Efficacy of novel agents against cellular models of familial platelet disorder with myeloid malignancy (FPD-MM). [Stranded mRNA-Seq]

Germline, mono-allelic mutations in RUNX1 cause familial platelet disorder (RUNX1-FPD) that evolves into myeloid malignancy (FPD-MM): MDS or AML. FPD-MM commonly harbors co-mutations in the second RUNX1 allele and/or other epigenetic regulators. Here we utilized patient-derived (PD) FPD-MM cells and established the first FPD-MM AML cell line (GMR-AML1). GMR-AML1 cells exhibited active super-enhancers of MYB, MYC, BCL2 and CDK6, augmented expressions of c-Myc, c-Myb, EVI1 and PLK1 and surface markers of AML stem cells. In longitudinally studied bone marrow cells from a patient at FPD-MM vs RUNX1-FPD state, we confirmed increased chromatin accessibility and mRNA expressions of MYB, MECOM and BCL2 in FPD-MM cells. GMR-AML1 and PD FPD-MM cells were sensitive to homoharringtonine (HHT or omacetaxine) or mebendazole-induced lethality, associated with repression of c-Myc, EVI1, PLK1, CDK6 and MCL1. Co-treatment with MB and the PLK1 inhibitor volasertib exerted synergistic in vitro lethality in GMR-AML1 cells. In luciferase-expressing GMR-AML1 xenograft model, MB, omacetaxine or volasertib monotherapy, or co-treatment with MB and volasertib, significantly reduced AML burden and improved survival in the immune-depleted mice. These findings highlight the molecular features of FPD-MM progression and demonstrate HHT, MB and/or volasertib as effective agents against cellular models of FPD-MM. Overall design: Paired end RNA-Seq of a leukemia cell line treated with homoharringtonine or mebendazole for 16/24 hours.

RUNX1的生殖系单等位基因突变可引发家族性血小板疾病（RUNX1-familial platelet disorder, RUNX1-FPD），该疾病可进展为髓系恶性肿瘤（FPD-MM），即骨髓增生异常综合征（Myelodysplastic Syndrome, MDS）或急性髓系白血病（Acute Myeloid Leukemia, AML）。FPD-MM通常伴随第二个RUNX1等位基因及/或其他表观遗传调控因子的共突变。本研究利用患者来源的（patient-derived, PD）FPD-MM细胞，建立了首个FPD-MM AML细胞系（GMR-AML1）。GMR-AML1细胞携带有MYB、MYC、BCL2及CDK6的活性超级增强子（super-enhancer），且c-Myc、c-Myb、EVI1及PLK1的表达水平显著上调，同时表达AML干细胞表面标志物。通过对一名患者处于FPD-MM与RUNX1-FPD阶段的骨髓细胞进行纵向研究，我们证实FPD-MM细胞中MYB、MECOM及BCL2的染色质可及性与mRNA表达水平均显著升高。GMR-AML1及患者来源的FPD-MM细胞对高三尖杉酯碱（homoharringtonine, HHT，又称奥沙他星omacetaxine）或甲苯咪唑（mebendazole, MB）诱导的杀伤作用敏感，该杀伤效应与c-Myc、EVI1、PLK1、CDK6及MCL1的表达抑制密切相关。联合应用MB与PLK1抑制剂伏拉塞替尼（volasertib），可在体外对GMR-AML1细胞产生协同杀伤作用。在表达荧光素酶的GMR-AML1异种移植模型中，单药使用MB、高三尖杉酯碱或伏拉塞替尼，或联合使用MB与伏拉塞替尼，均可显著降低免疫缺陷小鼠的AML负荷并延长其生存期。上述研究结果阐明了FPD-MM进展的分子特征，并证实高三尖杉酯碱、MB及/或伏拉塞替尼可作为靶向FPD-MM细胞模型的有效治疗药物。实验设计：对经高三尖杉酯碱或甲苯咪唑处理16/24小时的白血病细胞系进行双端RNA测序（paired end RNA-Seq）。