Khan Robin, Phely Laurent, Ehrenfeld Sophia, Schmitz Tatjana, Veratti Pia, Wolfes Jakob, Shoumariyeh Khalid, Andrieux Geoffroy, Martens Uta S, Bra Stephan de, Auer Martina, Schilling Oliver, Boerries Melanie, Speicher Michael, Illert Anna L, Duyster Justus, Miething Cornelius
Department of Medicine I, University Medical Center Freiburg, University of Freiburg, 79106 Freiburg, Germany.
Department of Pediatrics, University Hospital Würzburg, 97080 Würzburg, Germany.
Cancers (Basel). 2025 Jul 2;17(13):2226. doi: 10.3390/cancers17132226.
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 the cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation.
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 the and gene loci in murine cells.
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 novo translocations 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 the cDNA with Ba/F3 cells transformed by CRISPR-mediated translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the overexpression model, suggesting increased reactive changes induced by exogenous overexpression of . 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.
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（ALCL）是一种侵袭性T细胞淋巴瘤，其特征是表达由t(2;5)染色体重排诱导产生的间变性淋巴瘤激酶（ALK），进而导致NPM-ALK融合癌基因的表达。以往大多数ALK阳性ALCL的临床前模型都是基于从异源启动子过表达cDNA。由于是强制表达，这种方法容易出现因合成过表达、启动子竞争和插入变异而产生的假象。
为了改进现有的ALCL模型并更精确地重现ALK阳性ALCL中的致癌事件，我们采用基于CRISPR/Cas的染色体工程技术，在小鼠细胞中选择性地引入 和 基因座之间的易位。
通过在小鼠IL-3依赖的Ba/F3报告细胞系的11号和17号染色体的同系位点诱导精确的DNA切割，我们在体内产生了新的 易位，导致细胞在不依赖IL-3的情况下生长。为了验证有效的重组，我们分析了重组细胞中NPM-ALK融合蛋白的表达，并且还在不依赖IL-3的Ba/F3细胞中显示出t(11;17)。随后使用ALK抑制剂对这些细胞进行功能测试，结果显示它们对克唑替尼具有极高的反应性，表明对新产生的ALK融合癌蛋白有强烈依赖性。此外，将过表达 cDNA的Ba/F3细胞与通过CRISPR介导的 易位转化的Ba/F3细胞的基因表达模式进行比较，结果表明，虽然两者大致重叠，但包括未折叠蛋白反应途径在内的一组通路在 过表达模型中有所增加，这表明 的外源性过表达诱导了更多的反应性变化。此外，我们在基于CRISPR的新模型中观察到位于靠近断点的染色体区域中的基因表达发生聚集性变化，这表明易位事件介导了对基因表达的位置效应，而这在旧模型中并不存在。
因此，CRISPR介导的重组为模拟致癌易位提供了一种新颖且更可靠的方法，这可能有助于更好地理解ALCL的分子发病机制，并建立更准确的由致癌融合蛋白驱动的恶性肿瘤治疗模型。
