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（例如宫颈癌、睾丸癌）的癌症和脑或骨恶性肿瘤。 然而，对正常骨髓（BM）造血干细胞和祖细胞（HSPC）的不利影响 是放射治疗的主要局限。我们还面临着日益严重的恐怖主义或军事威胁， 使用放射性/核武器。尽管有大量的调查和需求，FDA还是没有治疗方法- 批准用于广泛的临床或紧急造血放射防护。鞘氨醇1-磷酸（S1 P）是一种 在血液中以高nM至低µM浓度发现的简单磷脂，并通过特异性G蛋白发出信号 偶联受体最好的特征是S1 P β，这是FDA批准的多发性硬化症药物的靶点， FTY 720（Gilenya）。我们最近发现，S1 P可以通过BM淋巴细胞祖细胞上的S1 P β 1发出信号， 调节它们的增殖，但在其他HSPC群体中，在稳态或稳态下，S1 P信号转导的作用 压力条件未知。初步数据：使用一种新的小鼠模型，报告S1 P 激活作为核GFP表达，我们发现暴露于单剂量750 rad gIR导致 增加HSPC S1 P β 1表达和信号传导。诱导型遗传性S1 P过表达小鼠模型的建立 （S1 P1 Tg）产生与≤ 900 rad gIR诱导的BM失败表型相似的BM失败表型。临床用药 IR前2小时的FTY 720相关剂量可保护BM衰竭和随后的致死性， 基因S1 P1 KO。基于这些数据，我们假设S1 P1表达和信号传导的增加是由于 驱动gIR诱导的细胞毒性和细胞凋亡的HSPC内在机制。DNA损伤， 线粒体功能障碍和活性氧是gIR的主要后果， 死亡，但S1 P β信号传导如何在这些效应途径中发挥作用尚不清楚。在以下具体 目的是，我们将利用这种新的GPCR信号转导体内模型和诱导型S1 P1 Tg和S1 P1 KO模型， 离体造血和体内设置，以严格检查的概念，S1 P-S1 P1信号轴是 控制HSPC对γ-IR的凋亡反应的主要途径：1）确定 gIR诱导造血S1 P β 1表达、信号传导和随后的细胞凋亡; 2）确定GIR诱导造血S1 P β 1表达、信号传导和随后的细胞凋亡。 增加的S1 P β 2信号传导导致HSPC死亡和BM衰竭的机制; 3）确定S1 P β 2信号传导是否增加， S1 P拮抗剂的辐射保护作用导致长期存活率增加，如果同时存在 白血病发病率增加。这些研究很可能通过扩大我们的研究范围而产生巨大的整体影响。 理解HSPC的基本凋亡机制和对γIR的反应，并表征HSPC的凋亡机制。 一种新的，治疗上易于处理的信号通路，作为预防辐射的潜在靶点， 诱发血液毒性。

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.