PO.TB10.09 · 肿瘤生物学
Brca2 缺陷促进前列腺癌中 cGAS-STING 通路活化增强及免疫抑制性肿瘤免疫微环境的形成
Brca2- deficiency promotes increased cGAS-STING pathway activation and an immunosuppressive tumor immune microenvironment in prostate cancer
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摘要 Abstract
中文摘要
背景：对晚期转移性前列腺癌进行的胚系与体细胞肿瘤联合分析显示，约 20% 的转移性肿瘤存在 DNA 修复基因（如 BRCA2）突变。BRCA2 胚系变异使个体易患侵袭性高危前列腺癌，并在诊断时表现为更晚期的疾病。BRCA2 对于通过同源重组（HR）修复 DNA 双链断裂（DSB）至关重要，其缺陷会导致基因组不稳定，而后者已被证明可通过 cGAS-STING 通路驱动炎症。前列腺癌中 BRCA2 功能缺失对免疫活化和肿瘤微环境（TME）的影响尚不清楚。这在设计有效靶向 HR 缺陷型前列腺癌免疫抑制性 TME 的疗法方面构成了重大的知识空白。
方法：我们利用 CRISPR/Cas9 构建了一个免疫功能健全的 Brca2 缺陷型前列腺癌模型，携带功能缺失的移码（fs）突变（Brca2 fs），以填补当前临床前模型中的关键空白。我们验证了 BRCA2 功能缺失，并表征了通过 cGAS-STING 通路的固有免疫信号传导。我们对来自免疫功能健全小鼠的 Brca2 WT 和 Brca2 fs 肿瘤进行了光谱流式细胞术和单细胞 RNA 测序（scRNA-seq），以评估 TME。对荷有 Brca2 fs 肿瘤的小鼠使用抗 CCR8 单克隆抗体（联合或不联合抗 PD-1）进行治疗，以检验在 Brca2 缺陷型前列腺肿瘤中靶向 CCR8 轴的有效性。
结果：与 Brca2 WT 细胞相比，Brca2 fs 细胞表现出 DNA 损伤增加和 cGAS-STING 通路活化增强的特征。对植入的 Brca2 fs 肿瘤的分析表明，与 Brca2 WT 肿瘤相比，CD4+ T 细胞和免疫抑制性 CCR8+ 调节性 T 细胞（Treg）富集。植入免疫功能健全小鼠的 Brca2 fs 肿瘤的 scRNA-seq 显示干扰素表达特征显著增加，这可能是肿瘤细胞中 cGAS-STING 通路活化增强的结果。此外，来自 Brca2 fs 肿瘤的肿瘤相关巨噬细胞（TAM）表现出更强的免疫抑制表型，M2 极化（促肿瘤）增加，并高表达 CCR8 受体配体 Ccl8。抗 CCR8 与抗 PD-1 联合治疗显著减少了荷有 Brca2 fs 肿瘤小鼠的肿瘤生长。
结论：我们的分析揭示了在 Brca2 缺陷型（而非 Brca2 功能完整型）前列腺癌肿瘤中富集免疫抑制性 CCR8+ Treg 和高表达 Ccl8 的 TAM。抗 CCR8 治疗（以清除 CCR8+ Treg）联合抗 PD-1 治疗，在我们的免疫功能健全小鼠模型中显著减少了 Brca2 fs 肿瘤的生长。这些发现提出了在 BRCA2 缺陷型前列腺肿瘤中靶向 CCR8 轴以克服这些免疫抑制机制并增强肿瘤反应性 T 细胞活性的诱人可能性。
查看英文原文 English abstract
Background : Combined germline and somatic tumor profiling of advanced metastatic prostate cancer has revealed that ~20% of metastatic tumors have mutations in DNA repair genes such as BRCA2 . Germline variants in BRCA2 predispose to aggressive high-risk prostate cancer and more advanced disease at diagnosis. BRCA2 is vital to the repair of DNA double-strand breaks (DSB) by homologous recombination (HR) and deficiency leads to genomic instability which has been shown to drive inflammation via the cGAS-STING pathway. The implications of loss of BRCA2 function in prostate cancer on immune activation and the tumor microenvironment (TME) are unknown. This represents a significant knowledge gap towards devising therapies that effectively target the immunosuppressive TME of HR-deficient prostate cancer.
Methods : We have engineered an immunocompetent Brca2 -deficient prostate cancer model with loss of function frame shift (fs) mutations ( Brca2 fs ) using CRISPR/Cas9 to address the critical gap in current preclinical models. We validated BRCA2 loss of function and characterized innate immune signaling through the cGAS-STING pathway. We performed spectral flow cytometry and single-cell RNA sequencing (scRNA-seq) on Brca2 WT and Brca2 fs tumors from immunocompetent mice to assess the TME. Mice bearing Brca2 fs tumors were treated with anti-CCR8 monoclonal antibodies with and without anti-PD-1 to test the effectiveness of targeting the CCR8-axis in Brca2 -deficient prostate tumors.
Results : Brca2 fs cells show hallmarks of increased DNA damage and cGAS-STING pathway activation compared to Brca2 WT cells. Analysis of implanted Brca2 fs tumors demonstrated enrichment of CD4+ T cells and immunosuppressive CCR8+ regulatory T cells (Treg) compared to Brca2 WT tumors. scRNA-seq of Brca2 fs tumors implanted in immunocompetent mice showed a significant increase in the interferon expression signature that is likely the result of increased cGAS-STING pathway activation in tumor cells. Additionally, tumor associated macrophages (TAMs) from Brca2 fs tumors showed a greater immunosuppressive phenotype with an increase in M2 polarization (pro-tumor) and high expression of Ccl8 , the CCR8 receptor ligand. Combination anti-CCR8 and anti-PD-1 therapy significantly reduced tumor growth in mice bearing Brca2 fs tumors.
Conclusions : Our analysis has revealed enrichment of immunosuppressive CCR8+ Tregs and Ccl8 -high expressing TAMs in Brca2 -deficient but not Brca2 -proficient prostate cancer tumors. Anti-CCR8 therapy, to deplete CCR8+ Tregs, in combination with anti-PD-1 therapy significantly reduced Brca2 fs tumor growth in our immunocompetent mouse model. These findings present the exciting possibility of targeting the CCR8-axis in BRCA2 -deficient prostate tumors to overcome these immunosuppressive mechanisms and enhance activity of tumor-reactive T cells.
利益披露 Disclosure
K. A. Rickman, None..
I. Q. Sun, None..
R. Parameswaran, None..
X. Gou, None..
M. E. Diolaiti, None.
H. N. Vasudevan,
Genentech Patent.
Eli Lilly Patent.
SpringWorks Independent Contractor, ).
GondolaBio Independent Contractor.
A. Ashworth,
Azkarra Therapeutics co-founder.
Kytarro co-founder.
Ovibio Corporation co-founder.
Tango Therapeutics co-founder.
Tiller Tx co-founder.
Cambridge Science Corporation g., Board of Directors, non-salaried role).
Cytomx g., Board of Directors, non-salaried role).
Ovibio Corporation g., Board of Directors, non-salaried role).
Ambagon scientific advisory board.
Bluestar/Clearnote Health scientific advisory board.
Circle scientific advisory board.
Deciphera scientific advisory board.
Genvivo scientific advisory board.
GLAdiator scientific advisory board.
HAP10 scientific advisory board.
Interdict Bio Inc scientific advisory board.
Earli scientific advisory board.
ORIC scientific advisory board.
Phoenix Molecular Designs scientific advisory board.
AstraZeneca Patent, holds patents on the use of PARP inhibitors held jointly with AstraZeneca from which he has benefited financially (and may do so in the future).