Hematopoietic stem and progenitor cells in experimental remission model of acute myeloid leukemia in vivo [ATAC-seq]

Inflammation is a hallmark of cancer and is linked to patient prognosis in acute myeloid leukemia (AML). Our recent study revealed that hematopoietic stem and progenitor cells (HSPCs) exhibit an inflammatory active state and serve as a cellular source of inflammatory cytokine secretion in AML. Recent studies in infection and chronic inflammation related disease models have revealed that inflammation can reprogram HSPCs to fuel chronic inflammation related co-mobidities. Building on our report of AML-associated inflammation in HSPCs, here we ask whether AML-experienced normal HSPCs are reprogrammed in AML. To investigate this, we utilized a chimeric model of AML, carrying both doxycycline-inducible leukemic and healthy wildtype hematopoietic fractions, to generate an AML remission model. To test whether AML-experienced HSPCs harbor inflammatory memory, mice were challenged with heterogenous inflammatory activation. Our transcriptomic and epigenetic results show for the first time that phenotypically normal, AML-experienced HSPCs carry a durable inflammatory memory whereby primary exposure to AML secreted factors alters their response to secondary stimuli. Our finding suggests that AML-experienced HSPCs are innate immune reprogrammed in the AML niche. AML remission model was generated by first co-transplanting non-induced whole bone marrow (WBM) from inducible MLL-AF9 (iMLL-AF9) mice (CD45.1; homozygous hMLL-AF9 and rtTA; 2E5 cells per recipient) together with WBM from healthy wildtype mice (double positive CD45.1+/2+; 8E5 cells per recipient), into lethally irradiated C57BL/6J recipients (CD45.2) and is termed "AF9+/+" mice. Control naive mice were generated by co-transplanting hMLL-AF9-null (CD45.1; hMLL-AF9-null, homozygous rtTA only; 2E5 cells per recipient) together with healthy wildtype mice (double positive CD45.1+/2+; 8E5 cells per recipient) and is termed "AF9-/-" mice. After 6-8 weeks after transplantation, AML remission state was generated by first subjecting mice to leukemogenesis (via doxycycline induction) for 5 weeks, followed by disease regression (via doxycycline withdraw) for 4 weeks. Mice were administered with either PBS or LPS to model baseline remission and inflammatory-challenged remission state. Mice were sacrificed 24 hours after the injection. Bone marrow hematopoietic stem and progenitor cells (HSPCs; CD45.1+/2+, Lin- cKit+ Sca1+) were fluorescent-activated cell sorting (FACS)-sorted for RNA-Seq and ATAC-Seq.

炎症是癌症的标志性特征，且与急性髓系白血病（acute myeloid leukemia, AML）患者的预后密切相关。我们的近期研究表明，在AML中，造血干祖细胞（hematopoietic stem and progenitor cells, HSPCs）呈现炎症活化状态，并可作为炎症细胞因子分泌的细胞来源。针对感染及慢性炎症相关疾病模型的既往研究已证实，炎症可对HSPCs进行重编程，进而加剧慢性炎症相关共病的发生发展。基于我们关于AML中HSPCs相关炎症的研究报道，本研究旨在探究经AML暴露的正常HSPCs是否会在AML微环境中发生重编程。

为探究这一科学问题，我们构建了携带多西环素（doxycycline）诱导性白血病组分与健康野生型造血组分的AML嵌合模型，以此制备AML缓解模型。为验证经AML暴露的HSPCs是否携带炎症记忆，我们通过异质性炎症激活对小鼠进行造模处理。我们的转录组学与表观遗传学研究结果首次证实，表型正常的经AML暴露的HSPCs具有持久性炎症记忆：初次接触AML分泌的细胞因子可改变其对二次刺激的应答反应。本研究结果提示，经AML暴露的HSPCs在AML微环境中发生了先天免疫重编程。

AML缓解模型的构建流程如下：首先将诱导型MLL-AF9（inducible MLL-AF9, iMLL-AF9）小鼠（CD45.1；纯合子hMLL-AF9与rtTA；每只受体鼠输注2×10^5个细胞）的未诱导全骨髓（whole bone marrow, WBM）与健康野生型小鼠的全骨髓（双阳性CD45.1+/2+；每只受体鼠输注8×10^5个细胞）共同移植至经致死剂量辐照的C57BL/6J受体鼠（CD45.2）体内，该模型命名为"AF9+/+"小鼠。对照野生型小鼠的构建方式为：将hMLL-AF9缺失型小鼠（CD45.1；仅携带纯合子rtTA；每只受体鼠输注2×10^5个细胞）的全骨髓与健康野生型小鼠的全骨髓（双阳性CD45.1+/2+；每只受体鼠输注8×10^5个细胞）共同移植，该模型命名为"AF9-/-"小鼠。

移植后6-8周，通过以下流程构建AML缓解状态：先给予小鼠多西环素诱导白血病发生，持续5周；随后停用多西环素促使疾病消退，持续4周。分别向小鼠注射磷酸盐缓冲液（phosphate-buffered saline, PBS）与脂多糖（lipopolysaccharide, LPS），以模拟基线缓解状态与炎症刺激后的缓解状态。于注射后24小时处死小鼠。我们通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）从骨髓中分离出造血干祖细胞（HSPCs；CD45.1+/2+, Lin- cKit+ Sca1+），用于RNA测序（RNA-Seq）与转座酶可及性染色质测序（ATAC-Seq）。