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

增强肿瘤免疫反应的一种策略是放射治疗(RT)。最近的数据支持RT- 诱导微核(MN)本质上是免疫刺激的，因为破裂的MN释放双链DNA (DsDNA)触发环状GMP-AMP合成酶(CGAS)和干扰素基因刺激物(STING)途径。 虽然在结合免疫检查点阻断(ICB)和RT方面取得了进展，但相对较少 已知RT诱导免疫刺激信号的物理机制以及它们如何被抑制 在DNA损伤和DNA修复抑制的情况下出现临床症状。高电离密度辐射 (或线性能量转移，LET)导致更多簇状DNA损伤，更多MN和更高的细胞杀伤率 低LET辐射。与光子和质子相比，α粒子的特征是它们的高LET，并且可以 是产生高水平的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。一种新的传递α粒子的方式最近成功地被证明是-- 在I期临床试验中使用了一种名为扩散阿尔法发射体放射疗法(DART)的方法。飞镖 由被α粒子发射体--Re-224包裹的间隙放射性种子组成。~(224)Re衰变 链条的独特之处在于，衰变产物也会释放α粒子并扩散，允许α粒子的剂量 离种子超过2-3毫米的地方。因此，将多个种子植入肿瘤中可以产生高LET的α粒子 在整个肿瘤体积内沉积的剂量。除了辐射，药物对DNA的抑制作用 修复影响MN的存在。这种对DNA修复的抑制可以通过药物产生，如共济失调Telan- 关节扩张和RAD3相关(ATR)抑制物(ATRI)。ATRI与α粒子诱导聚簇的结合 DSB损伤可协同增强MN的蓄积，最终增强免疫刺激信号。 纳尔。这些都将通过ICB进行研究，以确定如何协同增强RT诱导的抗肿瘤 豁免权。我们假设α粒子与ATRI相结合会产生更多的MN，导致更多的cGAs 与dsDNA结合，从而增强强大的抗肿瘤免疫力。我们建议：1)阐明机械-- 在颗粒+ATRI处理的细胞中cGAS与双链DNA结合的异常；2)阐明了cGAS与双链DNA结合的机制 -颗粒+ATRI诱导免疫信号传导；3)体内评价-颗粒+ATRI的抗肿瘤免疫作用。 我们的研究有可能将颗粒定义为增强抗肿瘤免疫反应的工具，尤其是 对于已知对ICB无效的肿瘤。我们提议的研究具有关键的相关性，以解决 预后不良与免疫寒冷的多发性晚期实体癌有关。我们的建议 这项工作是创新的，因为它的目标是定义颗粒诱导的簇状dna损伤对肿瘤的影响 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，这最终可能指导使用高LET辐射的医疗模式的组合合理使用-- 治疗(包括发射α的放射性药物)、脱氧核糖核酸修复抑制剂和治疗侵袭性癌症的ICB。

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.