C/EBPα confers dependence to fatty acid anabolic pathways and vulnerability to lipid oxidative stress-induced ferroptosis in FLT3-mutant leukemia [ATAC-seq]. C/EBPα confers dependence to fatty acid anabolic pathways and vulnerability to lipid oxidative stress-induced ferroptosis in FLT3-mutant leukemia [ATAC-seq]

While transcription factor C/AAT-enhancer binding protein α (C/EBPα) is critical for normal and leukemic differentiation, its role on cell and metabolic homeostasis is largely unknown in cancer. Here, multi-omics analyses uncovered a coordinated activation of C/EBPα and Fms-like tyrosine kinase 3 (FLT3) that increased lipid anabolism in vivo and in patients with FLT3-mutant acute myeloid leukemia (AML). Mechanistically, C/EBPα regulated FASN-SCD axis to promote fatty acid (FA) biosynthesis and desaturation. We further demonstrated that FLT3 or C/EBPα inactivation decreased mono-unsaturated FAs incorporation to membrane phospholipids through SCD downregulation. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress, which was exploited by combining FLT3 and glutathione peroxidase 4 inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells. Altogether, our study reveals a C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML to ferroptosis with promising therapeutic application. Overall design: shCTL and shC/EBPα MOLM14 cells (in biological duplicates) were FACS-sorted, and ATAC-seq was performed as previously described (94) with minor modifications. Briefly, 50 000 cells were lysed in ice-cold lysis buffer and the transposition reaction was performed using the Tn5 transposase at 37°C for 30 min. DNA was purified using the QIAGEN MinElute kit (QIAGEN). The libraries were prepared using Tagment DNA Enzyme and Buffer kits (Illumina), NEBNext High-Fidelity 2X PCR Master Mix (NEB, catalog # M0541S) with custom sequencing primers (95–98). The libraries were purified twice using AMPure XP beads (Beckman) following a double-sided protocol to remove primer dimers and large fragments. Libraries quality was assessed using the NGS High Sensitivity Kit on the Fragment Analyzer (Agilent Technologies, Santa Clara, USA). Only high-quality libraries were subsequently equimolarly pooled and sequencing was performed at the Pôle Technologique du CRCT – Plateau de Génomique et Transcriptomique (Inserm-UMR1037, Toulouse, France). The pool of libraries was quantified by qPCR using the KAPA Library Quantification Kit (Roche, Basel, Switzerland) to obtain an accurate quantification. Sequencing was then performed on one flowcell of the Illumina NextSeq 550 instrument (Illumina, San Diego, USA), using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles), and a paired-end 2 x 75 pb strategy. A minimum of 2x80 million raw reads were produced per sample.

转录因子C/AAT增强子结合蛋白α（C/EBPα）在正常造血分化与白血病分化过程中发挥关键调控作用，但其在癌症中对细胞稳态与代谢稳态的影响目前仍未明确。本研究通过多组学分析，揭示了C/EBPα与Fms样酪氨酸激酶3（FLT3）的协同激活效应，该效应可在体内及FLT3突变型急性髓系白血病（AML）患者体内增强脂质合成代谢。机制层面，C/EBPα可调控脂肪酸合成酶-硬脂酰辅酶A去饱和酶（FASN-SCD）轴，以促进脂肪酸（FA）的生物合成与去饱和修饰。我们进一步证实，FLT3或C/EBPα失活可通过下调SCD的表达，减少单不饱和脂肪酸向细胞膜磷脂的掺入。据此，抑制SCD可增强细胞对脂质氧化还原应激的敏感性；联合使用FLT3抑制剂与谷胱甘肽过氧化物酶4（glutathione peroxidase 4）抑制剂，可靶向触发脂质氧化应激，进而增强FLT3突变型AML细胞的铁死亡（ferroptosis）。综上，本研究揭示了C/EBPα在脂质稳态及氧化还原应激适应中的全新功能，同时发现FLT3突变型AML对铁死亡存在此前未被报道的易感特性，具备潜在的临床转化治疗价值。

整体实验设计：将携带短发夹RNA对照（shCTL）与靶向C/EBPα的短发夹RNA（shC/EBPα）的MOLM14细胞（设置2个生物学重复）进行荧光激活细胞分选（FACS），随后采用略有改良的已报道方法（文献94）开展转座酶可及性染色质测序（ATAC-seq）。具体实验流程为：取5×10⁴个细胞于冰预冷的裂解缓冲液中裂解，采用Tn5转座酶在37℃下进行转座反应30分钟；使用QIAGEN MinElute试剂盒（QIAGEN）纯化所得DNA。文库构建采用Illumina Tagment DNA Enzyme and Buffer试剂盒、NEBNext高保真2×PCR预混液（NEB, catalog # M0541S）及定制测序引物（文献95–98）。随后采用双侧磁珠纯化策略，使用AMPure XP磁珠（Beckman）对文库进行两轮纯化，以去除引物二聚体与大片段DNA片段。利用Fragment Analyzer搭载NGS高灵敏度试剂盒评估文库质量。仅合格的高质量文库将按摩尔浓度等量混合，随后在法国图卢兹Inserm-UMR1037的CRCT技术平台–基因组与转录组平台（Pôle Technologique du CRCT – Plateau de Génomique et Transcriptomique）进行测序。文库混合液采用KAPA Library Quantification试剂盒（Roche, Basel, Switzerland）通过定量PCR（qPCR）进行准确定量。测序在Illumina NextSeq 550测序仪（Illumina, San Diego, USA）的单个flowcell上开展，采用NextSeq 500/550 High Output Kit v2.5（150个循环）及双端2×75碱基对（2×75 bp）测序策略。每个样本至少产出2×80 million条原始测序reads。