Genetic or pharmacological inactivation of CREBBP sensitizes B-cell Acute Lymphoblastic Leukemia to Ferroptotic Cell Death upon BCL2 Inhibition [ChIP-seq]. Genetic or pharmacological inactivation of CREBBP sensitizes B-cell Acute Lymphoblastic Leukemia to Ferroptotic Cell Death upon BCL2 Inhibition [ChIP-seq]

B-cell acute lymphoblasTc leukemia (B-ALL) is a leading cause of death in childhood and outcomes in adults remain dismal. There is therefore an urgent clinical need for therapies that target the highest risk cases. MutaTons in the histone acetyltransferase CREBBP confer high-risk and increased chemoresistance in ALL. Performing a targeted drug-screen in isogenic human cell lines, we idenTfied a number of small molecules that specifically targeted CREBBP-mutated B-ALL, with the most potent the BCL2-inhibitor Venetoclax. Of note, this acted through a non-canonical mechanism resulTng in ferroptoTc rather than apoptoTc cell death. CREBBP-mutated cell lines showed differences in cell- cycle, metabolism, lipid composiTon and response to oxidaTve stress, predisposing them to ferroptosis, which were further dysregulated upon acquisiTon of Venetoclax resistance. Lastly, small- molecule inhibiTon of CREBBP pharmacocopies CREBBP-mutaTon, sensiTzing B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in-vitro and in-vivo, providing a potential novel drug combination for broader clinical translation in B-ALL. Overall design: ChIP-seq was performed using 2.5μg of anTbody (IgG, Proteintech, 30000-0-AP; H3, Cell Signalling Technology, 14269S and H3K27ac, AcTve moTf, 39133). Immunocomplexes were captured using the protein A (10002D) and protein G (10004D) Dynabeads (Invitrogen). Aäer 4h, the immunocomplexes were magneTzed and repeatedly washed in the following buffers in 7 million 697WT and 697KI

B细胞急性淋巴细胞白血病（B-cell acute lymphoblastic leukemia, B-ALL）是儿童期首要的致死性病因之一，成人患者的预后仍不容乐观。因此临床上迫切需要针对高风险病例的治疗策略。组蛋白乙酰转移酶（histone acetyltransferase）CREBBP的突变可在急性淋巴细胞白血病（acute lymphoblastic leukemia, ALL）中赋予肿瘤细胞高风险表型并增强化疗耐药性。我们在同基因人类细胞系中开展靶向药物筛选，鉴定出多种可特异性靶向CREBBP突变型B-ALL的小分子化合物，其中活性最强的为BCL2抑制剂维奈克拉（Venetoclax）。值得注意的是，该药物通过非经典机制发挥作用，诱导细胞发生铁死亡（ferroptosis）而非凋亡（apoptosis）。CREBBP突变细胞系在细胞周期、代谢、脂质组成以及氧化应激应答方面均存在特征性差异，使其易于发生铁死亡；而在获得维奈克拉耐药性后，这些特征进一步出现失调。最后，通过小分子靶向抑制CREBBP可模拟CREBBP突变的生物学表型，使无论基因型背景如何的B-ALL细胞，在体外与体内均对维奈克拉诱导的铁死亡敏感，这为B-ALL的更广泛临床转化提供了一种潜在的新型药物联合方案。

整体实验设计：采用2.5μg抗体进行染色质免疫共沉淀测序（ChIP-seq），所用抗体包括：IgG（Proteintech，30000-0-AP）、H3（Cell Signalling Technology，14269S）以及H3K27ac（Active Motif，39133）。使用蛋白A（10002D）和蛋白G（10004D）磁珠（Dynabeads，Invitrogen）捕获免疫复合物。孵育4小时后，对免疫复合物进行磁分离，并使用以下缓冲液反复洗涤，实验样本为700万697WT与697KI细胞