Project 1: Glutaminolytic GDH1 activation-dependent immunotherapy resistance in LKB1-mutant lung adenocarcinoma

项目总结 Emory肺癌P01应用的项目1专注于询问LKB1的代谢失调- 突变型肺腺癌(LUAD)克服免疫治疗耐药性。LKB1，25%失活 LUAD在调节细胞代谢方面起着重要作用。谷氨酰胺代谢升高对 癌症进展，但谷氨酰胺分解上调并有助于免疫的机制 逃生仍然难以捉摸。我们发现谷氨酸脱氢酶1(GDH1)是谷氨酰胺分解的关键酶 途径，诱导CAMKK2，它替代LKB1激活AMPK来管理能量动态平衡和 促进LKB1基因缺陷肺癌的转移。通过对激酶抑制剂的分析，我们发现了 黏附激酶(FAK，项目3的焦点)在Y135处使GDH1磷酸化，并在 LKB1突变LUAD。为了研究GDH1信号和抗肿瘤免疫之间的联系，我们进行了 免疫图谱和证明GDH1有助于促进激活的调节性T细胞和 体内肿瘤浸润性CD8T细胞的减弱。补充能量代谢对LKB1突变肺癌的作用 通过重新激活AMPK使GDH1缺失降低了增强的T细胞在体外的存活。通过基因组图谱 在免疫检查点因子中，我们还发现GDH1促进了程序性死亡配体1的表达 (PD-L1)和脊髓灰质炎病毒受体(PVR)及其潜在的转录因子CREB被发现激活 通过GDH1-CAMKK2-CamK4信号转导。此外，GDH1的缺失导致了蛋白表达的增加 干扰素基因刺激物(STING；项目2的重点)。新型谷氨酸脱氢酶抑制剂R162对GDH1的抑制作用 抑制肺癌进展和使LKB1突变的LUAD细胞易受原代激活的影响 免疫细胞。此外，R162与PD1抑制剂联合使用可进一步诱导癌细胞死亡 单药治疗，提示GDH1是克服免疫治疗耐药的一个有前途的靶点。 因此，我们的中心假设是FAK通过Y135磷酸化激活GDH1，特异性地在LKB1- 突变型LUAD，通过代谢、转录和蛋白质稳定性提供免疫治疗抵抗 监管。因此，GDH1与免疫治疗药物相结合是一种很有前途的抗癌靶点。 靶向PD-L1/PVR信号。提出了三个具体目标：(1)破译 FAK介导的GDH1的磷酸化和激活促进LKB1依赖的肿瘤进展 (2)确定GDH1如何通过提供代谢，转录， 以及通过AMPK、CREB和STING获得的蛋白质稳定性优势；(3)评价治疗效果 联合以-PD-L1/-TIGIT为基础的免疫治疗。以及项目2(STING) 和项目3(FAK)，实现我们项目的目标，有望开发出新的靶向GDH1 提高LKB1突变LUAD对免疫治疗反应的治疗方法。

PROJECT SUMMARY Project 1 of the Emory Lung Cancer P01 application focuses on interrogation of metabolic dysregulation in LKB1- mutant lung adenocarcinoma (LUAD) to overcome immunotherapy resistance. LKB1, which is inactivated in 25% of LUAD, plays an important role in regulating cell metabolism. Elevated glutamine metabolism is critical for cancer progression, but the mechanism by which glutaminolysis is upregulated and contributes to immune escape is still elusive. We found that glutamate dehydrogenase 1 (GDH1), the crucial enzyme in glutaminolysis pathway, induces CamKK2, which substitutes for LKB1 to activate AMPK to manage energy homeostasis and promote metastasis in LKB1-deficient lung cancer. Through kinase inhibitor profiling, we found that focal adhesion kinase (FAK; focus of Project 3) phosphorylates GDH1 at Y135 and activates GDH1 specifically in LKB1 mutated LUAD. To investigate the link between GDH1 signaling and antitumor immunity, we performed immune profiling and demonstrated that GDH1 contributes to promotion of activated regulatory T cells and attenuation of tumor infiltrated CD8 T cells in vivo. Replenishing energy metabolism in LKB1 mutated lung cancer with GDH1 loss by reactivating AMPK reduced the enhanced T cell survival in vitro. Through a genomic profiling of immune checkpoint factors, we also found that GDH1 promotes expression of programmed death-ligand 1 (PD-L1) and poliovirus receptor (PVR), and their potential transcription factor CREB was found to be activated by GDH1-CamKK2-CamK4 signaling. Furthermore, GDH1 loss resulted in increased protein expression of stimulator of interferon genes (STING; focus of Project 2). Inhibition of GDH1 using our novel GDH inhibitor R162 attenuated lung cancer progression and rendered LKB1 mutated LUAD cells vulnerable to activated primary immune cells. Moreover, R162 in combination with PD1 inhibitor further induced cancer cell death compared to single agent treatment, suggesting GDH1 as a promising target to overcome immunotherapy resistance. Thus, our central hypothesis is that FAK activates GDH1 by Y135 phosphorylation specifically in LKB1- mutant LUAD and provides immunotherapy resistance through metabolic, transcriptional, and protein stability regulation. Thus, GDH1 represents a promising anti-cancer target in combination with immunotherapy agents targeting PD-L1/PVR signaling. Three specific aims are proposed: (1) To decipher the mechanism by which FAK-mediated phosphorylation and activation of GDH1 promotes cancer progression in an LKB1-dependent manner; (2) To determine how GDH1 confers immunotherapy resistance by providing metabolic, transcriptional, and protein stability advantages through AMPK, CREB, and STING; and (3) To evaluate the therapeutic efficacy of targeting GDH1 in combination with -PD-L1/-TIGIT-based immunotherapy. Along with Project 2 (STING) and Project 3 (FAK), accomplishing the goals of our project is expected to develop novel GDH1-targeted therapeutic approaches to enhance the response of LKB1-mutant LUAD to immunotherapies.