Alpha particles combined with ATR inhibition to activate the immune system: mechanisms and pre-clinical translation

增强对肿瘤的免疫应答的一种策略是放射治疗（RT）。最近的数据支持RT- 诱导的微核（MN）本质上具有免疫刺激作用，因为破裂的MN释放双链DNA （dsDNA）引发循环GMP-AMP合酶（cGAS）和干扰素基因刺激物（STING）途径。 尽管在将免疫检查点阻断（ICB）与RT相结合方面取得了进展，但相对而言， 了解RT引发免疫刺激信号的物理机制以及它们如何被破坏， 在DNA损伤和DNA修复抑制的背景下临床上需要。高电离密度辐射 (or线性能量转移，LET）诱导更多的成簇DNA损伤，更多的MN和更高的细胞杀伤。 低LET辐射与光子和质子相比，α粒子的特征在于它们的高LET， 是产生高水平MN和下游增强免疫刺激信号激活的理想选择 通过cGAS-STING途径。最近，一种新的α粒子输送方式已经成功地被证明是可行的。 在I期临床试验中使用称为扩散α发射器放射治疗（DaRT）的方法进行了验证。DaRT 由涂有镭-224（一种α粒子发射体）的放射性粒子组成。镭-224衰变 链的独特之处在于衰变产物也会发射α粒子并扩散，从而使α粒子的剂量 沉积在离种子2-3毫米处。因此，将多个粒子植入肿瘤中可以使高LET α粒子 剂量沉积在整个肿瘤体积内。除了辐射，药物抑制DNA 修复影响MN的存在。这种对DNA修复的抑制可以通过药物来产生，如共济失调， 血管扩张和Rad 3相关（ATR）抑制剂（ATRi）。ATRi与α粒子诱导的聚集 DSB损伤可能协同增强MN的积累，最终增强免疫刺激信号。 nals。这些将与ICB一起研究，以确定如何协同增强RT诱导的抗肿瘤作用。 免疫力我们假设α粒子与ATRi结合产生更多MN，导致更多cGAS 与dsDNA结合，并因此增强稳健的抗肿瘤免疫力。我们建议：1）阐明机械- cGAS与dsDNA结合的机制; 2）阐明cGAS与dsDNA结合的机制， β-颗粒+ATRi诱导免疫信号传导;和3）在体内评价来自β-颗粒+ATRi的抗肿瘤免疫。 我们的研究有可能将抗肿瘤颗粒定义为增强抗肿瘤免疫反应的工具， 对ICB无反应的肿瘤我们提出的研究对解决 与免疫冷的多种晚期实体癌相关的不良预后。我们提出的 这项工作是创新性的，因为它的目的是确定在肿瘤中的γ-颗粒诱导的成簇DNA损伤的影响。 抗肿瘤免疫的背景。我们的发现将阐明免疫调节背后的机制， 高LET辐射，这可能最终指导使用高LET辐射的方式的合理组合使用， 放射性药物（包括α-放射性药物），DNA修复抑制剂和ICB用于侵袭性癌症。

One strategy to enhance the immune response to tumors is radiotherapy (RT). Recent data support that RT- induced micronuclei (MN) are intrinsically immunostimulatory, as ruptured MN releases double stranded DNA (dsDNA) eliciting the cycling GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) pathway. Although progress has been made in combining immune checkpoint blockade (ICB) with RT, relatively little is known about the physical mechanisms of RT that elicit immunostimulatory signals and how they can be har- nessed clinically in the context of DNA damage and DNA repair inhibition. Radiation with high ionization density (or linear energy transfer, LET) induces more clustered DNA lesions, more MN and higher cell kill compared to low-LET radiation. α-particles are characterized by their high-LET in contrast to photons and protons and may be ideal for creating high levels of MN and downstream enhanced activation of immunostimulatory signals through the cGAS-STING pathway. A novel modality to deliver α-particles has recently been successfully demon- strated in a phase I clinical trial using a method called diffusing alpha-emitters radiation therapy (DaRT). DaRT consists of interstitial radioactive seeds coated with radium-224, an α-particle emitter. The radium-224 decay chain is unique in that the decay products also emit α-particles and diffuse, allowing the α-particles’ dose to be deposited over 2-3 mm from the seed. Thus, multiple seeds implanted into a tumor allow the high-LET α-particle dose to be deposited within the entire tumor volume. In addition to radiation, pharmacologic inhibition of DNA repair affects the presence of MN. This inhibition of DNA repair can be created with drugs such as Ataxia telan- giectasia and Rad3 related (ATR) inhibitors (ATRi). The combination of an ATRi with α-particle-induced clustered DSB lesions may synergistically enhance the accumulation of MN, ultimately enhancing immunostimulatory sig- nals. These will be investigated with ICB to determine how to synergistically augment RT-induced antitumor immunity. We hypothesize that α-particles combined with an ATRi produces more MN, results in more cGAS binding to dsDNA and consequently potentiate robust antitumor immunity. We propose to: 1) Elucidate the mech- anisms by which cGAS binds to dsDNA in -particles+ATRi treated cells; 2) Elucidate the mechanisms by which -particles+ATRi induces immune signaling; and 3) Evaluate antitumor immunity from -particles+ATRi in vivo. Our research has the potential to define -particles as a tool to augment antitumor immune response, especially for tumors that are known to be unresponsive to ICB. Our proposed research is of critical relevance to address the poor prognosis associated with multiple advanced solid cancers that are immunologically cold. Our proposed work is innovative, in that it aims to define the effects of -particle-induced clustered DNA damage on tumors in the context of antitumor immunity. Our findings will elucidate the mechanisms behind immune modulation by high-LET radiation, which may ultimately guide the combined rational use of modalities that use high-LET radi- ation (including α-emitting radiopharmaceuticals), DNA repair inhibitors and ICB for aggressive cancers.