Orlikova-Boyer Barbora, Lorant Anne, Gajulapalli Sruthi Reddy, Cerella Claudia, Schnekenburger Michael, Lee Jin-Young, Paik Ji Yeon, Lee Yejin, Siegel David, Ross David, Han Byung Woo, Nguyen Thi Kim Yen, Christov Christo, Kang Hyoung Jin, Dicato Mario, Diederich Marc
Laboratoire de Biologie Moléculaire du Cancer, BAM3 Pavillon 2, 6A Rue Nicolas-Ernest Barblé, L-1210, Luxembourg, Luxembourg.
Department of Pharmacy, Research Institute of Pharmaceutical Sciences & Natural Products Research Institute, College of Pharmacy, Seoul National University, 1, Gwanak-Ro, Gwanak-Gu, Seoul, 08826, Republic of Korea.
Biomark Res. 2024 May 4;12(1):47. doi: 10.1186/s40364-024-00594-w.
Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.
Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.
Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients' stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.
Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.
尽管酪氨酸激酶抑制剂（TKIs）在慢性髓性白血病（CML）治疗方面取得了进展，但耐药性和不耐受性仍然是重大挑战。白血病干细胞（LSCs）和TKI耐药细胞依赖于改变的线粒体代谢和氧化磷酸化。靶向重新布线的能量代谢并诱导非凋亡性细胞死亡，以及释放损伤相关分子模式（DAMPs），可以增强针对CML的治疗策略和免疫疗法，并防止TKI耐药细胞的出现和LSC的持续存在。
使用CML患者干细胞和健康细胞的数据集进行转录组分析。通过荧光显微镜和流式细胞术评估DNA损伤。通过台盼蓝排斥试验、荧光显微镜、流式细胞术、集落形成试验和体内斑马鱼异种移植评估细胞死亡。通过测量NAD和NADH水平、用海马分析仪测定ATP产生速率以及细胞内ATP含量来确定能量代谢。通过流式细胞术测量线粒体膜电位、ROS和钙积累来估计线粒体健康状况，并通过透射电子显微镜观察形态。利用生物信息学分析、实时定量PCR、蛋白质印迹、化学反应预测和分子对接来鉴定药物靶点。通过高迁移率族蛋白盒（HMGB）1酶联免疫吸附测定、基于荧光素酶的细胞外ATP测定、流式细胞术检测异位钙网蛋白表达，并通过吞噬试验和使用同基因C57BL/6小鼠的体内疫苗接种试验进行验证。
转录组分析确定了CML患者的代谢改变和DNA修复缺陷特征。CML患者在免疫系统、DNA修复和代谢途径中表现出富集。与BRCA突变肿瘤相关的基因特征在CML数据集中富集，表明双链断裂修复途径存在缺陷。此外，与健康对照相比，CML患者干细胞中的聚（ADP-核糖）聚合酶（PARP）1显著上调。与CML患者DNA修复特征一致，用甲基化吲哚醌MAC681治疗可诱导DNA损伤、线粒体功能障碍、钙稳态破坏、代谢灾难和坏死性凋亡样细胞死亡。同时，MAC681导致PARP1降解，而3-氨基苯甲酰胺可阻止这种降解。MAC681处理的髓系白血病细胞释放DAMPs，并在C57BL/6小鼠中显示出产生免疫原性疫苗的潜力。MAC681和阿塞西尼布在杀死伊马替尼敏感和耐药的CML方面表现出协同作用，开启了新的治疗机会。
总体而言，通过PARP1降解和线粒体失调增加肿瘤突变负担使CML适合免疫治疗。
