Humanized NSG mouse model to study combinatorial leukemogenic effects of inherited ELANE and acquired CSF3R/RUNX1 mutations in congenital neutropenia

严重先天性中性粒细胞减少症(CN)是一种白血病前期的骨髓衰竭综合征，具有发展为白血病或骨髓增生异常综合征(MDS)的高风险。最近，我们发现在发展为白血病或MDS的CN患者中，RUNX1和CSF3R协同突变的频率非常高。CSF3R突变本身不能在CN患者或表达转基因突变G-CSFR(D715)的小鼠中诱发白血病，我们假设，在遗传Elane突变的背景下共获得RUNX1是CN白血病转化的关键步骤。此外，我们最近发现，在发生白血病的Elane突变CN患者中，有27%的人在p.C151Y位置存在相同的Elane突变。有趣的是，对携带C151Y Elane突变并发展为白血病的CN患者的造血细胞基因表达特征的序列分析显示，在共获得RUNX1突变后，CSF3R突变的造血祖细胞迅速转化为更原始的造血祖细胞。基于这些观察，我们推测，由于Elane突变，特别是C151Y突变的遗传，在病理背景下共同获得RUNX1和CSF3R突变，使造血分化程序向更原始的造血祖细胞转移，其增殖能力增强，髓系分化减少，最终导致白血病。在本研究中，我们旨在进一步研究协同遗传Elane和获得性CSF3R和RUNX1突变在人源化小鼠体内模型中对HSCs白血病转化的作用。我们计划研究获得RUNX1和CSF3R突变的时间序列在白血病转化中的重要性，以及遗传的CN特异性Elane突变(C151Y)在这一过程中的作用。我们还计划描述在ELANE、CSF3R和RUNX1突变存在的情况下，在HSC中激活的白血病细胞内信号转导通路/S。我们的长期目标是建立使用小分子纠正白血病致病因子/信号通路的方法，以预防或治疗携带Elane突变的CN患者的白血病。

Severe congenital neutropenia (CN) is a pre-leukemic bone marrow failure syndrome with a high risk of evolving into leukemia or myelodysplastic syndrome (MDS). Recently we demonstrated a very high frequency of cooperating RUNX1 and CSF3R mutations in CN patients who developed leukemia or MDS. CSF3R mutations alone are unable to induce leukemia in CN patients or in mice expressing a transgenic mutated G-CSFR (d715) and we hypothesized, that co-acquisition of RUNX1 on the background of inherited ELANE mutations is an essential step in the leukemogenic transformation in CN. Moreover, we recently found that 27 % of ELANE-mutated CN patients who developed leukemia harbor same ELANE mutation at the position p.C151Y. Interestingly, sequential analysis of the gene expression signature of hematopoietic cells of CN patients who harbors C151Y ELANE mutation and developed leukemia revealed rapid transformation of CSF3R-mutated hematopoietic progenitors into more primitive hematopoietic progenitors after co-acquisition of RUNX1 mutation. Based on these observations, we hypothesized, that co-acquisition of RUNX1 and CSF3R mutations on the pathological background due to inheritance of ELANE mutations, especially C151Y mutation, shifts the hematopoietic differentiation program towards more primitive hematopoietic progenitors with elevated proliferative capacity and reduced myeloid differentiation, which ultimately lead to leukemia. In the present study we aimed to further investigate the role of cooperative inherited ELANE and acquired CSF3R and RUNX1 mutations on the leukemogenic transformation of HSCs in humanized mouse model in vivo. We plan to study the importance of the time sequence of acquisition of RUNX1 and CSF3R mutations in leukemogenic transformation and the role of inherited CN-specific ELANE mutation (C151Y) in this process. We also plan to delineate leukemogenic intracellular signal transduction pathway/s activated in HSCs in the presence of ELANE, CSF3R and RUNX1 mutations. Our long-term aim is to establish methods of correction of the leukemogeinc factors/signaling pathways using small molecules in order to prevent or to treat leukemia in CN patients harboring ELANE mutations.