Genetic or pharmacological inactivation of CREBBP sensitizes B-cell Acute Lymphoblastic Leukemia to Ferroptotic Cell Death upon BCL2 Inhibition [CUT&RUN]

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. CUTnRUN was performed using the CUTANA CUT&RUN kit (Epicypher, SKU: 15-1016) according to the manufacturer’s protocol (CUTANA™ CUT&RUN Protocol v2.0) in 0.3 million cells per sample of 697WT and 697KI cell lines.

B细胞急性淋巴细胞白血病（B-cell acute lymphoblastic leukemia, B-ALL）是儿童死亡的首要诱因之一，成人患者的预后仍不容乐观。因此临床亟需针对高危病例的治疗策略。组蛋白乙酰转移酶（histone acetyltransferase）CREBBP的突变会使急性淋巴细胞白血病（acute lymphoblastic leukemia, ALL）患者出现高危表型并增强化疗耐药性。我们通过同基因人类细胞系开展靶向药物筛选，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（BCL2-inhibitor）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，诱导的细胞死亡类型为铁死亡（ferroptosis）而非凋亡（apoptosis）。CREBBP突变的细胞系在细胞周期、代谢、脂质组成以及氧化应激应答方面存在特征性差异，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，通过小分子抑制CREBBP可模拟CREBBP突变的表型，使无论基因型背景如何的B-ALL细胞在体外（in vitro）和体内（in vivo）均对维奈克拉诱导的铁死亡敏感，为B-ALL的更广泛临床转化提供了潜在的新型药物联合方案。本研究使用CUTANA CUT&RUN试剂盒（Epicypher, SKU: 15-1016），严格遵循制造商发布的"CUTANA™ CUT&RUN Protocol v2.0"，对697WT和697KI细胞系的每个样本（0.3×10^6个细胞）完成CUT&RUN实验。