探討TREX1抑制劑於抑止TREX1免疫沉默之功效

Abstract

在抗病毒及抗腫瘤免疫反應中，第一型干擾素的產生扮演了相當重要的角色。而第一型干擾素可以藉由pattern recognition receptor (PRR) 辨識pathogen-associated molecular patterns (PAMPs) 或damage-associated molecular patterns (DAMPs) 後活化下游訊息傳遞路徑來生成。Cyclic GMP-AMP synthase (cGAS) 做為識別細胞質中ssDNA 和dsDNA的PRR，可以辨識例如從細胞核和粒線體釋放的病毒DNA或自身DNA來合成cyclic GMP-AMP (cGAMP)。cGAMP接著會活化stimulator of interferon genes (STING)，最終使下游的轉錄因子interferon regulatory factor 3 (IRF3) 被磷酸化，從而促使第一型干擾素的表現。而被分泌出的第一型干擾素則會透過與interferon-α/β receptor (IFNAR) 結合，促進Interferon Stimulated Genes (ISGs) 的表達和T細胞的召集/活化。近年來，研究發現three prime repair exonuclease 1 (TREX1) 做為3'→5' 的DNA核酸外切酶，具有降解細胞質中DNA的功能，可以避免 cGAS-STING 訊息傳遞路徑產生不必要的活化。此外，也有研究指出細胞質中異常 DNA 的累積會上調TREX1基因剔除細胞株中第一型干擾素的表現。因此，我們假設 TREX1抑制劑可以促進由辨識細胞質 DNA傳遞路徑所誘導之第一型干擾素的表現。在本篇研究中，我們首先使用同時表現了TREX1、cGAS 和 STING 的 Calu-3 細胞作為細胞培養模型，以證明 TREX1 抑制劑能否增加 cGAS-STING介導之第一型干擾素的表現。並且，藉由測定細胞存活率來確認先前體外 (in vitro) 實驗中，透過抑制劑偶聯核酸酶活性測定所找到的TREX1 抑制劑是否會對 Calu-3 細胞造成細胞毒性。實驗結果發現， 所有TREX1 抑制劑皆具低細胞毒性，且其中三種抑制劑能夠增加由衣黴素 (tunicamycin) 誘導之第一型干擾素的表現。因此，我們初步選定此三種TREX1抑制劑用於進一步的研究。此外，我們也測定了TREX1抑制劑在乳腺癌細胞中的抑制效果。我們發現cGAS、STING 和 TREX1蛋白質的表現量在三種不同乳腺癌細胞系（包括MDA-MB-231、MCF-7 和HCC1395）中並不一致。在實驗中，我們證明了所篩選出的TREX1 抑制劑在這些乳腺癌細胞系中(除了MDA-MB-231無法測得第一型干擾素的表現)確實可以增加在UV誘導下由cGAS-STING介導之第一型干擾素的表現，藉此初步篩選出可能有效的TREX1抑制劑。而藉由比較異位表現野生型TREX1的細胞和表現核酸分解酶突變型TREX1 (hTREX1-D18N) 細胞中第一型干擾素的表現量，我們也驗證了所選之TREX1抑制劑的專一性。最終，我們篩選出兩種特定的TREX1抑制劑可開發為潛在的治療藥物，以增加cGAS-STING介導之第一型干擾素的表現，未來將應用在活體 (in vivo) 實驗中來驗證其抗腫瘤的治療效力。

The induction of type I interferon (IFN) plays important roles in antiviral as well as in anti-tumor immune responses. Type I IFN can be induced when pattern recognition receptors (PRRs) recognize the pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to activate the downstream signaling pathways. Cyclic GMP-AMP synthase (cGAS) is the PRR that recognizes cytoplasmic ssDNA and dsDNA, such as viral genomic DNAs or self-DNAs that released from the nucleus and mitochondria, to synthesize cyclic GMP-AMP (cGAMP). Stimulator of interferon genes (STING) is activated by cGAMP, the downstream transcription factor, interferon regulatory factor 3 (IRF3), will be phosphorylated and then promotes the expression of type I IFNs. The secreted type I IFNs will signal through interferon-α/β receptor (IFNAR) and drive the expression of Interferon Stimulated Genes (ISGs) and T cell recruitment/activation. Recently, three prime repair exonuclease 1 (TREX1) has been discovered as a 3’→5’ DNA exonuclease, which can degrade cytosolic DNA species to prevent the unnecessary activation of cGAS-STING pathways. In previous studies, type I IFNs have been found up-regulated in the TREX1 knock-out cells due to abnormal DNAs accumulation in the cytoplasm. We then hypothesize that TREX1 inhibitors can stimulate the production of type I IFNs driven by cytosolic DNA sensing pathways. We used Calu-3 cells which expresses all TREX1, cGAS, and STING at the protein level as our cell culture model to demonstrate whether TREX1 inhibitors may enhance cGAS-STING-mediated type I IFN induction. TREX1 inhibitors, which have been identified in vitro by inhibitor-coupled nuclease activity assay, were then determined their cytotoxicity in Calu-3 cells. We found that all TREX1 inhibitors have low cytotoxicity. Among them, three TREX1 inhibitors showed the ability to enhance tunicamycin-induced type I IFN expression and were selected for further studies. In addition, we determined the TREX1 inhibitory effects of these TREX1 inhibitors in breast cancer cells. The protein levels of cGAS, STING, and TREX1 were not ubiquitous among breast cancer cell-lines, including MDA-MB-231, MCF-7, and HCC1395. We demonstrated that selected TREX1 inhibitors could enhance cGAS-STING-mediated type I IFN induction in these breast cancer cell-lines. The specificity of selected inhibitors was determined by comparing the expression of type I IFN between ectopically expressing wild-type TREX1 or nuclease mutant TREX1 (hTREX1-D18N) cells. Finally, we selected two specific TREX1 inhibitors as therapeutic candidates to enhance cGAS-STING-mediated type I IFN expression, which will be validated in in vivo study for their anti-tumor effects.