Background/Objectives: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of theNPM-ALKcDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation. Methods: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between theNpm1andAlkgene loci in murine cells. Results: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novoNpm-Alktranslocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing theNpm-AlkcDNA with Ba/F3 cells transformed by CRISPR-mediatedNpm-Alktranslocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in theNpm-Alkoverexpression model, suggesting increased reactive changes induced by exogenous overexpression ofNpm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models. Conclusions: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins.
背景/目的：ALK阳性间变性大细胞淋巴瘤（ALK+ ALCL）是一种侵袭性T细胞淋巴瘤，其特征为表达由t(2;5)染色体易位诱导产生的间变性淋巴瘤激酶（ALK），该易位导致NPM-ALK融合癌基因的表达。既往大多数ALK+ ALCL的临床前模型均基于异源启动子过表达NPM-ALK cDNA。由于采用强制表达策略，该方法易因人工过表达、启动子竞争及插入变异而产生实验假象。方法：为改进现有ALCL模型并更精确模拟ALK+ ALCL的致癌事件，我们运用CRISPR/Cas介导的染色体工程技术，在小鼠细胞中选择性引入Npm1与Alk基因座间的易位。结果：通过对小鼠IL-3依赖性Ba/F3报告细胞系中11号和17号染色体同源区进行精准DNA切割，我们在体内成功构建了全新的Npm-Alk易位模型，该模型可诱导细胞获得IL-3非依赖性生长能力。为验证重组效率，我们检测了重组细胞中NPM-ALK融合蛋白的表达，并在IL-3非依赖性Ba/F3细胞中证实了t(11;17)易位的存在。后续使用ALK抑制剂对这些细胞进行功能测试显示，其对克唑替尼具有高度敏感性，表明细胞生长强烈依赖于新生成的ALK融合癌蛋白。此外，通过比较过表达Npm-Alk cDNA的Ba/F3细胞与CRISPR介导Npm-Alk易位转化的Ba/F3细胞的基因表达谱发现：虽然两者表达谱广泛重叠，但在Npm-Alk过表达模型中，包括未折叠蛋白反应通路在内的多条信号通路表达上调，提示外源性Npm-Alk过表达可能诱导更强的应激性改变。值得注意的是，在新构建的CRISPR模型中，我们观察到断裂点附近染色体区域基因呈现簇状表达变化，表明易位事件可通过位置效应调控基因表达，这一现象在传统模型中未被体现。结论：因此，CRISPR介导的重组技术为模拟致癌性染色体易位提供了全新且更精准的研究手段，有望深化对ALCL分子发病机制的理解，并为融合癌蛋白驱动型恶性肿瘤构建更精确的治疗模型。
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