Oncogenic Kras G12D activation in the non-hematopoietic bone marrow microenvironment causes myelodyplastic syndrom in mice

The bone marrow microenvironment is key player in the regulation and maintenance of hematopoiesis and is also involved in the pathogenesis of hematologic malignancies. We investigated the effect of KrasG12D expression in the non-hematopoietic bone marrow microenvironment. Therefore, we generated chimeric mice which express oncogenic Kras only in the non-hematopoietic system. Within 6-8 weeks, these mice developed a myelodysplastic syndrome characterized by cytopenia, myeloid expansion and signs of disturbed differentiation and marked dysplasia within the myeloid lineage. Gene expression analysis of KrasG12D expressing non-hematopoietic cells of the bone marrow microenvironment revealed a significant upregulation of multiple genes and pathways including proinflammatory signaling such as upregulation of the NLRP3 inflammasome and related genes. Rosa26Cre;LSL-KrasG12D mice were lethally irradiated and transplanted with bone marrow harvested from wild type mice. Single allele littermates were used as control. When the mice were fully engrafted, tamoxifen was administered to induce Cre-mediated KrasG12D expression in the non-hematopoietic tissues. Blood and bone marrow analyisis was performed 6-8 weeks after tamoxifen administration. In order to analyze gene expression changes in KrasG12D expressing bone marrow niche cells, Rosa26Cre;LSL-KrasG12D mice were treated with tamoxifen to ubiquitously induce KrasG12D expression. After 5.5 weeks, osteoblasts, mesenchymal stem and progenitor cells, endothelial cells as well as CXCL12 abundant reticular cells were isolated using fluorescence-activated cell sorting according to published cell surface marker characteristics. RNA was extracted for microarray based gene expression analysis.

骨髓微环境（bone marrow microenvironment）是调控与维持造血功能的核心参与者，同时也参与血液系统恶性肿瘤的发病机制。本研究探讨了KrasG12D在非造血性骨髓微环境中的表达效应。为此，我们构建了仅在非造血系统中表达致癌性Kras的嵌合小鼠模型。该类小鼠在造模后6~8周内便出现骨髓增生异常综合征（myelodysplastic syndrome），其特征为血细胞减少、髓系细胞扩增，同时伴有髓系谱系分化紊乱与显著发育异常表型。对骨髓微环境中表达KrasG12D的非造血细胞进行基因表达分析后发现，包括促炎信号通路在内的多个基因与通路均出现显著上调，其中NLRP3炎症小体（NLRP3 inflammasome）及其相关基因的上调尤为突出。将Rosa26Cre;LSL-KrasG12D小鼠施以致死剂量辐照后，移植野生型小鼠来源的骨髓细胞，以单等位基因同窝小鼠作为对照。待小鼠完全实现造血嵌合后，给予他莫昔芬以诱导非造血组织中Cre介导的KrasG12D表达。在他莫昔芬给药后6~8周，对小鼠血液与骨髓开展检测分析。为分析表达KrasG12D的骨髓龛细胞的基因表达变化，我们对Rosa26Cre;LSL-KrasG12D小鼠给予他莫昔芬以泛在性诱导KrasG12D的表达。造模5.5周后，依据已发表的细胞表面标志物特征，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离得到成骨细胞、间充质干细胞与祖细胞、内皮细胞以及CXCL12富集网状细胞（CXCL12 abundant reticular cells）。提取RNA后开展基于基因芯片的基因表达分析。