Supporting data for "Inhibition of Polo-Like Kinase 4 induced both cell intrinsic and non-cell intrinsic anti-leukemia effects on TP53 mutated acute myeloid leukemia"

Acute myeloid leukemia (AML) carrying TP53 mutations is a distinct subtype characterized by genomic and chromosomal instability and a complex and monosomy karyotype (CK/MK). TP53 mutated AML portends an extremely grave prognosis and is refractory to conventional chemotherapy and allogeneic hematopoietic stem cells transplantation (HSCT). There is an unmet clinical need to develop novel therapeutic strategy for this disease. In an in-silico analysis to identify specific gene signatures of TP53 mutated AML, Polo-Like Kinase 4 (PLK4) was found to be highly expressed in this AML subtype. PLK4 is the master regulator of centriole duplication and cytokinesis and has been investigated as a target for therapeutic intervention in cancers. We hypothesized that PLK4 inhibition may perturb the oncogenic pathway of TP53 mutated AML. Twelve-day treatment with PLK4 inhibitor CFI-400945 as well as gene knockout by CRISPR/Cas 9 suppressed cellular proliferation of TP53 mutated AML cell lines in vitro. CFI-400945 treatment for two days induced DNA damage as evident by γH2AX staining and cellular senescence by β-galactosidase (SA-β-Gal) staining. There was progressive increase in DNA ploidy and number of microtubule organizing center (MTOC), consistent with defective cytokinesis, which was demonstrable by time-lapsed microscopy. Cytoplasmic chromatin could be readily identified in the polyploid cells. Transcriptome analyses of TP53 mutated K052 cell line treated with CFI-400945 demonstrated induction of senescence-associated secretory phenotype (SASP), occurring before the onset of polyploidy. Quantitative RT-PCR and ELISA assay confirmed the increase of CCL2, CXCL8, IL10, IL6, TNF-α and IFN-γ upon CFI-400945 treatment. Both M1 macrophages and T-cells were activated upon co-culture with K052 in the presence of CFI-400945, as evident by the respective increases in phagocytic activity and intracellular IFN-γ/TNF-α. Mechanistically, CFI-400945 induced increase in STING dimers and cGAMP levels and cellular senescence could be ameliorated by STING and cGAS inhibitors. Therapeutically, CFI-400945 treatment in vivo significantly reduced bioluminescence of NSG mice transplanted with luciferase tagged K052 cells, resulting in prolonged survival of recipient animals. The in vivo effects could be potentiated by concomitant treatment with anti-CD47 monoclonal antibody (B6H12), which binds to human CD47 and blocks the interaction with macrophages receptor SIRPα, leading to phagocytosis of leukemic cells. Our results demonstrated that PLK4 inhibition exerted both cell intrinsic and non-cell intrinsic therapeutic effects on TP53 mutated AML with the latter being mediated by activation of cGAS/STING pathway hence both innate and adaptive immunity.

急性髓系白血病（AML）携带TP53突变是一种独特的亚型，其特征为基因组学和染色体不稳定性，以及复杂和单倍体核型（CK/MK）。TP53突变型AML预示着极其严重的预后，且对传统化疗和同种异体造血干细胞移植（HSCT）具有耐药性。针对此疾病，存在着未满足的临床需求，即开发新的治疗策略。通过计算机模拟分析以识别TP53突变型AML的特定基因特征，发现Polo样激酶4（PLK4）在此AML亚型中高表达。PLK4是中心体复制和细胞分裂的 master regulator，并已被研究作为癌症治疗干预的目标。我们假设PLK4的抑制可能干扰TP53突变型AML的致癌途径。使用PLK4抑制剂CFI-400945进行为期12天的治疗以及通过CRISPR/Cas 9进行基因敲除，在体外抑制了TP53突变型AML细胞系的增殖。CFI-400945治疗两天后，通过γH2AX染色观察到DNA损伤，通过β-galactosidase（SA-β-Gal）染色观察到细胞衰老。DNA倍性增加和微管组织中心（MTOC）数量逐渐增加，这与细胞分裂缺陷一致，可以通过时间推移显微镜观察到。多倍体细胞中可以轻易识别到细胞质染色质。对CFI-400945处理的TP53突变K052细胞系的转录组分析表明，在多倍性出现之前，诱导了衰老相关分泌表型（SASP）。定量RT-PCR和ELISA检测证实，在CFI-400945治疗后，CCL2、CXCL8、IL10、IL6、TNF-α和IFN-γ的浓度增加。与CFI-400945共培养K052细胞时，M1巨噬细胞和T细胞被激活，这可以通过吞噬活性和细胞内IFN-γ/TNF-α的增加得到证实。从机制上讲，CFI-400945诱导的STING二聚体和cGAMP水平增加，细胞衰老可以通过STING和cGAS抑制剂得到改善。在体内，CFI-400945治疗显著降低了携带荧光素酶标记的K052细胞的NSG小鼠的生物发光，延长了受体的生存时间。体内效应可以通过与抗CD47单克隆抗体（B6H12）的联合治疗得到增强，该抗体与人类CD47结合并阻断与巨噬细胞受体SIRPα的相互作用，导致白血病细胞的吞噬。我们的结果表明，PLK4抑制对TP53突变型AML具有细胞内在和非细胞内在的治疗作用，后者通过cGAS/STING途径的激活而介导，因此既涉及固有免疫也涉及适应性免疫。