Mechanisms of hematopoietic acute radiation syndrome induction and radioprotection through sphingosine 1-phosphate receptor 1 signal modulation

意义：电离辐射(IR)是一种经常用于各种癌症的治疗方式， 包括白血病、骨盆区域癌症(如宫颈、睾丸)以及脑或骨恶性肿瘤。 然而，对正常骨髓(BM)造血干细胞和祖细胞(HSPC)的不利影响 是放射治疗的一个主要限制。我们还面临着越来越多的恐怖主义或军事威胁。 放射性/核武器的使用。尽管进行了密集的调查和需求，但FDA没有治疗方法- 被批准用于广泛的临床或紧急造血放射防护。1-磷酸鞘氨醇(S1P)是一种 从高NM到低微米血液中发现的简单磷脂和通过特定G蛋白发出的信号 偶联受体。最具代表性的是S1P₁，它是FDA批准的多发性硬化症药物的靶点， FTY720(吉伦亚)。我们最近发现，S1P可以通过BM淋巴细胞前体细胞上的S1P₁1信号转导 调节其增殖，但S1P₁信号在其他HSPC群体中的作用，处于动态平衡或低于 压力条件，是未知的。初步数据：使用一种报告S1P₁的新型小鼠模型 作为核GFP表达的激活，我们发现单次暴露于750rad Gir会导致 HSPC S1P₁表达增加，信号转导增加。一种可诱导遗传性S1P₁过度表达的小鼠模型 (S1P1Tg)产生与≤900rad Gir相似的BM失效表型。临床给药 相关剂量的FTY720仅在IR前2小时即可保护BM故障和随后的致命性， 遗传性S1P1KO。基于这些数据，我们假设增加的S1P1表达和信号是 HSPC的内在机制驱动GIR诱导的细胞毒性和细胞凋亡。DNA损伤， 线粒体功能障碍和活性氧是GIR触发细胞的主要后果 死亡，但S1P₁信号如何在这些效应器通路中发挥作用尚不清楚。在以下具体内容中 目的，我们将利用这一新的体内GPCR信号模型和可诱导的S1P1Tg和S1P1KO模型在 体外造血和体内设置，以批判性地检查S1P-S1P1信号轴是 调控这些HSPC对γ-IR的凋亡反应的一个主要途径：1)决定了 GIR诱导造血细胞S1P₁1表达、信号转导和随后的细胞凋亡；2)确定 S1P₁信号增强导致HSPC死亡和BM失败的机制；3)确定 S1P₁拮抗剂的辐射防护作用可增加长期存活率，如果符合 白血病发病率的增加。这些研究可能会通过扩大我们的 了解HSPC凋亡的基本机制和对γIR的反应，并表征 新的，治疗上易处理的信号通路作为预防辐射的潜在靶点- 诱导的血液毒性。

Significance: Ionizing radiation (IR) is a frequently utilized treatment modality for a variety of cancers, including leukemias, cancers in the pelvic region (e.g. cervical, testicular), and brain or bone malignancies. However, detrimental effects on normal bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) are a major limitation of radiotherapy. We are also faced with the increasing threat of terrorist or military utilization of radioactive/nuclear weapons. Despite intense investigation and need, there is no therapy FDA- approved for broad clinical or emergency hematopoietic radioprotection. Sphingosine 1-phosphate (S1P) is a simple phospholipid found in high nM to low µM concentrations in blood and signals via specific G-protein coupled receptors. The best characterized is S1P₁, the target of an FDA-approved multiple sclerosis drug, FTY720 (Gilenya). We have recently shown that S1P can signal via S1P₁1 on BM lymphocyte progenitors to regulate their proliferation, but roles for S1P₁ signaling in other HSPC populations, at homeostasis or under stress conditions, are unknown. Preliminary data: With the use of a novel mouse model that reports S1P₁ activation as nuclear GFP expression, we found that exposure to a single dose of 750 rad gIR results in increased HSPC S1P₁ expression and signaling. A mouse model of inducible genetic S1P₁ over-expression (S1P1Tg) yields a BM failure phenotype similar to that induced with ≤ 900 rad gIR. Administration of clinically relevant doses of FTY720 as little as 2h pre-IR protects from BM failure and subsequent lethality, as does genetic S1P1KO. Based on these data, we hypothesize that increased S1P1 expression and signaling are an HSPC-intrinsic mechanism driving gIR-induced cytotoxicity and apoptosis. DNA damage, mitochondrial dysfunction, and reactive oxygen species are the primary consequences of gIR that trigger cell death, but how S1P₁ signaling plays a role in these effector pathways is unknown. In the following Specific Aims, we will utilize this novel in vivo model of GPCR signaling and models of inducible S1P1Tg and S1P1KO in ex vivo hematopoiesis and in vivo settings to critically examine the concept that the S1P-S1P1 signaling axis is a major pathway governing these HSPC apoptotic responses to γ-IR: 1) Determine the mechanisms by which gIR induces hematopoietic S1P₁1 expression, signaling, and subsequent apoptosis; 2) Determine the mechanism whereby increased S1P₁ signaling leads to HSPC death and BM failure; 3) Determine if the radioprotective effect of S1P₁ antagonism leads to increased long-term survival and if there is a coincident increase in leukemia incidence. These studies are likely to have a large overall impact by expanding our understanding of fundamental HSPC apoptotic mechanisms and responses to γIR, and characterizing a novel, therapeutically tractable signaling pathway as a potential target for prevention of radiation- induced hematotoxicity.