Reprogramming the Tumor Microenvironment in Pancreas Cancer to Enhance Immunotherapy

项目总结/摘要 胰腺癌（PDAC）中难以克服的治疗耐药的三个主要因素 是KRAS癌基因突变，存在致密促结缔组织增生基质，作为药物输送的屏障 和效应免疫细胞浸润，以及免疫抑制性肿瘤微环境（TME），使肿瘤 对免疫治疗无效。我们在靶向致癌RAS下游效应子方面的努力表明，MEK 抑制（MEKi）导致STAT 3信号传导的相互激活，这赋予治疗抗性，并且持续 PDAC细胞生长。JAK/STAT 3（STAT 3 i）和MEKi的组合抑制克服了以下治疗抗性： 通过平行反馈环激活介导的RAS抑制。我们现在发现了一种新的机制 显示组合的MEKi和STAT 3 i也抑制肿瘤纤维化并增强CD 8+细胞毒性T细胞（CTL）， 肿瘤浸润，同时下调免疫抑制调节性T细胞（TCLs）和髓源性T细胞（TCLs）。 抑制细胞（MDSC）在TME中，导致肿瘤负荷减少，并提高了基因工程治疗的生存率。 PDAC的小鼠模型（GEM）。此外，我们发现MEKi和STAT 3 i的肿瘤抑制作用是T细胞增殖抑制， 依赖。然而，TME的这种变化伴随着持续的PD-L1/PD-1和CTLA-4表达。我们 初步结果进一步表明，MEKi和STAT 3 i与PD-1抑制的组合可以利用 免疫检查点抑制剂，用于增强抗肿瘤反应。重新编程肿瘤的治疗策略 激活T细胞抗肿瘤免疫和逆转免疫耐受基质是至关重要的，因为它们 有可能彻底改变胰腺癌的治疗方法并改善临床结果。 我们的中心假设是MEKi和STAT 3 i将重编程PDAC TME的细胞组分， 刺激CD 8 + CTL的浸润并克服PDAC的免疫抑制环境，以增强 检查点抑制（CPI），以实现持久和持续的抗肿瘤反应。这将通过以下具体的 目的：目的1：确定用MEKi和STAT 3 i抑制检查点是否会改善PDAC的GEM中的存活。 将确定MEKi/STAT 3 i和抗PD 1和/或抗CTLA-4抗体治疗应答的安全性和功效 在PDAC的两个不同的GEM中。目的2：确定基质和免疫微环境是否发生变化 由MEKi/STAT 3 i诱导和检查点抑制导致持久和持续的抗肿瘤免疫 PDAC在体内的反应。为此，将使用多重流式细胞术检测细胞类型的变化， 活化表型，以确定治疗前和治疗后的差异是否预测响应。目标3：确定 MEK和/或STAT 3信号传导的PDAC细胞特异性和CAFs特异性敲低对细胞内信号传导的变化的影响 PDAC中基质和免疫微环境。这一目标将阐明细胞特异性敲低的机制 MEK和/或STAT 3介导的变化，导致CD 8 + T细胞浸润增加， 抑制性MDSC和TGF 1 α。这项工作不仅将评估一种新的PDAC治疗策略， 对检查点抑制剂反应的潜在生物标志物。

PROJECT SUMMARY/ABSTRACT Three major contributors to therapeutic resistance that have been difficult to overcome in pancreatic cancer (PDAC) are mutations in the KRAS oncogene, the presence of a dense desmoplastic stroma that acts as a barrier to drug delivery and effector immune cell infiltration, and the immunosuppressive tumor microenvironment (TME) that renders the tumor ineffective to immunotherapy. Our efforts at targeting downstream effectors of oncogenic RAS, have shown that MEK inhibition (MEKi) results in reciprocal activation of STAT3 signaling, which confers therapeutic resistance and continued PDAC cell growth. Combined inhibition of JAK/STAT3 (STAT3i) and MEKi overcomes therapeutic resistance following RAS inhibition that is mediated through parallel feedback loop activation. We have now identified a novel mechanism showing that combined MEKi and STAT3i also inhibits tumor fibrosis and enhances CD8+ cytotoxic T cell (CTL) infiltration to the tumor while downregulating immunosuppressive regulatory T cells (Tregs) and myeloid derived suppressor cells (MDSCs) in the TME, resulting in reduced tumor burden and improved survival in genetically engineered mouse models (GEMs) of PDAC. In addition, we show that the tumor suppressive effects of MEKi and STAT3i are T cell dependent. This change in the TME, however, is accompanied by sustained PD-L1/PD-1 and CTLA-4 expression. Our preliminary results further show that combined MEKi and STAT3i with PD-1 inhibition can harness the effects of immune checkpoint inhibitors for an enhanced anti-tumor response. Therapeutic strategies that reprogram the tumor stroma to activate T-cell anti-tumor immunity and reverse immune tolerance are of paramount importance as they have the potential to revolutionize treatment for pancreatic cancer and improve clinical outcomes. Our central hypothesis is that MEKi and STAT3i will reprogram cellular components of the PDAC TME to stimulate infiltration of CD8+ CTLs and overcome the immunosuppressive milieu of PDAC to enhance the effects of checkpoint inhibition (CPI) for a durable and sustained anti-tumor response. This will be proven by the following specific aims: Aim 1: Determine if checkpoint inhibition with MEKi and STAT3i will improve survival in GEMs of PDAC. Safety and efficacy of MEKi/STAT3i and anti-PD1 and/or anti-CTLA-4 antibodies treatment response will be determined in two different GEMs of PDAC. Aim 2: Determine if changes in the stromal and immune microenvironment induced by MEKi/STAT3i and checkpoint inhibition result in a durable and sustained anti-tumor immune response in PDAC in vivo. In this aim, multiplex flow cytometry will be used to detect the changes in the cell types and activation phenotypes to determine if the differences pre- and post-treatment predict response. Aim 3: Determine the effects of PDAC cell specific and CAFs specific knockdown of MEK and/or STAT3 signaling on changes in the stromal and immune microenvironment in PDAC. This aim will elucidate the mechanism of cell-specific knockdown of MEK and/or STAT3 mediated changes in that result in increased infiltration CD8+ T cells and suppression of suppressive MDSCs and Tregs. This work will not only evaluate a novel treatment strategy for PDAC, but may uncover potential biomarkers of response to checkpoint inhibitors.