Reprogramming the Tumor Microenvironment in Pancreas Cancer to Enhance Immunotherapy

项目摘要/摘要 胰腺癌(PDAC)难以克服的三大治疗耐药因素 是KRAS癌基因的突变，即致密的促结缔组织间质的存在，它是药物传递的障碍 和效应免疫细胞的渗透，以及导致肿瘤的免疫抑制肿瘤微环境(TME) 对免疫治疗无效。我们针对致癌RAS下游效应分子的努力表明，MEK 抑制(Meki)导致STAT3信号的相互激活，从而产生治疗耐药并持续 PDAC细胞生长。联合抑制JAK/STAT3(STAT3i)和Meki克服以下治疗耐药 RAS抑制是通过平行反馈环激活介导的。我们现在已经确定了一种新的机制 显示Meki和STAT3i联合应用还能抑制肿瘤纤维化并增强CD8+细胞毒性T细胞(CTL) 免疫抑制调节性T细胞(Tregs)和髓系来源的免疫抑制T细胞(Tregs)下调时肿瘤的侵袭 TME中的抑制细胞(MDSCs)，从而降低了肿瘤负担并提高了基因工程患者的生存率 PDAC的小鼠模型(宝石)。此外，我们还发现Meki和STAT3i的抑瘤作用是T细胞。 依附的。然而，TME的这种变化伴随着PD-L1/PD-1和CTLA-4的持续表达。我们的 初步结果进一步表明，将Meki和STAT3i与PD-1抑制相结合可以利用 增强抗肿瘤反应的免疫检查点抑制剂。对肿瘤重新编程的治疗策略 基质激活T细胞抗肿瘤免疫和逆转免疫耐受至关重要，因为它们 有可能给胰腺癌的治疗带来革命性的变化，改善临床结果。 我们的中心假设是Meki和STAT3i将重新编程PDAC TME的细胞组件以 刺激CD8+CTL的渗透并克服PDAC的免疫抑制环境以增强其疗效 检查点抑制(CPI)，以实现持久和持久的抗肿瘤反应。这将通过以下具体的证明来证明 目的：目的1：确定使用Meki和STAT3i抑制检查点是否能提高PDAC的存活率。 Meki/STAT3i和抗PD1和/或抗CTLA-4抗体治疗反应的安全性和有效性将被确定 在PDAC的两个不同的宝石中。目标2：确定基质和免疫微环境是否发生变化 Meki/STAT3i诱导和检查点抑制导致持久的抗肿瘤免疫 体内对PDAC的反应。为此，将使用多重流式细胞术来检测细胞类型和 激活表型以确定治疗前后的差异是否可以预测反应。目标3：确定 PDAC细胞特异性和CAFS特异性敲除MEK和/或STAT3信号对细胞周期变化的影响 PDAC的间质和免疫微环境。这一目标将阐明细胞特异性击倒的机制。 MEK和/或STAT3介导的改变导致CD8+T细胞浸润增加和抑制 抑制性MDSCs和Tregs。这项工作不仅将评估PDAC的一种新的治疗策略，而且可能发现 潜在的生物标志物对检查点抑制剂的反应。

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.