DataSheet_3_Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.pdf

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

携带21号染色体拷贝数增加、21号染色体内部扩增（iAMP21）或唐氏综合征（DS）的急性淋巴细胞白血病（ALL）患者，其21号染色体唐氏综合征关键区域（DSCR）内的基因表达水平显著升高，其中包括高迁移率族核小体结合蛋白1（HMGN1）。唐氏综合征患儿易罹患血液系统恶性肿瘤，这为阐明21号染色体在白血病发生发展中的作用提供了重要研究线索。白血病细胞中X/Y染色体假常染色体区域存在320kb的片段缺失，该缺失会引发嘌呤能受体与细胞因子受体样因子2形成融合基因（P2RY8::CRLF2），这一分子特征在约60%的唐氏综合征相关ALL（DS-ALL）患者和约40%的iAMP21患者中均较为常见，提示21号染色体与P2RY8::CRLF2融合基因的发生存在关联。在澳大利亚队列的38例携带P2RY8::CRLF2融合基因的儿童B细胞急性淋巴细胞白血病患者中，8例存在21号染色体拷贝数增加（+21），2例携带iAMP21，上述患者的HMGN1表达水平显著升高。研究人员使用诱导型CRISPR/Cas9系统构建P2RY8::CRLF2融合基因的细胞模型，以探究其与HMGN1的协同效应，并验证HMGN1作为P2RY8::CRLF2发生发展影响因子的作用。利用Cas9在假常染色体区域切割DNA且不进行定向修复时，相较于不表达HMGN1的细胞，表达HMGN1的细胞更易发生修复事件，进而形成P2RY8::CRLF2融合基因。表达HMGN1的CRISPR/Cas9构建的P2RY8::CRLF2细胞，其增殖能力、胸腺基质淋巴细胞生成素受体（TSLPR）表达水平以及JAK/STAT信号通路活性均出现上调，这与携带P2RY8::CRLF2的患者来源细胞的表型一致。综合患者的表达数据与独特的CRISPR/Cas9建模结果，研究表明HMGN1可提升P2RY8::CRLF2融合基因发生的易感性。这一发现对于唐氏综合征、+21或iAMP21相关患者具有重要的临床意义。