Background/Objectives: Approved for treatment of acute leukemia, gemtuzumab ozogamicin (GO) and inotuzumab ozogamicin (InO) are antibody–drug conjugates (ADCs) that deliver a toxic calicheamicin (CLM) derivative. The resistance mechanisms to GO/InO remain incompletely understood. Methods: We performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen for CLM sensitivity genes, and then performed confirmatory cytotoxicity assays. Results: Several DNA damage pathway regulation genes were identified, most notablyTP53. Across 13 acute leukemia cell lines, the sixTP53-mutant cell lines (TP53MUT) were indeed 10- to 1000-fold less sensitive to CLM than the sevenTP53WTcell lines. In fiveTP53WT/KOsyngeneic cell line pairs we generated,TP53KOcells were significantly less sensitive to CLM than theirTP53WTcounterparts. InTP53WTbut notTP53MUTcells, the MDM2 inhibitor and p53 activator, idasanutlin, enhanced CLM cytotoxicity, demonstrating that decoupling of cells from MDM2-p53 regulation sensitizes leukemia cells to CLM. The ATM inhibitors AZD1390 and lartesertib also significantly enhanced CLM efficacy but did so independent of theTP53status. In contrast, neither an ATR inhibitor, Chk1/Chk2 inhibitor, Chk2 inhibitor, or a PARP inhibitor significantly impacted CLM-induced cytotoxicity across the thirteen cell lines. Together, our studies identify ATM, MDM2, andTP53—which are in the same cellular response to DNA damage pathway—as key modulators of CLM-induced cytotoxicity in acute leukemia cells. Conclusions: These results support further evaluation of combination therapies with corresponding small-molecule inhibitors (currently pursued for therapy of other cancers) toward clinical testing as novel strategies to increase the efficacy of CLM-based ADCs such as GO and InO.
背景/目的：吉妥珠单抗奥佐米星（GO）与奥英妥珠单抗（InO）是已获批用于治疗急性白血病的抗体药物偶联物（ADC），可递送毒性卡奇霉素（CLM）衍生物。目前对GO/InO的耐药机制尚未完全阐明。方法：我们通过全基因组CRISPR/Cas9筛选技术鉴定CLM敏感性相关基因，并开展验证性细胞毒性实验。结果：研究识别出多个DNA损伤通路调控基因，其中TP53最为显著。在13种急性白血病细胞系中，6个TP53突变型细胞系（TP53MUT）对CLM的敏感性较7个TP53野生型细胞系（TP53WT）低10-1000倍。在我们构建的5对TP53WT/KO同源细胞系中，TP53KO细胞对CLM的敏感性显著低于TP53WT细胞。在TP53WT细胞中（TP53MUT细胞无此现象），MDM2抑制剂兼p53激活剂idasanutlin可增强CLM的细胞毒性，表明解除MDM2-p53调控通路能提高白血病细胞对CLM的敏感性。ATM抑制剂AZD1390和lartesertib同样显著增强CLM疗效，且该作用与TP53状态无关。相比之下，ATR抑制剂、Chk1/Chk2抑制剂、Chk2抑制剂及PARP抑制剂在13种细胞系中均未显著影响CLM诱导的细胞毒性。综上，我们的研究证实ATM、MDM2与TP53——三者同属DNA损伤应答通路——是调控CLM诱导急性白血病细胞毒性的关键因子。结论：这些结果为联合疗法研究提供了理论依据，支持进一步评估相应小分子抑制剂（目前正用于其他癌症治疗）与CLM类ADC药物（如GO和InO）的联合治疗方案，为提升此类ADC疗效的新型临床策略奠定基础。
肿瘤(癌症)患者之家