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 first in class CRM1 inhibitor recently approved for multiple myeloma and diffuse large B-cell lymphoma (DLBCL), has been limited by substantial adverse effects (AEs). To address this clinical problem, we focused on identifying novel strategies to boost the potency, reduce toxicity, and broaden the applicability of CRM1 inhibitors to a wider range of malignancies. We discovered that salicylates could markedly enhance the anti-tumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. KPT-330 was chosen as the prototypical CRM1 inhibitor given its current FDA approval status and characterized pharmacokinetics; and choline salicylate (CS) was chosen as the prototypical salicylate given its favorable pharmacokinetics and reduced antiplatelet, renal, neurological and gastrointestinal AEs in humans compared to other salicylates. By using cell lines belonging to different hematologic malignancies and solid tumors, we demonstrated ex vivo that the combination of KPT-330 and CS (K+CS) could induce unique and significant antitumor effect at much lower dose of KPT-330 (at 25% of the dose used in the clinic), thereby potentially mitigating prohibitive clinical AEs (Figure 1a-d, and e-g). This significant synergetic antitumor effect observed with K+CS ex vivo was also validated in vivo by using an NSG mouse model of mantle cell lymphoma (Figure 1h). Moreover, the K+CS combination did not show this potent toxic effect on non-malignant cells in vivo and was safe without inducing toxicity to normal organs in NSG mice. Mechanistically, protein profiling through mass spectroscopy revealed that K+CS uniquely affects the cellular pathways of DNA damage repair, DNA synthesis and cell cycle progression. Studies involving immunoblotting, cell cycle analysis, immunofluorescence microscopy assessing nucleocytoplasmic molecular export and DNA damage, reporter assay to assess homologous recombination repair proficiency and immunohistochemistry showed that, compared to KPT-330 treatment alone, K+CS decreased the expression of CRM1, Rad51 and thymidylate synthase proteins in vitro and in vivo, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, and importantly a unique cell cycle arrest in S-phase, thus leading to cell apoptosis. These effects on cellular proteins and pathways were unique to K+CS treatment and were not observed with KPT-330 or CS single agent treatment. Pathway analyses through RNA sequencing also paralleled the findings of proteomic studies thus further validating the unique effect of K+CS treatment on the aforementioned cellular pathways. Importantly, K+CS treatment exerted unique and significant antitumor effect ex vivo on primary malignant cells obtained from patient's with high-risk hematologic malignancies such as double hit DLBCL, BTK and BCL2 inhibitor resistant mantle cell lymphoma, TP53 deleted/mutated chronic lymphocytic leukemia and high-risk multiple myeloma thus signifying its broader applicability as a treatment option for these aggressive hematologic malignancies where there is a dire need to find new treatment strategies (Figure 1i). In summary, we describe a unique all-oral drug combination with a novel constellation of mechanisms of action on cellular pathways that are exploited by cancer cells. K+CS represents a new class of therapy for multiple cancer types and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for therapy of blood cancers. Disclosures Witzig: Karyopharm Therapeutics: Research Funding; Acerta: Research Funding; Incyte: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Research Funding; MorphSys: Consultancy; Immune Design: Research Funding; Spectrum: Consultancy.

中文翻译：

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水杨酸盐通过 S 期阻滞和受损的 DNA 损伤修复增强和扩大 CRM1 抑制剂的抗肿瘤活性

染色体区域维持蛋白1 (CRM1) 介导细胞核蛋白输出，是抗癌治疗的新靶点。KPT-330（selinexor）是最近获批用于治疗多发性骨髓瘤和弥漫性大 B 细胞淋巴瘤（DLBCL）的首个 CRM1 抑制剂，其更广泛的应用受到严重不良反应（AE）的限制。为了解决这一临床问题，我们专注于确定新的策略来提高效力、降低毒性并扩大 CRM1 抑制剂对更广泛的恶性肿瘤的适用性。我们发现水杨酸盐可以通过将作用机制扩展到 CRM1 抑制之外，从而显着增强 CRM1 抑制剂的抗肿瘤活性。鉴于 KPT-330 目前的 FDA 批准状态和特征药代动力学，KPT-330 被选为原型 CRM1 抑制剂；与其他水杨酸盐相比，胆碱水杨酸盐 (CS) 被选为原型水杨酸盐，因为它具有良好的药代动力学，并能减少人体中的抗血小板、肾脏、神经和胃肠道 AE。通过使用属于不同血液系统恶性肿瘤和实体瘤的细胞系，我们在体外证明了 KPT-330 和 CS (K+CS) 的组合可以在低得多的 KPT-330 剂量（25%）下诱导独特而显着的抗肿瘤作用临床使用的剂量），从而可能减轻禁止性临床 AE（图 1a-d 和例如）。使用 K+CS 离体观察到的这种显着的协同抗肿瘤作用也通过使用套细胞淋巴瘤的 NSG 小鼠模型在体内得到验证（图 1h）。而且，K+CS 组合在体内对非恶性细胞没有显示出这种有效的毒性作用，并且是安全的，不会对 NSG 小鼠的正常器官产生毒性。从机制上讲，通过质谱进行的蛋白质分析表明，K+CS 独特地影响 DNA 损伤修复、DNA 合成和细胞周期进程的细胞途径。涉及免疫印迹、细胞周期分析、评估核质分子输出和 DNA 损伤的免疫荧光显微镜检查、评估同源重组修复能力的报告基因分析和免疫组织化学的研究表明，与单独使用 KPT-330 治疗相比，K+CS 降低了 CRM1、Rad51 和胸苷酸合成酶蛋白在体外和体内，导致更有效地抑制 CRM1 介导的核输出，损害 DNA 损伤修复，嘧啶合成减少，重要的是独特的细胞周期停滞在 S 期，从而导致细胞凋亡。这些对细胞蛋白和通路的影响是 K+CS 治疗所独有的，在 KPT-330 或 CS 单药治疗中未观察到。通过 RNA 测序进行的通路分析也与蛋白质组学研究的结果相一致，从而进一步验证了 K+CS 治疗对上述细胞通路的独特影响。重要的是，K+CS 治疗对从患有高危血液系统恶性肿瘤（如双重打击 DLBCL、BTK 和 BCL2 抑制剂耐药套细胞淋巴瘤）的患者获得的原发性恶性细胞离体发挥独特而显着的抗肿瘤作用，TP53 缺失/突变的慢性淋巴细胞白血病和高危多发性骨髓瘤，因此表明其作为这些侵袭性血液系统恶性肿瘤的治疗选择具有更广泛的适用性，其中迫切需要寻找新的治疗策略（图 1i）。总之，我们描述了一种独特的全口服药物组合，具有对癌细胞利用的细胞途径的新型作用机制。K+CS 代表了一种针对多种癌症类型的新疗法，并将激发未来的研究，以利用 DNA 损伤修复和核质转运来治疗血癌。Witzig：Karyopharm Therapeutics：研究经费；Acerta：研究经费；Incyte：咨询；艾伯维：咨询；新基（Celgene）：咨询，研究经费；MorphSys：咨询；免疫设计：研究经费；频谱：咨询。