CRM1 inhibitor anti-tumor activity is enhanced with salicylates by S-phase arrest and impaired DNA-damage repair. CRM1 inhibitor anti-tumor activity is enhanced with salicylates by S-phase arrest and impaired DNA-damage repair

Chromosome region maintenance protein1 (CRM1) mediates protein export from the nucleus and is a new target for anti-cancer therapeutics. Broader application of KPT-330 (Selinexor), a CRM1 inhibitor approved for myeloma, has been limited by toxicity. We found that combining choline salicylate (CS) with low-doses of KPT-330 (K+CS) had potent synergistic activity across blood and solid organ cancers ex-vivo and in-vivo. Mechanistically, K+CS enhances CRM1 degradation, induces potent inhibition of CRM1 mediated nuclear export, and arrests cells in S-phase with simultaneous reduction of Rad51 causing impaired DNA damage repair. K+CS represents a potential new class of therapy for multiple cancer types. Overall design: JEKO-1 cells were treated with the drug conditions KPT-330, choline salicylate(CS), KPT-330+CS (K+CS) or DMSO control for 48 hours. RNA was extracted using the AllPrep DNA/RNA FFPE kit (Qiagen, Germany). Sequencing libraries were prepared using TruSeq RNA Library Prep kit V2 (Illumina, San Diego, CA) and analyzed on a HiSeq 4000 sequencer (Illumina, San Diego, CA). Sequenced reads were aligned to the hg38 reference using the previously published MAP-RSeq pipeline slightly modified to use the STAR aligner. Gene-level read counts based on Ensembl version 78 were analyzed using edgeR to find differentially expressed proteins. For this, gene counts were normalized using TMM method to remove batch effects. Normalized read counts were compared across experimental groups using a negative binomial generalized log-linear model. A total of four group comparisons were performed: KPT-330 vs. control, CS vs. control, K+CS vs. control and CS vs. KPT-300. For each comparison, genes with an adjusted p-value (Benjamini-Hochberg) ≤ 0.05 and an absolute log2 (fold change)>2.0 were considered as significantly differentially expressed and saved for further analysis.

染色体区域维持蛋白1（Chromosome region maintenance protein1, CRM1）可介导蛋白质的核输出，是抗肿瘤治疗的新型靶点。已获批用于多发性骨髓瘤治疗的CRM1抑制剂KPT-330（塞利尼索，Selinexor），其临床应用范围因毒性反应而受到限制。本研究发现，将水杨酸胆碱（CS，choline salicylate）与低剂量KPT-330联合使用（下称K+CS），在体外及体内实验中对血液系统肿瘤与实体脏器肿瘤均展现出强效的协同抗肿瘤活性。机制层面，K+CS可促进CRM1降解，强效抑制CRM1介导的核输出过程，使细胞阻滞于S期，同时下调Rad51的表达，进而导致DNA损伤修复功能受损。K+CS有望成为针对多种癌症类型的新型治疗方案。整体实验设计：将JEKO-1细胞分别以KPT-330、水杨酸胆碱（CS）、KPT-330联合CS（K+CS）及二甲基亚砜（DMSO）对照处理48小时。采用德国凯杰（Qiagen）公司的AllPrep DNA/RNA FFPE试剂盒提取RNA。使用美国加利福尼亚州圣地亚哥Illumina公司的TruSeq RNA文库制备试剂盒V2构建测序文库，并在HiSeq 4000测序仪（Illumina，美国加州圣地亚哥）上完成测序。通过经轻微修改、采用STAR比对器的已发表MAP-RSeq流程，将测序读段比对至hg38参考基因组。基于Ensembl 78版本的基因水平读段计数，采用edgeR软件分析差异表达蛋白。该分析中，采用TMM标准化方法对基因计数进行标准化以消除批次效应。使用负二项广义对数线性模型对各组间的标准化读段计数进行比较分析。共开展4组比较：KPT-330 vs. 对照组、CS vs. 对照组、K+CS vs. 对照组以及CS vs. KPT-300。对于每一组比较，校正后P值（本雅明尼-霍赫贝格法）≤0.05且绝对log₂(倍数变化)>2.0的基因被视为显著差异表达基因，用于后续分析。