demonstrated that the degree of global splicing pattern in AML patients could predict treatment outcomes independently of other well-established prognostic factors. These findings suggest that global AS aberrations may play a role in leukemogenesis, reflecting transcriptome complexity and instability, with potential implications for distinct clinical outcomes—a possible link between AS in specific genes and the pathogenesis of myeloid malignancies. Further investigation into the Nuclear Transcription Factor Y Subunit Alpha (NFYA) gene in AML, which has two major isoforms: NFYA-L (long form, with all 10 exons) and NFYA-S (short form, skipping exon 3), revealed that the NFYA-L/NFYA-S ratios were higher in AML cells compared to CD34+ normal hematopoietic stem cells (HSCs) from cord blood samples. Patients with NFYA-L predominance (higher NFYA-L and lower NFYA-S expression) had worse prognostic features and clinical outcomes after standard intensive chemotherapy compared to those with NFYA-S predominance (higher NFYA-S and lower NFYA-L expression). This prognostic effect was consistent regardless of age and the 2022 European LeukemiaNet (ELN) risk classification, as validated by The Cancer Genome Atlas (TCGA) cohort. Transcriptome analysis showed that NFYA-S predominance was associated with upregulated cell cycle-related genes, similar to those in active HSCs, indicating chemosensitivity. Conversely, NFYA-L predominance, as seen in KMT2A-rearranged leukemia, was linked to chemoresistance. This finding was supported by experiments showing enhanced cell proliferation and increased vulnerability to cytarabine in OCI-AML3 cells with NFYA-S overexpression. This study highlights the clinical significance of NFYA gene AS in AML, suggesting its potential as a prognostic biomarker with distinctive biological pathways in AML cells.