GATA3 rs3824662遗传变异增加B急性淋巴细胞白血病（B-ALL）不良预后的易感性，而其易感性机制至今仍不明确。申请人预实验发现：rs3824662等位基因A顺式激活GATA3转录并与化疗后白血病细胞残留高度相关；GATA3激活细胞自噬引起B-ALL细胞对门冬酰胺酶（L-Asp）耐药。初步机制显示：GATA3可通过JAK-STAT活化自噬。据此提出：rs3824662等位基因A顺式激活GATA3转录，进而活化JAK-STAT-自噬信号通路，最终导致B-ALL细胞对L-Asp耐药。本项目拟利用反向ChIP-质谱、染色体构象捕获和RNA干扰技术阐明rs3824662顺式调控GATA3转录的机制；通过高通量测序，结合JAK-STAT干预与FRET传感器明确GATA3导致L-Asp耐药的分子机制。项目有助于推动儿童ALL的遗传咨询工作，科学指导L-Asp的临床应用和ALL精准危险度分层。

Evidence from our previous study and other groups have identified that inherited GATA3 rs3824662 genetic variation strongly associated with poor prognosis and minimal residual disease (MRD) positivity in children with B-lymphocytic leukemia (B-ALL). However, the mechanism of how this inherited variant affect MRD remains unclarified. In our pilot study, we found that GATA3 rs3824662 located in a hematopoiesis specific enhancer region. The rs3824662 A allele was associated with MRD positivity after induction remission therapy. Also, the rs3824662 A allele increased the enhancer activity and cis-activated GATA3 transcription. In vitro cellular assays showed that higher GATA3 expression conferred B-ALL cells resistance to L-Asparaginase (L-Asp). Preliminary mechanistic study suggested that GATA3 induced L-Asp resistance might be due to GATA3-induced autophagy activation. Building upon these findings, we raised the hypothesis that inherited GATA3 rs3824662 variation promotes GATA3 transcription, which in turn activates JAK-STAT signaling and then induces L-Asp resistance in B-ALL cells via autophagy activation. To address this scientific question, we plan to apply reverse ChIP, LC-MS, Capture C, ChIP-qPCR and TA clone to find out the mechanism of rs3824662 cis-regulating GATA3 transcription, and then using ShRNA to further validate the mechanism. Further, we will perform ChIP-Seq, RNA-Seq and FRET sensors assays to explore that how GATA3 activates JAK-STAT and autophagy-induced L-Asp resistance. Finally, we will pretreat GATA3OE leukemic cells with JAK-STAT activation and inhibition, to confirm the mechanism of GATA3-induced autophagy activation. Completion of this study would definitely benefit genetic counseling and testing, better risk stratification, precision L-Asp resistance therapy in childhood ALL.