β-actin

引言 近年来白血病治疗已经取得了显著进展，但是MLL（mixed-lineage leukemia, 也称为MLL1、ALL-1或KMT2A）基因重排引起的白血病仍然难以治疗，患者对现有疗法预后不佳。由于染色体重排导致的MLL基因融合在急性白血病中尤为常见，特别是在婴幼儿急性白血病中。这些重排与极差的预后相关，其中35-50%的儿童急性髓性白血病（AML）病例具有MLL重排的特征。这一患者群体对标准治疗的反应不佳，凸显了发展其他有效疗法的紧急性和必要性。 2017年，闻宏、万里玲等发现ENL（也称为MLLT1或YEATS1），一种组蛋白乙酰化阅读器蛋白对MLL基因重排白血病的生长是必需的【1】。ENL作为转录共激活因子，主要与SEC复合物（Super elongation complex）和DOT1L复合物（组蛋白H3K79甲基转移酶）相结合，在转录延伸过程中发挥关键作用。复合物中的ENL通过其YEATS结构域识别组蛋白H3赖氨酸乙酰化，促进招募和稳定相关的转录机器，进而实现激活目的基因。基因敲除ENL或YEATS结构域突变均显示出抑制MLL基因重排白血病生长的潜力，表明了ENL作为MLL基因重排白血病治疗靶点的前景。 蛋白质降解靶向嵌合体（PROTACs）是一类异双功能小分子，它们通过介导目标蛋白与E3泛素连接酶复合物的结合，从而在蛋白酶体（proteasome）中引发配体依赖性的蛋白质降解【2】。这一技术在生物医学和药物发现领域引起了极大关注。此前关于传统ENL小分子抑制剂的研究为ENL PROTAC降解剂的开发奠定了基础【3, 4】，但ENL PROTAC降解剂这一领域的进展仍然有限。 近日，Van Andel研究所闻宏课题组和西奈山Icahn医学院金坚课题组合作在Science Advances 发表了题为A potent and selective ENL degrader suppresses oncogenic gene expression and leukemia progression的研究论文。该研究开发了一种高效且特异的ENL蛋白PROTAC降解剂MS41，并详细阐述了MS41抑制白血病细胞生长的分子机制。同时，通过小鼠肿瘤模型验证了其在体内的抑制效果。 研究者使用了PFI-6（一种特异性结合ENL/AF9的YEATS结构域的小分子抑制剂）【4】以及VHL E3泛素连接酶的经典配体VHL1-Me进行化合物的设计，开发出ENL蛋白PROTAC降解剂MS41（图1A）。MS41在多种人类白血病细胞系中均可以高效的降解ENL蛋白，在MV4;11和Jurkat细胞系中的半数蛋白降解浓度（DC50) 分别为3.50 nM 和 3.03 nM，最大降解率（Dmax）均超过93% (图1B和1C)。对使用100 nM MS41处理2小时的MV4;11细胞进行定量蛋白质组学分析，结果显示ENL蛋白是唯一显著减少的蛋白，显示出MS41的特异性（图1D）。进而，研究者发现MS41可以显著抑制多种人类MLL重排白血病细胞系的生长（图1E）。MS41在MV4;11和RS4;11细胞中的半数生长抑制浓度（GI50）分别为21.28 nM和25.06 nM。有趣的是，一种携带AML1-ETO易位的白血病细胞系KASUMI对MS41也非常敏感。MS41对不依赖于ENL的白血病细胞系Jurkat和K-562的生长则没有影响，体现了它在细胞水平的特异性。 图1.MS41是高效且特异的ENL蛋白降解剂。（A）MS41的化学结构以及降解ENL蛋白的示意图。（B）在MV4;11和Jurkat细胞中检测ENL和β-Actin表达水平的免疫印迹。（C）MS41在MV4;11和Jurkat细胞中的DC50和Dmax值。（D）MV4;11细胞中的定量蛋白质组学分析。（E）MS41在人类白血病细胞系中的生长抑制曲线。（Credit: Science Advances） 研究者进一步探索了MS41抑制白血病细胞生长的分子机制。对MS41处理后的MV4;11细胞进行RNA-seq，分析结果显示关键癌基因（如MYC）和白血病干细胞基因（如HOXA9、HOXA10、MEIS1和MYB）的表达显著降低，而髓系分化标志物（如CD11B）的表达增加。ENL通过与多个染色质相关蛋白复合物相互作用来发挥转录调控功能，特别是SEC复合物和DOT1L复合物。这两个复合物都与MLL重排引起的白血病相关，研究者之前的研究表明，ENL在将这些复合物招募到染色质上的过程中发挥了关键作用【1】。为了研究MS41介导的ENL降解是否影响这些转录相关复合物在染色质上的结合，研究者对SEC（AFF1和CDK9）、DOT1L以及延伸型RNA聚合酶（Pol II S2P）进行了ChIP-seq。分析结果表明，SEC 和 DOT1L复合物成分以及延伸型RNA Pol II在染色质上的结合与ENL在染色质上的结合高度正相关，它们在ENL结合密集的基因上也结合更为密集。在MS41处理之后，这些转录相关蛋白在染色质上的结合均明显减少，在ENL结合密集基因上的减少尤为显著。最后，研究者使用白血病异种移植（xenograft）的小鼠模型验证了MS41在体内的作用效果。MS41处理显著延迟了异种移植小鼠的白血病进展，并延长了生存期。小鼠的中位生存期从对照组的37天延长至MS41组的49.5天。 综上所述，该研究开发出了一种高效且特异的降解ENL蛋白的PROTAC降解剂MS41,进而在细胞中和小鼠模型中验证了MS41抑制白血病细胞生长的分子机制。该研究结果强调了药物性降解ENL在治疗ENL依赖性癌症中的治疗潜力。因此，MS41不仅是一个有价值的化学探针，还可能成为进一步开发的抗癌治疗药物。 参考文献 1. Wan L, Wen H, Li Y, Lyu J, Xi Y, Hoshii T, Joseph JK, Wang X, Loh YE, Erb MA, Souza AL, Bradner JE, Shen L, Li W, Li H, Allis CD, Armstrong SA, and Shi X. ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia. Nature, 2017; 832 543, 265-269. 2. Dale B, Cheng M, Park KS, Kaniskan HÜ, Xiong Y and Jin J. 2021. Advancing targeted protein degradation for cancer therapy. Nature Reviews Cancer, 2021; 21(10), pp.638-654. 3. Ma XR, Xu L, Xu S, Klein BJ, Wang H, Das S, Li K, Yang KS, Sohail S, Chapman A, Kutateladze TG, Shi X, Liu WR, and Wen H. Discovery of selective small-molecule inhibitors for the ENL YEATS domain. Journal of medicinal chemistry, 2021; 64(15), pp.10997-11013. 4. Raux B, Buchan KA, Bennett J, Christott T, Dowling MS, Farnie G, Fedorov O, Gamble V, Gileadi C, Giroud C, Huber KVM, Korczynska M, Limberakis C, Narayanan A, Owen DR, Saez LD, Stock IA, and Londregan AT. Discovery of PFI-6, a small-molecule chemical probe for the YEATS domain of MLLT1 and MLLT3. 2023; Bioorg Med Chem Lett, 129546. https://www.science.org/doi/10.1126/sciadv.ado1432 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

引言 在真核细胞中，核小体是染色质的基本组成单元，其可以通过改变自身的定位、共价修饰组蛋白及置换组蛋白变体的方式来改变染色质的结构和调控基因表达。核小体的这些调控主要由染色质重塑复合物和组蛋白修饰复合物来执行。人源TIP60复合物（hTIP60）是一个具有 17 个亚基的超大型复合物（分子量约为1.7 MDa），它的独特之处在于同时拥有组蛋白变体置换和组蛋白乙酰化活性。根据酶活和分子组成上的相似性，hTIP60被认为是酵母中 SWR-C复合物（ySWR-C）和 NuA4复合物（yNuA4）的融合物。之前的研究表明，hTIP60在多种生物学过程中发挥重要作用，包括转录调控、DNA修复、细胞周期控制和胚胎发育等，同时hTIP60的突变被认为与多种癌症相关。 过往的研究报道了hTIP60 复合物的同源物如 ySWR-C、 yNuA4、hSRCAP-C等复合物的高分辨冷冻电镜结构，从结构生物学的角度阐明了这些复合物结合底物核小体并发挥活性的机制，另外也有一些研究通过晶体学、核磁共振波谱（NMR）等技术对 hTIP60 复合物的部分亚基如YL1、YEATS4、MRG15-MRGBP、EPC1-MBTD1等进行了结构分析，而hTIP60 复合物的完整结构信息一直处于空白状态，因此目前该复合物的组装及其结合核小体的模式仍然很不清楚。 2024年8月17日，复旦大学徐彦辉团队在Nature Communications杂志上在线发表了题为Structure of the human TIP60 complex的研究论文。该研究利用单颗粒冷冻电镜技术，结合交联质谱及体外生化功能分析阐明了hTIP60复合物精细的分子组装机制，并提出了该复合物作用于核小体的模型。 根据已有的关于hTIP60复合物的报道及其与yNuA4和ySWR-C复合物的同源性研究，由17个亚基组成的hTIP60复合物可被划分为5个模块，其分别为含有Snf2家族ATPase结构域的motor 模块、调节性的ARP模块、发挥乙酰化功能的HAT模块、含有多个组蛋白修饰阅读器的TINTIN（Trimer Independent of NuA4 for Transcription Interactions with Nucleosomes）模块、以及用于结合转录激活因子的TRRAP模块（图1）。 图1. 人源TIP60复合物的模块化组织和结构域示意图(Credit: Nature Communications) 徐彦辉课题组的这项工作报道了hTIP60复合物单体状态下的冷冻电镜结构，9.4 Å的整体冷冻电镜密度图表明该复合物主要由两大部分构成，这两部分被称为核心亚复合物（包括motor 模块及ARP模块）和 TRRAP模块，它们通过一段柔性区域连接（图1a），随后研究人员将这两部分的分辨率均细化至3.2 Å（图1b）。这些结构表明EP400亚基作为hTIP60复合物的骨架蛋白，整合了复合物的motor 模块、ARP（actin-related protein）模块和TRRAP 模块。在motor模块中，RUVBL1-RUVBL2六聚体是EP400亚基的ATPase结构域和YL1亚基组装的刚性核心。在ARP模块中，ACTL6A-ACTB异二聚体和一个额外的ACTL6A 亚基通过疏水作用与EP400 亚基的HSA螺旋进行接触；而亚基 DMAP1、EPC1和EP400之间的相互作用网络增强了ACTL6A-ACTB异二聚体和EP400-HSA之间的结合。在TRRAP模块中，EP400亚基的 SANT结构域及其C端区域与TRRAP亚基存在广泛的相互作用界面。总体来看，ARP模块与motor模块之间的连接非常稳定，而其与TRRAP模块之间的连接非常灵活，这是hTIP60复合物特有的结构特征。 除了上述已经确定的模块外，hTIP60 复合物的整体冷冻电镜密度图中TRRAP模块和核心亚复合物之间还存在了一些较弱的密度，这些密度可能属于hTIP60复合物其他的模块，包括HAT模块和TINTIN模块（图2a）。因此在hTIP60复合物中，其HAT模块、TINTIN模块和TRRAP模块都以灵活的方式连接到核心亚复合物中，这种结构上的可塑性可能有助于该复合物在细胞中识别复杂的染色质环境并找到合适的底物。 图2. 人源TIP60复合物单体的结构：(a). hTIP60 复合物在低轮廓水平（low contour level，左）和高轮廓水平（high contour level，右）的整体冷冻电镜密度图。(b). 人源TIP60复合物单体状态下的冷冻电镜重构密度图及三维模型。(Credit: Nature Communications) 此外，该研究还报道了与核小体结合的hTIP60复合物的较低分辩率的冷冻电镜结构，该结构显示核小体结合在hTIP60复合物的核心亚复合物和TRRAP模块之间（图3），值得注意的是，在对hTIP60-核小体复合物的冷冻电镜数据进行仔细的分析后，研究人员并未观察到hTIP60的核心亚复合物像其他重塑复合物那样（即催化亚基的ATPase结构域抓住核小体的DNA以进行染色质重塑）结合核小体的构象。 图3. 核小体结合状态下的人源TIP60复合物的冷冻电镜重构密度图及模型(Credit: Nature Communications) 综上所述，该研究揭示了hTIP60 复合物的 motor 模块、ARP 模块和 TRRAP 模块高分辨的结构信息，阐明了该复合物的分子组装机制及各模块之间的相互作用模式；并首次报道了和核小体结合的hTIP60复合物的冷冻电镜重构密度图，从结构生物学的角度对该复合物如何与核小体作用提供了见解，为继续探索hTip60复合物作用于染色质并调控基因表达的机制提供了线索。 参考文献 https://www.nature.com/articles/s41467-024-51259-z 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

LEIDEN, Netherlands & CAMBRIDGE, Mass., April 22, 2024 (GLOBE NEWSWIRE) -- ProQR Therapeutics NV (Nasdaq: PRQR) (ProQR), a company dedicated to changing lives through transformative RNA therapies, today announced it will present new preclinical data for its proprietary Axiomer™ RNA editing technology platform, including the first preclinical data for its AX-0810 pipeline program for cholestatic diseases targeting NTCP at the American Society of Gene & Cell Therapy (ASGCT) Annual Meeting, May 7-11, 2024, in Baltimore, Maryland. “The in vitro and in vivo data that we will be presenting at ASGCT further build upon our experience with Axiomer and highlight the significant potential we see with the platform, including now preclinical proof of concept for cholestatic diseases targeting NTCP,” said Gerard Platenburg, Chief Scientific Officer and co-founder, ProQR. “We continue to be impressed by the robust editing efficiency of Axiomer editing oligonucleotides and look forward to leveraging these data as we advance AX-0810 toward the clinic.” Wednesday, May 8, 2024 poster presentations: P-705: “Robust and Durable RNA Editing In Vivo with Axiomer™ Editing Oligonucleotides in Non-Human Primates” Building on its robust platform data, the Company will present additional non-human primate (NHP) data including with ACTB and NTCP and initial in vitro and in vivo preclinical proof of concept for its AX-0810 program targeting NTCP for cholestatic diseases, including hepatic editing in NHP with strong correlating impact on disease-relevant biomarkers. P-726, partner poster presentation by Eli Lilly: “Complex Metabolism and Prolonged PK/PD of a GalNAc-Conjugated Editing Oligonucleotide (EON) in Mice” Following presentation at ASGCT, the posters will be available via ProQR’s website in the Presentations and Publications section. About Axiomer™ ProQR is pioneering a next-generation RNA base editing technology called Axiomer™, which could potentially yield a new class of medicines for diverse types of diseases. Axiomer “Editing Oligonucleotides”, or EONs, mediate single nucleotide changes to RNA in a highly specific and targeted way using molecular machinery that is present in human cells called ADAR (Adenosine Deaminase Acting on RNA). Axiomer EONs are designed to recruit and direct endogenously expressed ADARs to change an Adenosine (A) to an Inosine (I) in the RNA – an Inosine is translated as a Guanosine (G) – correcting an RNA with a disease-causing mutation back to a normal (wild type) RNA, modulating protein expression, or altering a protein so that it will have a new function that helps prevent or treat disease. About ProQR ProQR Therapeutics is dedicated to changing lives through the creation of transformative RNA therapies. ProQR is pioneering a next-generation RNA technology called Axiomer™, which uses a cell’s own editing machinery called ADAR to make specific single nucleotide edits in RNA to reverse a mutation or modulate protein expression and could potentially yield a new class of medicines for both rare and prevalent diseases with unmet need. Based on our unique proprietary RNA repair platform technologies we are growing our pipeline with patients and loved ones in mind. Learn more about ProQR at www.proqr.com. Forward Looking Statements for ProQR This press release contains forward-looking statements. All statements other than statements of historical fact are forward-looking statements, which are often indicated by terms such as “continue,” "anticipate," "believe," "could," "estimate," "expect," "goal," "intend," "look forward to", "may," "plan," "potential," "predict," "project," "should," "will," "would" and similar expressions. Such forward-looking statements include, but are not limited to, statements regarding our Axiomer™ technology, including the continued development and advancement of our Axiomer platform, the therapeutic potential of our Axiomer RNA editing oligonucleotides and our ability to expand preclinical in vivo and in vitro data, the timing, progress and results of our preclinical studies and other development activities, including the release of data related thereto, and the potential of our technologies and product candidates . Forward-looking statements are based on management’s beliefs and assumptions and on information available to management only as of the date of this press release. Our actual results could differ materially from those anticipated in these forward-looking statements for many reasons, including, without limitation, the risks, uncertainties and other factors in our filings made with the Securities and Exchange Commission, including certain sections of our most recent annual report filed on Form 20-F. These risks and uncertainties include, among others, the cost, timing and results of preclinical studies and clinical trials and other development activities by us and our collaborative partners whose operations and activities may be slowed or halted shortage and pressure on supply and logistics on the global market; the likelihood of our preclinical and clinical programs being initiated and executed on timelines provided and reliance on our contract research organizations and predictability of timely enrollment of subjects and patients to advance our clinical trials and maintain their own operations; our reliance on contract manufacturers or suppliers to supply materials for research and development and the risk of supply interruption or delays from suppliers or contract manufacturers; the potential for future data to alter initial and preliminary results of early-stage clinical trials; the unpredictability of the duration and results of the regulatory review of applications or clearances that are necessary to initiate and continue to advance and progress our clinical programs; the ability to secure, maintain and realize the intended benefits of collaborations with partners, including the collaboration with Eli Lilly and Company; the possible impairment of, inability to obtain, and costs to obtain intellectual property rights; possible safety or efficacy concerns that could emerge as new data are generated in research and development; general business, operational, financial and accounting risks, and risks related to litigation and disputes with third parties; and risks related to macroeconomic conditions and market volatility resulting from global economic developments, geopolitical instability and conflicts. Given these risks, uncertainties and other factors, you should not place undue reliance on these forward-looking statements, and we assume no obligation to update these forward-looking statements, even if new information becomes available in the future, except as required by law. ProQR Therapeutics N.V. Investor contact:Sarah KielyProQR Therapeutics N.V.T: +1 617 599 6228skiely@proqr.comorPeter KelleherLifeSci AdvisorsT: +1 617 430 7579 pkelleher@lifesciadvisors.com Media contact:Robert StanislaroFTI ConsultingT: +1 212 850 5657robert.stanislaro@fticonsulting.com