Only hematopoietic stem and progenitor cells from cord blood are susceptible to malignant transformation by MLL-AF4 translocations. Only hematopoietic stem and progenitor cells from cord blood are susceptible to malignant transformation by MLL-AF4 translocations

MLL rearrangements (MLLr) play a crucial role in leukemogenesis. Dependent on age, major differences exist regarding disease frequency, main fusion partners and prognosis. In infants, up to 80% of acute lymphoid leukemia (ALL) bear t(4;11) resulting in a poor prognosis. In contrast, in adults only 10% of acute myeloid leukemia (AML) bear t(9;11) with an intermediate prognosis. The reasons for these differences are only poorly understood. Recently, we established an efficient human patient-specific CRISPR/Cas9-based MLLr model in hematopoietic stem and progenitor cells (HSPCs) derived from human cord blood (huCB) faithfully mimicking the underlying biology of the disease. Here, we transfer this model into an adult system using HSPCs from adult bone marrow (huBM) allowing us to investigate the impact of the cell of origin and fusion partner on disease development. RNA-Seq uncovered an absent downregulation of FFAR2 in t(4;11) huBM, an epigenetic tumor suppressor, potentially responsible for the inability of MLL-AF4 to immortalize adult cells under myeloid conditions. Overall design: huCB and huBM CD34+ cells were engineered to bear endogenous MLL-AF4 or MLL-AF9 translocations. Generated cells were cultured to a purity of 100 translocated cells. Control cells were maintained in culture for an equal period of time. RNA sequencing was performed using NextSeq mRNA Stranded Sequencing (Illumina) of two biological replicates comparing tranlocated and control cells in regard to translocation partners and cell of origin.

混合谱系白血病重排（MLL rearrangements，MLLr）在白血病发生过程中发挥关键调控作用。依据发病年龄的不同，此类重排相关疾病在发病频率、主要融合伴侣及预后层面均存在显著差异。在婴儿群体中，高达80%的急性淋巴细胞白血病（acute lymphoid leukemia，ALL）病例携带t(4;11)易位，且预后极差；与之相反，成人患者中仅10%的急性髓系白血病（acute myeloid leukemia，AML）病例携带t(9;11)易位，预后处于中等水平。目前学界对这类差异的成因尚不甚明晰。

近期，本团队基于CRISPR/Cas9技术，在源自人脐带血（human cord blood，huCB）的造血干祖细胞（hematopoietic stem and progenitor cells，HSPCs）中构建了高效的患者特异性MLLr模型，该模型可忠实模拟该疾病的核心生物学特性。本研究将该模型拓展至成人体系，采用成人骨髓（adult bone marrow，huBM）来源的HSPCs，以此探究细胞起源与融合伴侣对疾病发生发展的影响。

RNA测序（RNA-Seq）分析显示，在携带t(4;11)易位的huBM细胞中，作为表观遗传抑癌基因的FFAR2未出现表达下调，这一特征可能是MLL-AF4无法在髓系培养条件下永化成体细胞的关键原因。

整体实验设计：对源自huCB与huBM的CD34+细胞进行基因工程改造，使其携带内源性MLL-AF4或MLL-AF9易位；将构建得到的细胞培养至易位阳性细胞纯度达100%，同时设置同期培养的对照细胞。采用Illumina NextSeq mRNA链特异性测序技术，对两组生物学重复样本进行RNA测序，对比不同易位伴侣及细胞起源条件下，易位细胞与对照细胞的转录组差异。