[Hi-C] Transcriptional regulation of CD19 antigen abundance in B-cell malignancy

Recent improvements in relapse/refractory B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) outcome can be attributed, in part, to the emergence of CD19-directed immune-based therapies including chimeric antigen receptor (CAR)-T cell therapy and bi-specific T cell engager (BiTE) therapy. Longitudinal BCP-ALL studies have highlighted individual cases of progressive surface CD19 antigen reduction associated with disease persistence following CAR-T infusion. CD19 genomic deletions and mRNA alternative splicing have been previously shown to confer CAR-T and BiTE resistance, however gene regulatory programs that modulate BCP-ALL CD19 surface antigen abundance remains poorly understood. To address this, we performed genome-wide CRISPR/Cas9 screening to identify positive and negative regulators of surface CD19 abundance in human BCP-ALL as compared to mature B cell neoplasms, chronic lymphocytic leukemia and B cell lymphoma. Here, we show DNA binding protein, ZNF143, occupies the CD19 promoter and transcriptionally regulates CD19 mRNA expression in human B-cell malignancies. Conversely, we show that RNA-binding protein, NUDT21, limits surface CD19 abundance on BCP-ALL. NUDT21 acts to regulate CD19 3' UTR length and CD19 mRNA stability. NUDT21 mRNA displays high concordance with CD19 mRNA expression in primary healthy and transformed human B cell progenitors. Using primary BCP-ALL patient single cell data, we show that reduction of CD19 mRNA expression following CAR-T therapy coincides with increased NUDT21 mRNA expression. Importantly, NUDT21 ablation sensitizes BCP-ALL to BiTE and CD19 CAR-T killing ex vivo. These findings reveal previously unknown modulators of CD19 antigen abundance that can be applied to therapeutically enhance or determine resistance to CD19-based therapies in B-cell leukemias. Overall design: Three dimensional chromatin structure profiling of three BCP-ALL cell lines (Reh, NALM6 and 697) transduced with sgROSA (control) or sgZNF143. B11;sgROSA, B6;sgZNF143

复发难治性B细胞祖细胞急性淋巴细胞白血病（relapse/refractory B-cell progenitor acute lymphoblastic leukemia，BCP-ALL）的预后改善，部分归功于CD19靶向免疫治疗的问世，包括嵌合抗原受体（chimeric antigen receptor, CAR）T细胞疗法与双特异性T细胞衔接蛋白（bi-specific T cell engager, BiTE）疗法。纵向BCP-ALL研究已报道多例CAR-T输注后疾病持续存在、伴随表面CD19抗原进行性丢失的病例。此前已有研究证实，CD19基因缺失与mRNA可变剪接可介导CAR-T及BiTE治疗耐药，但调控BCP-ALL细胞表面CD19抗原丰度的基因调控程序仍不甚明晰。为解决这一科学问题，本研究开展全基因组CRISPR/Cas9筛选，在人类BCP-ALL细胞中鉴定调控表面CD19丰度的正负向调控因子，并以成熟B细胞肿瘤、慢性淋巴细胞白血病及B细胞淋巴瘤作为对照。本研究发现，DNA结合蛋白ZNF143可结合CD19启动子，并在人类B细胞恶性肿瘤中转录调控CD19 mRNA的表达。与之相反，RNA结合蛋白NUDT21可降低BCP-ALL细胞表面CD19的丰度：NUDT21通过调控CD19的3'非翻译区（3' untranslated region, 3' UTR）长度及CD19 mRNA的稳定性发挥作用。在原代健康及转化的人类B细胞祖细胞中，NUDT21 mRNA的表达与CD19 mRNA的表达呈高度正相关。利用原代BCP-ALL患者的单细胞数据，本研究发现CAR-T治疗后CD19 mRNA表达下调与NUDT21 mRNA表达上调同步发生。重要的是，体外实验中敲除NUDT21可使BCP-ALL细胞对BiTE及CD19 CAR-T细胞杀伤的敏感性显著增强。本研究揭示了此前未被阐明的CD19抗原丰度调控因子，可为提升B细胞白血病患者对CD19靶向治疗的疗效，或辅助判断治疗耐药性提供新的策略。实验整体设计：对3株转导了sgROSA（对照组）或sgZNF143的BCP-ALL细胞系（Reh、NALM6及697）进行三维染色质结构分析，样本标注为B11;sgROSA、B6;sgZNF143。