复发性或难治性 B 细胞急性淋巴细胞白血病 (B-ALL) 是儿童癌症相关死亡的主要原因。复发特异性突变并不能解释 B-ALL 患者的所有化疗失败，这表明存在其他耐药机制。通过挖掘配对诊断/复发儿童 B-ALL 样本的 RNA-seq 数据集，我们发现了与复发相关的普遍选择性剪接 (AS) 模式，并影响糖皮质激素、抗叶酸剂和硫嘌呤耐药的驱动因素。大多数剪接变异代表盒式外显子跳跃、“毒害”外显子包含和内含子保留、表型复制有据可查的功能丧失突变。相比之下，NT5C2 mRNA 的复发相关 AS 产生了具有功能上未表征的框内外显子 6a 的同种型。将 8 个氨基酸序列 SQVAVQKR 掺入该酶中，创建了一个假定的磷酸化位点，并导致核苷酶活性升高，这是 NT5C2 中功能获得性突变的已知结果，也是 6-巯基嘌呤 (6-MP) 的常见决定因素） 反抗。与这一发现一致的是，NT5C2ex6a 和 R238W 热点变体在体外和体内均在 B-ALL 细胞中赋予了相当水平的 6-MP 耐药性。此外，NT5C2ex6a 和 R238W 变体均诱导对肌苷单磷酸脱氢酶 (IMPDH) 抑制剂咪唑立宾的附带敏感性。这些结果归因于剪接扰动在复发性 B-ALL 化疗耐药中的重要作用，并表明 IMPDH 抑制剂，包括常用的免疫抑制剂吗替麦考酚酯，可能是治疗硫嘌呤耐药性白血病的有价值的治疗选择。

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.