State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
BMC Cancer. 2019 Nov 8;19(1):1072. doi: 10.1186/s12885-019-6207-y.
DNMT3A R882H, a frequent mutation in acute myeloid leukemia (AML), plays a critical role in malignant hematopoiesis. Recent findings suggest that DNMT3A mutant acts as a founder mutation and requires additional genetic events to induce full-blown AML. Here, we investigated the cooperation of mutant DNMT3A and NRAS in leukemogenesis by generating a double knock-in (DKI) mouse model harboring both Dnmt3a R878H and Nras G12D mutations.
DKI mice with both Dnmt3a R878H and Nras G12D mutations were generated by crossing Dnmt3a R878H knock-in (KI) mice and Nras G12D KI mice. Routine blood test, flow cytometry analysis and morphological analysis were performed to determine disease phenotype. RNA-sequencing (RNA-seq), RT-PCR and Western blot were carried out to reveal the molecular mechanism.
The DKI mice developed a more aggressive AML with a significantly shortened lifespan and higher percentage of blast cells compared with KI mice expressing Dnmt3a or Nras mutation alone. RNA-seq analysis showed that Dnmt3a and Nras mutations collaboratively caused abnormal expression of a series of genes related to differentiation arrest and growth advantage. Myc transcription factor and its target genes related to proliferation and apoptosis were up-regulated, thus contributing to promote the process of leukemogenesis.
This study showed that cooperation of DNMT3A mutation and NRAS mutation could promote the onset of AML by synergistically disturbing the transcriptional profiling with Myc pathway involvement in DKI mice.
DNMT3A R882H 是急性髓系白血病（AML）中的一种常见突变，在恶性造血中起着关键作用。最近的研究结果表明，DNMT3A 突变体充当创始突变体，并需要额外的遗传事件来诱导完全性 AML。在这里，我们通过生成同时携带 Dnmt3a R878H 和 Nras G12D 突变的双敲入（DKI）小鼠模型，研究了突变型 DNMT3A 和 NRAS 在白血病发生中的合作。
通过将 Dnmt3a R878H 敲入（KI）小鼠和 Nras G12D KI 小鼠杂交，生成同时携带 Dnmt3a R878H 和 Nras G12D 突变的 DKI 小鼠。进行常规血液检查、流式细胞术分析和形态学分析以确定疾病表型。进行 RNA 测序（RNA-seq）、RT-PCR 和 Western blot 以揭示分子机制。
与单独表达 Dnmt3a 或 Nras 突变的 KI 小鼠相比，DKI 小鼠发展出更具侵袭性的 AML，其寿命明显缩短，白血病细胞比例更高。RNA-seq 分析表明，DNMT3A 和 NRAS 突变协同导致一系列与分化阻滞和生长优势相关的基因异常表达。Myc 转录因子及其与增殖和凋亡相关的靶基因上调，从而有助于促进白血病发生过程。
本研究表明，DNMT3A 突变与 NRAS 突变的合作通过协同扰乱转录谱并涉及 Myc 通路，在 DKI 小鼠中促进 AML 的发生。
