BioArt按
日前，拉斯克基礎醫學獎授予了兩位傑出的
表觀遺傳學家
，表彰他們所研究的內容拓展了人們對基因表達調控的認識。經過近20年的發展，當前表觀遺傳領域無論是基礎研究還是臨床轉化方面都取得了很大成績。有許多基於重要表觀蛋白的靶向抑制劑目前在臨床上表現較好，例如早期被FDA批准的DNMT1抑制劑（5-azacytidine）以及近年來臨床試驗表現良好的EZH2抑制劑、HDAC抑制劑、DOT1L抑制劑、BRD4抑制劑等，許多已經進入2期或3期臨床試驗了，再加上最近靶向表觀蛋白與腫瘤免疫治療抗體的聯合使用在臨床前試驗中顯現出了較好的效果，基於表觀遺傳的靶向藥物開發受到了廣泛的重視。基於組蛋白甲基轉移酶EZH2開發的抑制劑目前進入臨床試驗的儘管至少有8種，但是目前仍然面臨著推進速度慢的系列問題。剛剛在Cell在線發表的這篇來自中科院上海藥物所的文章聚焦EZH2抑制劑對部分實體瘤無效的問題，提出了可能的協同抑制表觀遺傳交互調控解決方案，這對加快EZH2的臨床試驗具有重要的意義。有鑑於此，BioArt特別邀請到了長期從事EZH2/PRC2相關研究工作的吳旭東教授解讀，並特別邀請到了哈佛大學
張毅教授和中科院上海藥物所蔣華良院士做精彩點評，以饗讀者！
Cell論文共同通訊作者：耿美玉研究員（中）、丁健院士（左）譚敏佳研究員（右）
解讀丨吳旭東 教授 （天津醫科大學）
點評丨張 毅 教授 （哈佛大學）；
蔣華良 院士 （中科院上海藥物所）
腫瘤進展過程中遺傳學變化和表觀遺傳學變化密切相關、相輔相承【1】。與遺傳學變化不同的是，表觀遺傳學變化往往是可逆的，以染色質修飾相關蛋白作為治療靶點，可使異常的表觀基因組得以糾正。Polycomb家族基因EZH2在白血病和淋巴瘤病人中較多地發生活性增強的突變，在實體瘤中往往高表達，並且與腫瘤惡性程度和不良預後顯著相關。目前，EZH2抑制劑在一部分血液系統腫瘤的治療中表現不錯，然而在實體瘤中，EZH2抑制劑僅僅對少數具有SWI/SNF成員基因突變或者BAP1突變的腫瘤表現出一定的效果【2】 , 在膠質母細胞瘤中長期抑制EZH2的表達甚至會導致細胞激活多能性調控網絡，增強細胞增殖和提高DNA損傷修復能力，反而促進腫瘤進展【3】。為了進一步擴大EZH2抑制劑在實體瘤的臨床應用，有必要深入瞭解腫瘤細胞對EZH2抑制劑不敏感的分子機制。
9月13日，中國科學院上海藥物研究所耿美玉、丁健和譚敏佳聯合研究團隊在Cell上發表了題為Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors的研究成果，揭示了EZH2抑制劑對大部分實體瘤治療無效的分子機制並提供了可能的協同抑制表觀遺傳交互調控解決方案，並對當前腫瘤的靶向治療提出了示範性方案，對於當前EZH2抑制劑臨床研究的困境破局具有重要的指導意義。
在這項研究中，研究人員系統地檢測了83株EZH2高表達的腫瘤細胞對兩種EZH2抑制劑（EPZ-6438和GSK126）的敏感性（IC50）。為了系統地鑑定與EZH2抑制劑敏感性相關的表觀遺傳標記，他們選取兩株敏感細胞和4株不敏感細胞，通過基於SILAC的質譜分析比較了111種組蛋白標記在EZH2抑制劑處理前後的變化，發現所有細胞在EZH2抑制劑處理之後H3K27me2和H3K27me3的水平都顯著下降，然而兩類細胞中H3K27ac水平的變化表現出最大的差異：敏感細胞的H3K27ac水平幾乎不變或者略有下調，而不敏感細胞的H3K27ac水平急劇升高。對43種代表性腫瘤細胞進行分析，他們發現H3K27ac水平增加的程度與細胞IC50顯著正相關，說明EZH2抑制劑誘導激活的H3K27ac對細胞耐藥起著重要作用。與此一致的是，敲低或者抑制負責催化H3K27ac的p300/CBP的確能使EZH2抑制劑不敏感的細胞變得敏感。然而，p300表達水平與敏感性無關，作者然後發現p300的募集蛋白MLL1在不敏感細胞中顯著高表達，而敏感的淋巴瘤細胞幾乎不表達MLL1；緊接著他們證實了MLL1敲低也能使EZH2抑制劑不敏感的細胞變得敏感，MLL1抑制劑的作用還有待確認。
為了系統探究H3K27me3逆轉成為H3K27ac導致腫瘤細胞對EZH2抑制劑耐藥的機制，作者對一株敏感細胞和兩株不敏感細胞進行了H3K27ac ChIP-seq、RNA-seq和蛋白質組學分析，發現在不敏感細胞中EZH2被抑制之後H3K27ac水平的增加導致了多種致癌信號通路的激活，體內成瘤模型甚至顯示某些腫瘤在EZH2抑制劑處理的情況下反而進展更快。為了干預H3K27ac水平升高導致的EZH2抑制劑耐藥，作者嘗試了聯用BRD4抑制劑（
下圖），體外體內實驗都證實BRD4抑制劑能恢復不敏感細胞對EZH2抑制劑的敏感性，抑癌效果顯著增強。
EZH2抑制劑聯合BRD4抑制劑作用模式圖
儘管如此，二聯療法在一部分腫瘤中的抑瘤作用仍然不夠顯著。質譜分析顯示，二聯療法顯著地激活了這些腫瘤細胞中的某些致瘤信號通路，比如MAPK通路。據此，作者在二聯療法基礎上再加入ERK抑制劑，極強地抑制了腫瘤的生長，而且安全劑量情況下對動物的毒性較小。這種三聯療法阻斷了多種致瘤途徑，因此，儘管某些腫瘤細胞中EZH2可能同時發揮酶活性不依賴的作用
【4】,該療法可能仍然有效。此外，本文體內實驗模型所用的是免疫缺陷小鼠，考慮到EZH2抑制劑也會提高殺傷性T細胞浸潤【5】和NK細胞殺傷活性【6】，這一三聯療法在正常免疫環境下的抗腫瘤療效依然很有希望。因此，這項突破性工作不僅揭示了EZH2抑制劑對大部分實體瘤治療無效的分子機制，同時提供了可能的解決方案，使EZH2抑制劑的臨床應用範圍得以擴大，而且也昭示著對錶觀遺傳調控更深入細緻的理解有望帶來更特異、更有效的抗癌靶向治療手段。
參考文獻
1. Shen H, Laird PW. Interplay between the cancer genome and epigenome. Cell. 2013;153(1):38-55. 2. Helin K, Minucci S. The role of chromatin-associated proteins in cancer. Annu Rev Cancer Biol . 2017; 1: 355-77.
3. de Vries NA, Hulsman D, Akhtar W, de Jong J, Miles DC, Blom M, et al. Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression. Cell Rep. 2015;10(3):383-97.
4. Kim K, Kim W, Howard T, Vazquez F, Tsherniak A, Wu J, et al. SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nat Med. 2015; 21(12): 1491-96.
5. Peng D, Kryczek I, Nagarsheth N, Zhao L, Wei S, Wang W, et al. Epigenetic silencing of TH1-type chemokines shapes tumour immunity and immunotherapy. Nature. 2015;527(7577):249-53.
6. Bugide S, Green MR, Wajapeyee N. Inhibition of Enhancer of zeste homolog 2 (EZH2) induces natural killer cell-mediated eradication of hepatocellular carcinoma cells. Proc Natl Acad Sci U S A. 2018;115(15):E3509-E18.
專家點評
張毅（哈佛大學教授，HHMI研究員）
Polycomb group (PcG) proteins have long been known to be part of the cellular memory system involved in maintaining the spatial patterns of homeotic box (Hox) gene expression during embryonic development (Francis and Kingston, 2001). The Polycomb genes were originally named based on genetic screens in which gene mutations cause the generation of multi-sexcomb in flies. Due to his pioneering work in identifying genes (including PcG genes) controlling body patterning, Edward B. Lewis received the 1995 Nobel Prize in Physiology or Medicine (https://www.nobelprize.org/prizes/medicine/1995/lewis/facts/). Nevertheless, how PcG proteins maintaining the “off state” of Hox genes to control body patterning during embryonic development was not known till the discoveries that the two major PcG protein complexes PRC1 and PRC2 respectively possess histone H2A uniquitylation and histone H3 lysine 27 methylation (H3K27me3) activities to mediate Hox gene silencing (Cao et al., 2002; Wang et al., 2004). Around the same time, it was also found that the catalytic subunit of the PRC2, EZH2, is overexpressed in prostate cancer (Varambally et al., 2002). Subsequent studies indicated that overexpression of EZH2 is generally associated with various cancers (Comet et al., 2016) raising the possibility that EZH2 might be a promising target for cancer treatment. The biotech company Epizyme and the pharmaceutical company GSK were the first to develop small chemical inhibitors specifically targeting EZH2 with efficacy for hematological cancers (Knutson et al., 2012; McCabe et al., 2012). Despite the promising clinic results of these inhibitors in hematological cancers, whether these inhibitors are effective for solid tumors overexpressing EZH2 remains to be determined.
In this study, Huang et al. tested the EZH2 inhibitors (EZH2is) in a panel of 83 cancer cell lines that include both hematological and solid tumors and found that hematological tumor cell lines are relatively more sensitive to the inhibitors compared to the solid tumor cell lines. To determine why solid tumor cell lines are less sensitive to EZH2i, the authors analyzed 111 histone marks of EZH2i sensitive and insensitive cells and observed that the insensitive cells have a much higher H3K27 acetylation (H3K27ac) levels compared to those of the sensitive cells. Interestingly, the EZH2i sensitivity and drug-induced change in H3K27ac exhibited a reciprocal relationship suggesting that increased H3K27ac might contribute to cell’s resistance to EZH2 inhibition. Consistent with this notion, the authors found that the EZH2i insensitive cell lines express multiple oncogenes at a higher level which correlates with high levels of H3K27ac. Given previous studies demonstrating that H3K27ac is “write” by p300/CBP and “read” by the Bromodomain protein BRD4, the authors used a BRD4 inhibitor to block the H3K27ac function so that the EZH2i insensitive cells can be sensitized. Indeed, this combinational treatment significantly improved the efficacy of EZH2i in both cell culture and mouse xenograft models, supporting the author’s hypothesis as well as extending EZH2i’s therapeutic potential. Despite the use of BRD4 inhibitor improved the efficacy of EZH2i, a subset of cancer cells still escape the combinational treatment. To find out why this combined approach does not work for these cancer cells, the authors performed comparative ChIP-seq, RNA-seq, and proteomic analysis which identified a few oncogenic pathways, including the MAPK pathway, that escape the combined treatment. This observation prompted the authors to interfere the oncogenic MAPK pathway to further expand the therapeutic potential to a number of tumor models, including pancreatic and lung cancers.
In summary, using a comprehensive approaches, the authors demonstrate the existence of a complex network of cross talks of epigenetic modifications that regulate cancer cells’ response to EZH2i treatment. In addition, the study provides a strategy for patient stratification and therapeutic intervention. Specifically, for EZH2+/MLL- patients, EZH2i alone might be effective. However, for EZH2+/MLL+ patients, a combined EZH2i and BRD4i regiment might be needed. If this is still not effective, a triple combination that include the MAPK inhibitor might be considered. Given the generally encouraging xenograft results, clinic trial might be expected. Thus, this study provides a strong scientific bases for expanding the efficacy of EZH2i through combinational therapies.
References
Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R.S., and Zhang, Y. (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298, 1039-1043.
Comet, I., Riising, E.M., Leblanc, B., and Helin, K. (2016). Maintaining cell identity: PRC2-mediated regulation of transcription and cancer. Nat Rev Cancer 16, 803-810.
Francis, N.J., and Kingston, R.E. (2001). Mechanisms of transcriptional memory. Nature reviews Molecular cell biology 2, 409-421.
Knutson, S.K., Wigle, T.J., Warholic, N.M., Sneeringer, C.J., Allain, C.J., Klaus, C.R., Sacks, J.D., Raimondi, A., Majer, C.R., Song, J., et al. (2012). A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nature chemical biology 8, 890-896.
McCabe, M.T., Ott, H.M., Ganji, G., Korenchuk, S., Thompson, C., Van Aller, G.S., Liu, Y., Graves, A.P., Della Pietra, A., 3rd, Diaz, E., et al. (2012). EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature 492, 108-112.
Varambally, S., Dhanasekaran, S.M., Zhou, M., Barrette, T.R., Kumar-Sinha, C., Sanda, M.G., Ghosh, D., Pienta, K.J., Sewalt, R.G., Otte, A.P., et al. (2002). The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419.
Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst, P., Jones, R.S., and Zhang, Y. (2004). Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873-878.
專家點評
蔣華良（中國科學院院士、中科院上海藥物所所長）
表觀遺傳領域是腫瘤治療研究的前沿陣地，特別組蛋白甲基轉移酶EZH2，因其在多種血液系統腫瘤和實體瘤中廣泛存在基因擴增、激活突變或過表達，並與腫瘤發生發展密切相關，一直是腫瘤學基礎研究及各大藥廠新藥研發追逐的熱點（Kim and Roberts, 2016）。然而，在腫瘤學研究及抑制劑發現齊頭並進的同時，對於EZH2抑制劑的療效響應機制和敏感群體的認識卻大大滯後，成為EZH2抑制劑臨床轉化的瓶頸。2018年8月，當前臨床研究進展最快的EZH2抑制劑Tazemetostat（Epizyme公司研發）單藥治療EZH2突變的瀰漫性大B細胞淋巴瘤（Diffuse large B cell lymphoma）宣佈失敗（NCT01897571），更加凸顯深入系統認識EZH2抑制劑臨床治療背後的生物學機制的重要性與緊迫性。
中科院上海藥物研究所耿美玉研究員、丁健院士和譚敏佳研究員的團隊長期從事抗腫瘤藥物敏感群和耐藥機制研究。團隊研究人員通過梳理以往關於敏感群體的研究發現，腫瘤中EZH2自身突變、EZH2功能相對的表觀遺傳調控複合物功能（或其底物的）異常，如SWI-SNF成員失活突變（Kim et al., 2015）和H3K27突變（Mohammad et al., 2017）會導致該群體對EZH2抑制敏感。已有認識將上述群體中EZH2抑制劑的治療效應發揮歸結於其底物組蛋白H3的27位賴氨酸甲基化（H3K27me）水平狀態的變化。但是，一個顯而易見的矛盾點是，EZH2抑制劑能普遍降低所有腫瘤的H3K27me水平，而治療響應迥異。
對近百株腫瘤細胞EZH2抑制劑的敏感性和表觀遺傳動態修飾變化進行系統篩查，結合組蛋白修飾全景的檢測分析方法，團隊研究人員發現EZH2抑制劑可改變數十種組蛋白修飾狀態、重塑腫瘤細胞內蛋白修飾格局。尤為值得關注的是，EZH2被抑制後，可導致H3K27位甲基化與乙酰化（H3K27ac）修飾的轉換，且恰是這種修飾模式的轉換決定了EZH2抑制劑治療的不敏感。有關抑制EZH2可介導H3K27ac上調這一現象早在2010年就被報道（Pasini et al., 2010），但是，由於該研究及後繼相關研究主要將該現象歸結為蛋白修飾佔位效應的體現，屬繼發效應，故並未引起領域的重視。研究團隊通過組蛋白修飾譜系統分析揭示，H3K27me與H3K27ac的轉換具顯著有特異性；且在EZH2抑制劑引發的數十種組蛋白修飾改變中，只有H3K27ac變化與藥物敏感性相關。H3K27me與H3K27ac作為基因轉錄開關的兩個重要標誌，其在轉錄調調控中的對立關係深為領域所知。這一關聯性的存在提示，
EZH2抑制劑在引發基因轉錄關閉的同時，還通過影響H3K27ac控制轉錄開啟，具有雙重角色。而H3K27ac開啟的癌基因轉錄，是造成EZH2抑制劑不敏感的核心機制。接下來的問題是，為什麼EZH2抑制劑引起的H3K27me與H3K27ac轉換主要在大部分的實體瘤中發生？團隊研究人員發現，TrxG（Trithorax group）家族成員MLL1的表達水平是核心制約因素。在大多數實體瘤中，EZH2和MLL1均高表達，抑制EZH2的同時，MLL1招募p300和CBP形成複合物並催化H3K27ac修飾上調。反之，在MLL1低表達細胞中，抑制EZH2導致 H3K27me下調，卻難以觸發H3K27ac發生。這一發現提出了基於MLL1的H3K27me與H3K27ac的相互牽制決定EZH2抑制劑生物學效應的工作模型，揭開了PRC2和TrxG兩大轉錄開關家族之間高度依存關係的新篇章。因此，EZH2抑制劑、MLL抑制劑以及p300和BRD4抑制劑，這些原本看似功能相悖的抑制劑，以MLL1募集功能為紐帶，有了聯合使用的可能。進一步研究證實EZH2和BRD4抑制劑聯合用藥在大多數EZH2高表達的腫瘤中療效顯著，為上述機制模式提供了有力的支持。
有意思的是，研究團隊通過組蛋白修飾譜-轉錄譜-蛋白質譜-磷酸化譜的協同差異化分析發現，在一小部分腫瘤中，MAPK通路的異常激活是導致表觀遺傳修飾協同抑制仍然不能完全阻止腫瘤生長的重要因素。在此基礎上，針對性聯合抑制MAPK通路，可實現對肝癌、胰腺癌等難治性腫瘤的分群體組合治療，效果顯著。上述發現證實，
協同抑制表觀遺傳交互調控的方案，儘管可實現對腫瘤轉錄異常的控制，但有些腫瘤依舊存留或被反饋激活的激酶信號通路，仍然可能是腫瘤細胞生存的驅動力量，這可能是表觀遺傳抑制劑獲得性耐藥產生的又一不可忽視的因素。總之，該項研究著眼全局，全景分析了在廣泛存在EZH2異常的腫瘤中，EZH2抑制劑如何誘發組蛋白修飾相互轉變，如何與激酶信號通路的交互影響，並據此實現了對EZH2高表達腫瘤的精準治療分層，並針對性提出了遞進式個性化治療方案，對於當前EZH2抑制劑臨床研究的困境破局具有重要的指導意義，同時對於其他表觀遺傳類靶點藥物敏感群體選擇和耐藥機制發現及聯合用藥策略制定，也均具有重要的示範作用。
參考文獻
Kim, K.H., Kim, W., Howard, T.P., Vazquez, F., Tsherniak, A., Wu, J.N., Wang, W., Haswell, J.R., Walensky, L.D., Hahn, W.C., et al. (2015). SWI/SNF-mutant cancers depend on catalytic and non-catalytic activity of EZH2. Nature medicine 21, 1491-1496.
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