Division of Cancer Pathobiology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.
Institute for Cancer Genetics, Columbia University, New York, New York.
Cancer Res. 2024 Oct 15;84(20):3327-3336. doi: 10.1158/0008-5472.CAN-23-3804.
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 sequencing 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, antifolates, 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 resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.
复发性或难治性 B 细胞急性淋巴细胞白血病 (B-ALL) 是导致儿科癌症相关死亡的主要原因。复发特异性突变并不能解释所有接受化疗的 B-ALL 患者的失败，这表明存在其他耐药机制。通过挖掘配对诊断/复发儿科 B-ALL 样本的 RNA 测序数据集，我们发现与复发相关并影响糖皮质激素、抗叶酸剂和硫嘌呤耐药的普遍存在的可变剪接 (AS) 模式。大多数剪接变化代表盒式外显子跳过、“有毒”外显子包含和内含子保留，模拟了经过充分证实的功能丧失突变。相比之下，与复发相关的 NT5C2 mRNA 的 AS 产生了一种具有无功能特征的内含子 6a 的同工型。将 8 个氨基酸序列 SQVAVQKR 纳入该酶中产生了一个假定的磷酸化位点，并导致核酶活性升高，这是 NT5C2 获得性功能突变的已知后果，也是 6-巯基嘌呤耐药的常见决定因素。与这一发现一致的是，NT5C2ex6a 和 R238W 热点变体在体外和体内均赋予 B-ALL 细胞对 6-巯基嘌呤的相当水平的耐药性。此外，NT5C2ex6a 和 R238W 变体都诱导对肌苷单磷酸脱氢酶抑制剂米力农的附带敏感性。这些结果将剪接扰动归因于复发 B-ALL 中化疗耐药的重要作用，并表明肌苷单磷酸脱氢酶抑制剂，包括常用的免疫抑制剂霉酚酸酯，可能是治疗硫嘌呤耐药白血病的有价值的治疗选择。意义：可变剪接是复发性/难治性急性淋巴细胞白血病获得性耐药的一种有效机制，对缺乏已知化疗耐药基因突变的患者具有诊断和治疗意义。
