Supplementary Material for: The Study of SRSF1 Regulates Abnormal Alternative Splicing of BCL2L11 and the Role in Refractory Acute Ayeloid Leukemia

Introduction: Abnormalities in splicing factors, such as mutations or deregulated expression, can lead to aberrant splicing of target genes, potentially contributing to the pathogenesis of acute myeloid leukemia (AML). Despite this, the precise mechanism underlying the abnormal alternative splicing induced by SRSF1, a splicing factor associated with poor AML prognosis, remains elusive.
Methods: Using strict splicing criteria, we globally screened for alternative splicing(AS) events in NPMc-positive and NPMc-negative AML samples from TCGA. An AS network associated with AML prognosis was then established. Functional assays, including CCK-8, flow cytometry, and Western blot, were conducted on K562 and THP-1 cells overexpressing SRSF1. Cell viability following 72-hour Omipalisib treatment was also assessed. To explore the mechanism of SRSF1-induced AS, we created a BCL2L11 miniGene with a site-specific mutation at its branch point. The AS patterns of both wild-type and mutant miniGenes were analyzed following SRSF1 overexpression in HEK-293T, along with the subcellular localization of different spliceosomes.
Results: SRSF1 was significantly associated with AML prognosis. Notably, its expression was markedly upregulated in refractory AML patients compared to those with a favorable chemotherapy response. Overexpression of SRSF1 promoted THP-1 cell proliferation, suppressed apoptosis, and reduced sensitivity to Omipalisib. Mechanistically, SRSF1 recognized an aberrant branch point within the BCL2L11 intron, promoting the inclusion of a cryptic exon 3, which in turn led to apoptosis arrest.
Conclusions: Overexpression of SRSF1 and the resulting abnormal splicing of BCL2L11 are associated with drug resistance and poor prognosis in AML.

引言：剪接因子的异常（如突变或表达失调）可导致靶基因发生可变剪接异常，进而可能参与急性髓系白血病（acute myeloid leukemia, AML）的发病进程。尽管如此，由与AML不良预后相关的剪接因子SRSF1所诱导的异常可变剪接的确切分子机制仍不明确。
方法：本研究采用严格的剪接判定标准，从癌症基因组图谱（The Cancer Genome Atlas, TCGA）的NPMc阳性与NPMc阴性AML样本中，全局筛选可变剪接（alternative splicing, AS）事件。随后构建了与AML预后相关的可变剪接调控网络。我们在过表达SRSF1的K562与THP-1细胞中开展了包括CCK-8实验、流式细胞术以及蛋白质印迹（Western blot）在内的功能学检测，同时评估了经Omipalisib处理72小时后的细胞活力。为探究SRSF1诱导可变剪接的分子机制，我们构建了在分支点处携带位点特异性突变的BCL2L11迷你基因（miniGene）。在HEK-293T细胞中过表达SRSF1后，我们分析了野生型与突变型迷你基因的剪接模式，并检测了不同剪接体的亚细胞定位。
结果：SRSF1的表达水平与AML预后显著相关。值得注意的是，相较于化疗应答良好的患者，难治性AML患者体内的SRSF1表达水平显著上调。过表达SRSF1可促进THP-1细胞增殖、抑制细胞凋亡，并降低其对Omipalisib的药物敏感性。从分子机制层面分析，SRSF1能够识别BCL2L11内含子内的异常分支位点，促进隐蔽外显子3的纳入，进而引发细胞凋亡阻滞。
结论：SRSF1过表达及其介导的BCL2L11异常剪接与AML的耐药性及不良预后密切相关。