Exploring PARP biology and therapy response via metabolic flux analysis and a novel chemical proteomics workflow

聚-ADP-核糖聚合酶(PARP)是一种NAD依赖的酶，催化蛋白质上ADP-核糖修饰的形成，对DNA损伤反应、染色质重塑和在RNA剪接中发挥新的作用至关重要(Matveeva等人，2016)。在目前确定的17个PAP中，我们缺乏对每个酶优先修饰的蛋白质组子集的详细了解，部分原因是定义全球PARylome的分析挑战。PARP(I/II/III)抑制剂在HR缺乏的癌症中具有综合致命性，正成为卵巢癌重要的维持治疗方法。PARP抑制剂的疗效与PARP被损伤DNA‘捕获’的程度密切相关(Murai等人，2012年)。NAD辅因子对于PARP活性和通过自动PAR化去除捕获的PARP是必不可少的；然而，癌细胞中代谢重新编程和NAD的可用性对PARylome或PARP抑制剂反应的影响尚未被系统地探索。我们(Keun)观察到，KRAS突变的卵巢癌细胞通过化学抑制NAD挽救酶NAMPT(FK866)而选择性地对NAD耗竭敏感(图1)。这与KRAS驱动的代谢重新编程一致，导致了经典的Warburg状态，对胞浆中的NAD+保持高糖酵解率以及对NADPH用于生物合成和抗氧化保护都有很高的要求。我们假设，在KRAS突变的卵巢癌细胞中，对NAD供应的额外需求改变了全球PARylome，随后改变了PARylated蛋白的下游功能。4利用DiMaggio实验室开发的一种新的定量化学蛋白质组学工作流程，包括多个可点击的NAD+前体的代谢整合和TMT等压标记(在综述中，《自然化学生物学》)，我们意外地观察到，在乳腺癌细胞中，RNA剪接因子显示出最大程度的PAR化降低和PARP抑制(图2)。因此，我们还假设，NAD代谢可能通过PARP活性调节剪接体的功能，这可能是以前未见过的作用。这一假设得到了最近的CRISPR筛查的支持，该筛查揭示了核糖核酸酶是PARP抑制剂反应的新的合成致命决定因素(Zimmermann等人，2018年)。在这项研究中，共有73个高置信度基因在三个细胞系中被鉴定出来，当这些基因发生突变时，会导致对PARP抑制剂的敏感性增加。一些RNA剪接因子，如DDX46和SRSF11，都在这些基因中，但没有进一步研究。值得注意的是，DDX46和SRSF11也被我们的化学蛋白质组学工作流程确定为在MDA-MB-231细胞中使用PARP抑制剂处理后去PAR化程度最高的蛋白质之一。

Poly-ADP-ribose polymerases (PARPs) are NAD-dependent enzymes catalysing the formation of ADP-ribose modifications on proteins and are critical for DNA damage response, chromatin remodelling and with emerging roles in RNA splicing (Matveeva et al., 2016). Across the 17 PARPs currently identified we lack a detailed understanding of the subset of the proteome preferentially modified by each enzyme partly due to the analytical challenge of defining the global PARylome. PARP (I/II/III) inhibitors are synthetically lethal in HR-deficient cancer and are becoming an important maintenance therapy in ovarian cancer. The efficacy of PARP inhibitors is closely linked to the extent to which PARP is 'trapped' on damaged DNA (Murai et al., 2012). The NAD cofactor is essential for PARP activity and removal of trapped PARP by autoPARylation; however the impact of metabolic reprogramming in cancer cells and NAD availability on the PARylome or PARP inhibitor response has not been systematically explored We (Keun) have observed that KRAS mutant ovarian cancer cells are selectively sensitive to NAD depletion via chemical inhibition (FK866) of the NAD salvage enzyme NAMPT (Figure 1). This is consistent with KRAS-driven metabolic reprogramming leading to a classic Warburg state with a high requirement for both NAD+ in the cytosol to maintain a high glycolytic rate and NADPH for biosynthesis and antioxidant protection. We hypothesise that extra demands on NAD supply in KRAS mutant ovarian cancer cells alter the global PARylome, and subsequently the downstream function of PARylated proteins. 4 Using a novel quantitative chemical proteomics workflow involving metabolic incorporation of multiple clickable NAD+ precursors combined with TMT isobaric tagging developed in the DiMaggio lab (in review, Nature Chemical Biology), we unexpectedly observed that RNA splicing factors showed the greatest reduction in PARylation with PARP inhibition in breast cancer cells (Figure 2). Therefore we also hypothesise that NAD metabolism may have a previously uncharacterised role in regulating function of the spliceosome via PARP activity. This hypothesis is supported by a recent CRISPR screen that revealed ribonucleases are novel synthetically lethal determinants of PARP inhibitor response (Zimmermann et al., 2018). In this study a total of 73 high-confidence genes were identified across three cell lines that when mutated resulted in increased sensitivity to PARP inhibitors. A number of RNA splicing factors, such as DDX46 and SRSF11, were among these genes but not investigated further. Notably, DDX46 and SRSF11 were also identified by our chemical proteomics workflow as one of the most de-PARylated proteins after treatment with PARP inhibitors in MDA-MB-231 cells .