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

项目摘要 埃默里肺癌P01申请的项目1侧重于询问LKB 1中的代谢失调， 突变型肺腺癌（LUAD），以克服免疫治疗耐药性。LKB 1，25%失活 在调节细胞代谢中起重要作用。谷氨酰胺代谢升高对于 癌症进展，但其机制，其中β-内酰胺酶上调，并有助于免疫 逃脱仍然是难以捉摸的。我们发现谷氨酸脱氢酶1（GDH 1）是谷氨酸分解的关键酶 途径，诱导CamKK 2，其取代LKB 1激活AMPK以管理能量稳态， 促进LKB 1缺陷型肺癌的转移。通过激酶抑制剂分析，我们发现， 粘附激酶（FAK;项目3的焦点）在Y135处磷酸化GDH 1，并特异性激活GDH 1， LKB 1突变的LUAD。为了研究GDH 1信号传导和抗肿瘤免疫之间的联系，我们进行了 免疫分析，并证明GDH 1有助于促进活化的调节性T细胞， 体内肿瘤浸润的CD 8 T细胞的衰减。LKB 1突变肺癌的能量代谢补充 通过重新激活AMPK而导致GDH 1丢失，降低了体外增强的T细胞存活。通过基因组分析 我们还发现，GDH 1促进程序性死亡配体1的表达， 发现PD-L1和脊髓灰质炎病毒受体（PVR）及其潜在的转录因子CREB被激活 通过GDH 1-CamKK 2-CamK 4信号传导。此外，GDH 1的缺失导致蛋白表达增加， 干扰素基因的刺激物（STING;项目2的焦点）。使用我们的新型GDH抑制剂R162抑制GDH 1 减弱肺癌进展，并使LKB 1突变的LUAD细胞对活化的原发性 免疫细胞此外，与对照组相比，R162与PD 1抑制剂的组合进一步诱导癌细胞死亡。 单剂治疗，表明GDH 1作为克服免疫疗法抗性的有希望的靶标。 因此，我们的中心假设是FAK通过Y135磷酸化激活GDH 1，特异性地在LKB 1中， 突变LUAD，并通过代谢、转录和蛋白质稳定性提供免疫治疗抗性 调控因此，GDH 1代表了与免疫治疗剂组合的有希望的抗癌靶点 靶向PD-L1/PVR信号传导。提出了三个具体目标：（1）破译的机制， FAK介导的GDH 1磷酸化和激活促进LKB 1依赖性肿瘤细胞的癌症进展 （2）确定GDH 1如何通过提供代谢，转录， 通过AMPK、CREB和STING分析蛋白质稳定性优势;（3）评价治疗效果 靶向GDH 1与基于PD-L1/PD-TIGIT的免疫疗法的组合。沿着项目2（STING） 和项目3（FAK），完成我们的项目的目标，预计将开发新的GDH 1靶向 增强LKB 1突变型LUAD对免疫疗法的应答的治疗方法。

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.