Characterizing the biochemistry and dynamics of the immune suppressive CXCL12 coat in pancreatic cancer

项目摘要/摘要 胰腺导管腺癌是美国癌症相关死亡的第三大原因。 五年存活率为7-8%。这种较差的生存能力可以部分归因于较晚的检测， 然而，PDAC治疗仍然停滞不前，这放大了患者的存活率与 PDAC和其他癌症。免疫检查点阻断(ICB)使某些恶性疾病的治疗发生了革命性变化。 但大多数癌症，包括PDAC，仍然是难治性的。Fearon实验室已经确定这些化学物质- Okine(C-X-C基序)配体12(CXCL12)“包裹”PDAC小鼠模型中的癌细胞，并阻断 CXCL12与趋化因子(C-X-C基序)受体4的相互作用导致T细胞 进入癌细胞巢和对ICB的反应。此外，我们发现CXCL12与角蛋白是共价连接的 19(KRT19)通过异肽键形成酶转谷氨酰胺酶2(TGM2)作用于癌细胞表面， 而Krt19或Tgm2基因的敲除会导致T细胞进入癌细胞巢并对ICB产生反应。此数据 提示CXCL12-KRT19结合物是肿瘤免疫抑制的主要介体，然而，我们的 对其形成的生化过程以及其形成和分解的动力学的理解是 缺乏这一点将为未来试图调节这些过程的研究提供洞察力。因此，我将去- 在体内终止KRT19与CXCL12共价结合所需的谷氨酰胺并检测其突变 导致CXCL12涂层的丢失和对ICB的响应。此外，我将量化形成的速度和 拆解CXCL12外壳并表征伴随而来的免疫细胞活性的变化。 在目标1中，我将使用TGM2在体外生成CXCL12-KRT19结合物，并对其进行质量检测 光谱分析鉴定CXCL12和KRT19之间的交联残基。我会产生回弹- 含有发现与CXCL12交联的残基的Q到N突变的双链KRT19，并验证 CXCL12不能再链接到KRT19。然后我将在小鼠PDA 1242细胞中表达突变体KRT19，并 验证CXCL12在体内未能与癌细胞连接，并确定这种失败是否会导致怀疑- 对ICB的认可。在目标2中，我将向C57BL/6J小鼠注射1242细胞的Matrigel悬浮液以促进早期肿瘤 用免疫荧光组织染色技术获取和定量CXCL12涂层的形成速度 英。我将使用多西环素关闭的KRT19表达系统来研究CXCL12涂层的拆解率和 使用RNA测序进行免疫细胞图谱分析，以确定浸润性T细胞对衣原体疾病的反应 桑布利。这项建议将研究CXCL12涂层是如何形成的，其形成和分解的速度，以及 强调调节CXCL12涂层可以相互改变抗肿瘤免疫活性。另外。这 奖学金将为我通过实验室学习翻译肿瘤免疫学的纵向目标提供培训 在冷泉港实验室和石溪大学进行培训、研讨会、会议和临床工作。

Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related deaths in the United States, with a five-year survival rate of 7-8%. This poor survivability can be partially attributed to late detection, however PDAC therapies have remained stagnant which amplifies the discordance between the survivability of PDAC and other cancers. Immune checkpoint blockade (ICB) has revolutionized the treatment of certain malig- nancies, but most carcinomas, including PDAC, remain refractory. The Fearon Lab has identified that the chem- okine (C-X-C motif) ligand 12 (CXCL12) “coats” cancer cells in mouse models of PDAC, and that blocking the interaction between CXCL12 and chemokine (C-X-C motif) receptor 4, the CXCL12 receptor, results in T cell influx into cancer cell nests and response to ICB. Additionally, we find that CXCL12 is covalently linked to keratin 19 (KRT19) on the surface of cancer cells by the isopeptide bond forming enzyme transglutaminase 2 (TGM2), and knockout of either Krt19 or Tgm2 results in T cell entry into cancer cell nests and response to ICB. This data indicates that the CXCL12-KRT19 conjugate is a primary mediator of tumor immune suppression, however, our understanding of the biochemical process of its formation, and kinetics of its formation and disassembly, are lacking yet would provide insight for future studies attempting to regulate these processes. Therefore, I will de- termine the glutamine of KRT19 required to covalently bond to CXCL12 in vivo and determine if its mutation results in loss of CXCL12 coating and response to ICB. Additionally, I will quantify the rate of formation and disassembly of the CXCL12 coat and characterize the accompanying changes to immune cell activity. In Aim 1 I will generate the CXCL12-KRT19 conjugate using TGM2 in vitro and subject this complex to mass spectrometry analysis to identify the crosslinked residues between CXCL12 and KRT19. I will generate recom- binant KRT19 containing a Q to N mutation of the residue found to be crosslinked to CXCL12 and validate that CXCL12 can no longer be linked to KRT19. I will then express the mutant KRT19 in mouse PDA 1242 cells and validate that CXCL12 fails to become linked to cancer cells in vivo and determine if this failure results in suscep- tibility to ICB. In Aim 2 I will inject Matrigel suspensions of 1242 cells into C57BL/6J mice to facilitate early tumor harvesting and quantification of the rate of formation of the CXCL12 coat using immunofluorescent tissue stain- ing. I will use a Doxycycline-Off KRT19 expression system to study the rate of CXCL12 coat disassembly and perform immune cell profiling using RNA sequencing to determine how infiltrative T cells respond to coat disas- sembly. This proposal will study how the CXCL12 coat forms, the rate of its formation and disassembly, and highlight that modulating the CXCL12 coat reciprocally modifies anti-tumor immune activity. Additionally. this fellowship will provide training for my longitudinal goal of studying translational tumor immunology through in-lab training, workshops, meetings, and clinical work at Cold Spring Harbor Laboratory and Stony Brook University.