Clinical Translation of Nuclear Export Inhibitor in Metastatic Pancreatic Cancer

摘要：胰腺导管腺癌 (PDAC) 仍然是一种致命疾病，迫切需要更新的药物 治疗方式。 PDAC 肿瘤具有非常异质性，许多关键通路均发生改变 并需要能够激活多种肿瘤抑制蛋白 (TSP) 的广泛疗法 同时。我们已经证明，在 PDAC 中，核输出蛋白 Exportin 1 过度表达 (XPO1) 通过错误定位导致 TSP（例如 FOXO3a、p27、Par-4）功能失活。 我们是第一个证明 CRISPR/Cas9 对 XPO1 的抑制验证了选择性抑制剂 Nuclear Export (SINE) selinexor 和类似物可以恢复多种 TSP 的抗肿瘤功能，从而导致 原位模型中的 PDAC 细胞死亡和肿瘤抑制。更重要的是，我们的研究结果表明 SINE 与吉西他滨 (GEM) 和白蛋白结合型紫杉醇协同作用，增强 PDAC 生长抑制、细胞凋亡、 逆转上皮间质转化 (EMT) 和 PDAC 衍生癌症的球体崩解 干细胞（CSC）。观察到的协同作用部分归因于 TSP 的核定位增强和 抑制 CSC 和 EMT 标记。通路分析显示细胞死亡信号传导增强 抑制 CSC 维持网络。最重要的是，我们观察到成纤维细胞特异性细胞的激活 死亡途径强调了 XPO1 在维持成纤维细胞生长中尚未被探索的作用，即 公认支持 PDAC 中的高促纤维增生反应 (DR)。我们的多模型临床前工作取得了 涉及 GEM-nab-paclitaxel-selinexor 治疗转移性 PDAC 的 Ib/II 期临床研究 (NCT02178436)。在这个 我们假设 XPO1 抑制将增强 TSP 的核保留，恢复其抗- 肿瘤功能导致 GEM-nab-紫杉醇在 PDAC 中的细胞毒性功效增强。为了验证我们的 假设，我们建议在初级中整合高通量 RNA-Seq 和 SILAC-MS 方法 细胞和转基因小鼠模型以及将 selinexor 与 Gem-nab-紫杉醇联合使用的 Ib/II 期试验 PDAC 患者。我们预计我们的整体分析将首次描述 XPO1 的影响 抑制 PDAC CSC 和 DR 网络的重新编程，从而增强 GEM-nab-紫杉醇在肿瘤中的作用。在 Aim1 中，我们将证明 GEM-nab-紫杉醇和 使用高通量方法在初级 PDAC 模型中使用 selinexor。在 Aim2 中，我们将演示 GEM-nab-紫杉醇-selinexor 在 LSL-KrasG12D/ ;LSL-Trp53R172H/ ;Pdx-1-Cre 小鼠中的功效和协同作用 模型。这些研究将确定敏感性和/或耐药性标记物，以支持我们的 Ib/II 期试验 目标 3，我们打算确定 selinexor 与 GEM-nab 联合使用时的安全性和有效性 紫杉醇治疗转移性 PDAC 患者。该试验的活检将用于验证锁定 患者中 selinexor 靶点参与的药效学标志物。临床影响：成功 完成我们的研究将提出一种新的疗法来提高 PDAC 患者的生存率。

Summary: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease in urgent need of newer therapeutic modalities. PDAC tumors are very heterogeneous carrying alterations in many critical pathways and require a broad form of therapy that can activate multiple tumor suppressor proteins (TSPs) simultaneously. We have shown that in PDAC over-expression of the nuclear exporter protein Exportin 1 (XPO1) leads to functional inactivation of TSPs (such as FOXO3a, p27, Par-4) through their mislocalization. We were the first to demonstrate that inhibition of XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) selinexor and analogs can restore the anti-tumor function of multiple TSPs leading to PDAC cell death and tumor inhibition in orthotopic models. More significantly, our findings show that SINE synergize with gemcitabine (GEM) and nab-paclitaxel leading to enhanced PDAC growth inhibition, apoptosis, reversal of epithelial-to-mesenchymal transition (EMT) and spheroid disintegration of PDAC derived cancer stem cells (CSCs). The observed synergy was due in part to enhanced nuclear localization of TSPs and suppression of CSC and EMT markers. Pathway analysis showed enhancement in cell death signaling and suppression of CSC sustaining networks. Most importantly we observed activation of fibroblast specific cell death pathways highlighting an as of yet unexplored role of XPO1 in sustaining fibroblast growth that is recognized to support high desmoplastic reaction (DR) in PDAC. Our multi-model pre-clinical work has led to a Phase Ib/II clinical study involving GEM-nab-paclitaxel-selinexor for metastatic PDAC (NCT02178436). In this proposal we hypothesize that XPO1 inhibition will enforce the nuclear retention of TSPs, restore their anti- tumor function leading to enhancement of cytotoxic efficacy of GEM-nab-paclitaxel in PDAC. To validate our hypothesis, we propose to integrate high-throughput RNA-Seq and SILAC-MS methodologies in primary cellular and transgenic mice models and in a Phase Ib/II trial combining selinexor with Gem-nab-paclitaxel in PDAC patients. We anticipate that our holistic analyses will, for the first time, delineate the impact of XPO1 inhibition in re-programming of PDAC CSC and DR networks that leads to the enhancement in the efficacy of GEM-nab-paclitaxel in tumors. In Aim1 we will demonstrate in vitro synergy between GEM-nab-paclitaxel and selinexor in primary PDAC models using high-throughput methodologies. In Aim2 we will demonstrate the efficacy and synergy of GEM-nab-paclitaxel-selinexor in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice model. These studies will identify sensitivity and/or resistance markers that will support our phase Ib/II trial in Aim3 in which we intend to determine the safety and efficacy of selinexor when combined with GEM-nab- paclitaxel in patients with metastatic PDAC. Biopsies from this trial will be used to validate locked pharmacodynamic markers of selinexor target engagement in patients. Clinical Impact: The successful completion our studies will bring forward a new therapy to improve the survival of patients with PDAC.