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

聚ADP-核糖聚合酶（PARP）是催化蛋白质上ADP-核糖修饰形成的NAD依赖性酶，并且对于DNA损伤反应、染色质重塑和RNA剪接中的新兴作用至关重要（Matveeva等人，2016）。在目前确定的17个PARP中，我们缺乏对蛋白质组子集的详细了解，这些蛋白质组子集优先被每种酶修饰，部分原因是定义全局PARylome的分析挑战。PARP（I/II/III）抑制剂在HR缺陷癌症中具有合成致死性，并正在成为卵巢癌的重要维持疗法。PARP抑制剂的功效与PARP被“捕获”在受损DNA上的程度密切相关（Murai等人，2012年）。NAD辅因子对于PARP活性和通过自体PAR化去除捕获的PARP是必不可少的;然而，尚未系统地探索癌细胞中代谢重编程和NAD可用性对PARylome或PARP抑制剂反应的影响。我们（Keun）观察到KRAS突变卵巢癌细胞通过化学抑制（FK 866）NAD补救酶NAMPT对NAD消耗选择性敏感（图1）。这与KRAS驱动的代谢重编程一致，导致经典的瓦尔堡状态，其对细胞溶质中的NAD+和NADPH都有很高的需求，NAD+用于维持高糖酵解速率，NADPH用于生物合成和抗氧化保护。我们假设KRAS突变卵巢癌细胞对NAD供应的额外需求改变了整体PARylome，并随后改变了PARylated蛋白的下游功能。 4使用迪马吉奥实验室开发的新型定量化学蛋白质组学工作流程，涉及多种可点击的NAD+前体的代谢掺入与TMT同量异位素标记相结合（在《自然化学生物学》评论中），我们意外地观察到RNA剪接因子显示出最大的减少。乳腺癌细胞中PARP抑制的PARylation（图2）。因此，我们还假设NAD代谢可能在通过PARP活性调节剪接体功能中具有先前未表征的作用。这一假设得到了最近的CRISPR筛选的支持，该筛选揭示了核糖核酸酶是PARP抑制剂应答的新型合成致死决定子（Zimmermann et al. 2018年）。在这项研究中，在三种细胞系中共鉴定了73个高置信度基因，这些基因突变时会导致对PARP抑制剂的敏感性增加。一些RNA剪接因子，如DDX 46和SRSF 11，在这些基因中，但没有进一步研究。值得注意的是，DDX 46和SRSF 11也被我们的化学蛋白质组学工作流程鉴定为MDA-MB-231细胞中用PARP抑制剂处理后最多去PAR化的蛋白质之一。

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 .