CD73诱导的腺苷堆积在LKB1突变型肺癌“免疫沙漠型”微环境中的作用和机制研究

Patients with LKB1 mutant lung cancer tend to be primarily resistant to immunotherapy, which is closely associated with its “immune desert” tumor microenvironment (TME). Adenosine may exert negative influences on the TME, but its specific role on the formation of TME in LKB1-mutant lung cancer still needs to be investigated. CD73 is the critical enzyme in the production of adenosine. We previously found that, adenosine level was significantly increased in both LKB1-mutant lung cancer tissues and supernatants of LKB1-mutant cell lines. And negative effects on the TME from adenosine might include regulation of both endothelial cells and PBMCs. Furthermore, we found that CD73 expression level was significantly upregulated in LKB1-mutant cell lines, and possible mechanism may be the direct influence on the degradation of CD73 through AMPK. Therefore our hypothesis is that CD73 upregulation induced adenosine accumulation may be the primary reason for the formation of “immune desert” microenvironment in LKB1-mutant lung cancer. Here, we aim to further confirm that LKB1 is dependent on the direct control from AMPK to CD73 and their subtle regulation mechanism; and to demonstrate the negative impact on the TME through activation of CD73-adenosine pathway in vitro and in vivo. Finally we design to explore whether integrating AMPK agonists with PD-1 inhibitors is able to break through the dilemma of primary resistance of immunotherapy in lung cancer. We hope to unveil a new mechanism of the formation of immune desert microenvironment in LKB1-mutant lung cancer and discover new combination targets, and our study is therefore of profound significance.

LKB1突变型肺癌往往存在免疫治疗原发耐药，可能与其“免疫沙漠型”微环境形成相关，腺苷对免疫微环境具有较强的负调节作用，但其在LKB1突变型肺癌微环境形成中的作用尚缺少研究。CD73是催化腺苷生成过程中的关键限速酶。我们前期发现，LKB1突变型肺癌组织及细胞上清中，腺苷水平明显升高，且升高的腺苷可能通过影响内皮细胞及PBMCs等对微环境产生负向调控作用。进一步分析发现，LKB1突变型较野生型细胞CD73表达水平明显上调，可能是通过下游AMPK影响了CD73的降解过程。由此我们推测，CD73表达上调引起的腺苷水平升高可能是LKB1突变型肺癌特征性“免疫沙漠型”微环境形成的原因。本研究拟在体外及LKB1敲除小鼠模型中验证LKB1依赖于AMPK对CD73的精细调控机制及CD73-腺苷通路的活化对免疫微环境的负调节效应，并探讨联合AMPK激动剂逆转免疫治疗原发耐药可行性，具备极大的转化医学价值。

既往研究发现LKB1突变型肺腺癌表现为免疫治疗原发耐药，与其“免疫沙漠型”微环境形成相关。腺苷作为重要的免疫微环境负调因子，其对LKB1突变型肺腺癌微环境形成的作用尚缺少研究。我们实验结果表明：LKB1突变型肺腺癌组织及细胞上清中腺苷水平明显升高。LKB1突变导致的腺苷堆积促进“免疫沙漠型”微环境形成，导致免疫治疗原发耐药。进一步研究发现，微环境中PMN-MDSCs嘌呤代谢异常，是腺苷增多的关键因素。基于前期研究结果，我们提出科学假设：LKB1突变肿瘤通过调控PMN-MDSCs的嘌呤代谢，促成腺苷堆积和免疫逃逸，最终导致免疫治疗耐药。为验证假说，我们通过体内外实验证明了LKB1突变对PMN-MDSCs浸润数量和ENTPD1/NT5E表达量的影响；其次，通过对转录组测序数据进行分析明确了mTOR-HIF1α为LKB1突变诱导腺苷堆积的关键信号通路；最后，我们探讨了mTOR通路抑制剂逆转LKB1突变肺腺癌免疫治疗耐药的可能性。