Loss of JAK1 Function Causes G2/M Cell Cycle Defects Vulnerable to KIF18A Inhibition [Cal27 CRISPR Screen]

Therapeutic resistance to DNA damage is a significant challenge in oncology. To gain insight into biological mechanisms that cause DNA damage resistance and to inform strategies for achieving synergy with therapeutic radiation, we performed parallel pooled genetic CRISPR-Cas9 screening for survival in high-risk head and neck squamous cell carcinoma (HNSCC) subtypes. Surprisingly, in addition to known mediators of radiotherapy response, including ATM, DNAPK, and NF-?B signaling, loss of JAK1 was identified as a driver of tumor cell radioresistance. Knockout of JAK1 in HNSCC increased cell survival by enhancing the DNA damage-dependent G2/M cell cycle arrest and delaying progression to radiation-induced mitotic catastrophe. In line with this finding, both JAK1 knockout and kinase inhibition with abrocitinib prevented subsequent formation of radiation-induced micronuclei. Loss of JAK1 function did not affect canonical CDK1 signaling but instead reduced activation of PLK1 and AURKA, two kinases with auxiliary roles in the regulation of G2 and M phase progression. Correspondingly, using both EdU labelling and live cell imaging techniques, JAK1 loss was found to cause prolonged metaphase, mitotic slippage, and progression to tetraploidy. Targeting the mitotic kinesin KIF18A with the small molecule sovilnesib exacerbated mitotic stress and enhanced the efficacy of radiation. These studies establish KIF18A inhibition as a strategy to counteract the protective G2/M cell cycle arrest induced by DNA damage and to thus enhance tumor cell sensitivity to radiation therapy. Overall design: We performed a kinome-focused (Brunello gRNA library) CRISPR screen on the Cal27 HNSCC cell line with radiation as a selection pressure. Three seperate experimental replicates of the Cal27 cells were either irradiated or treated with a control regimen. Sequencing was then performed to determine gRNAs that were enriched or depeletd in the irradiated samples compared to unirradiated samples.

DNA损伤治疗抗性是肿瘤学领域面临的重大挑战。为阐明DNA损伤治疗抗性的生物学机制，并为制定放疗协同治疗策略提供依据，我们针对高风险头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）亚型的细胞存活情况，开展了平行池化遗传CRISPR-Cas9筛选。令人意外的是，除了已知的放疗响应介导因子（如ATM、DNAPK以及核因子κB（NF-κB）信号通路）外，Janus激酶1（JAK1）基因缺失被鉴定为肿瘤细胞辐射抗性的驱动因素。HNSCC细胞中JAK1基因敲除可通过增强DNA损伤依赖性G2/M细胞周期阻滞，延缓辐射诱导的有丝分裂灾难进程，进而提升细胞存活率。与上述发现一致，JAK1基因敲除以及使用阿布昔替尼（abrocitinib）进行激酶抑制，均能阻断辐射诱导的微核形成。JAK1功能缺失并不会影响经典的细胞周期蛋白依赖性激酶1（CDK1）信号通路，反而会降低Polo样激酶1（PLK1）与Aurora激酶A（AURKA）的激活水平——这两种激酶在G2期与M期的进程调控中发挥辅助作用。相应地，通过EdU（5-乙炔基-2'-脱氧尿苷）标记与活细胞成像技术，研究发现JAK1缺失会导致细胞中期延长、有丝分裂滑移，并最终进展为四倍体。使用小分子抑制剂索维奈塞布（sovilnesib）靶向有丝分裂驱动蛋白KIF18A，可加剧有丝分裂应激并增强放疗疗效。本研究确立了KIF18A抑制策略，可抵消DNA损伤诱导的保护性G2/M细胞周期阻滞，从而提高肿瘤细胞对放疗的敏感性。整体实验设计：我们以辐射作为筛选压力，对Cal27 HNSCC细胞系开展激酶组靶向CRISPR筛选（采用Brunello向导RNA（gRNA）文库）。将3组独立生物学重复的Cal27细胞分别接受辐射处理或对照处理。随后通过测序，对比未辐射样本与辐射样本，鉴定出在辐射样本中富集或缺失的gRNA。