The cell-nonautonomous function of ID1 supports AML progression from the microenvironment via ANGPLT7

Bone marrow microenvironment (BMM) can maintain and regulate HSC and LSC via secreted components or direct interaction. Increasing evidence indicates that dissecting and targeting BMM-driven mechanisms of LSC might provide key adjuvant therapies to eradicate leukemia. ID1, a key transcriptional regulator in LSC previously identified by us, controls cytokine/chemokine production in the BMM, but the role of ID1 in AML-BMM remains obscure. Here, we report that ID1 is highly expressed in AML-BMM, especially in bone marrow mesenchymal stem cell (BMSC), and deletion of Id1 in BMM resulted in impaired AML progression in AML mouse model. Mechanistically, we found that Id1 deficiency markedly reduced the protein level of SP1 in mesenchymal cells co-cultured with AML cells. Through Id1-interactome analysis, we discovered Id1 interacts with Rnf4, the ubiquitin E3 ligase, and led to decreased Sp1 degradation. Disrupting ID1-RNF4 interaction by truncates in mesenchymal cells significantly decreased SP1 protein level and retarded the AML cell proliferation. We identified the target of Sp1, Angptl7, was the primary differentially expression protein factor in Id1-/- bone marrow supernatant fluid (BMSF) to regulate AML progression in mice. Taken together, our study highlights the critical role of ID1 in AML-BMM and assists development of diagnostic/therapeutic strategies for AML. Overall design: To evaluate the non-cell-autonomous function of Id1 on LICs in vivo, we employed an AML1-ETO9a(AE9a)/MLL-AF9 driven AML mouse transplantation model. After 6 weeks transplantation, Id1+/+ and Id1-/- mouse BMSC were harvested and cultured in a-MEM supplemented with 10% FBS. After the adherent MSCs are seen under the microscope, the total RNA is extracted. RNA-seq library prepared and sequenced by novogene, Beijing, China.

骨髓微环境（Bone marrow microenvironment, BMM）可通过分泌组分或直接相互作用维持并调控造血干细胞（Hematopoietic Stem Cell, HSC）与白血病干细胞（Leukemia Stem Cell, LSC）。越来越多的研究证据表明，解析并靶向BMM驱动的LSC调控机制，可能为根除白血病提供关键的辅助治疗策略。本团队此前已鉴定出LSC中的关键转录调控因子分化抑制因子1（Inhibitor of Differentiation 1, ID1），其可调控BMM内细胞因子/趋化因子的产生，但ID1在急性髓系白血病相关骨髓微环境（Acute Myeloid Leukemia-associated Bone Marrow Microenvironment, AML-BMM）中的作用仍不明确。

本研究发现，ID1在AML-BMM中高表达，尤其在骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cell, BMSC）内；在BMM中敲除Id1可显著削弱急性髓系白血病小鼠模型中的AML进展。机制层面研究显示，与AML细胞共培养的间充质细胞中，Id1缺失会显著降低特异性蛋白1（Specificity Protein 1, SP1）的蛋白水平。通过Id1互作组分析，本团队发现Id1可与泛素连接酶E3环指蛋白4（Ring Finger Protein 4, RNF4）相互作用，进而减少SP1的降解。在间充质细胞中通过截短体破坏ID1-RNF4的相互作用，可显著降低SP1的蛋白水平并阻滞AML细胞增殖。本研究还鉴定出SP1的靶基因血管生成素样蛋白7（Angiopoietin-like 7, Angptl7）是Id1敲除小鼠骨髓上清液（Bone Marrow Supernatant Fluid, BMSF）中调控小鼠AML进展的主要差异表达蛋白因子。

综上，本研究阐明了ID1在AML-BMM中的关键调控作用，可为急性髓系白血病的诊断与治疗策略开发提供参考。

整体实验设计：为体内评估Id1对白血病起始细胞（Leukemia Initiating Cell, LIC）的非细胞自主功能，本研究采用了AML1-ETO9a(AE9a)/MLL-AF9驱动的AML小鼠移植模型。移植6周后，收集Id1野生型（Id1+/+）与Id1敲除型（Id1-/-）小鼠的BMSC，并用添加10%胎牛血清（Fetal Bovine Serum, FBS）的α-最低必需培养基（α-Minimum Essential Medium, α-MEM）进行培养。待显微镜下观察到贴壁间充质干细胞后，提取总RNA。构建RNA测序文库并由中国北京诺禾致源（Novogene）完成测序。