BUB1

使用抗PD-L1抗体的癌症免疫疗法已被用于多种临床应用，并取得了一定的效果。然而，自身免疫性、肿瘤过度进展、患者总体反应率较低等局限性阻碍了其进一步的临床应用。越来越多的证据表明，PD-L1 不仅存在于肿瘤细胞膜中，还存在于细胞质、外泌体，甚至细胞核中。其中，PD-L1在肿瘤中的动态和空间异质性表达是导致PD-L1抗体疗效不理想的主要原因。今天小编和大家一起学习一下这篇今年11月发表在Advanced Science（IF 15.84分）上的综述，文章创造性地提出了PD-L1空间异质性表达的概念，涵盖了各种PD-L1的结构和生物学功能。然后深入分析了 PD-L1 的调控机制和潜在治疗靶点，力求为今后的研究提供坚实的基础。此外，文章还总结了药物的现状，尤其是针对这些新靶点开发的小分子调节剂，为潜在的 PD-L1 治疗策略提供了新的视角。挑战与机遇并存：癌症治疗中 PD-L1 的空间异质性表达一、研究背景免疫疗法的发明具有革命性意义，在2018年获得了诺贝尔奖。PD-1/PD-L1 通路的激活与肿瘤细胞免疫逃避机制有关，但由于 PD-L1 在空间上的异质性表达，其研究前景远大于其受体 PD-1。最近的研究发现，PD-L1 除了定位于膜外，还存在于细胞质和其他亚细胞结构中。因此，它通过 PD-1 依赖性途径对免疫抑制功能产生影响，同时也通过独立于 PD-1 的机制促进肿瘤的侵袭和增殖。因此，开发 PD-L1 小分子调节剂，对于阻断相关的 PD-L1 过程和逆转肿瘤微环境中的免疫抑制至关重要。据报道，抗PD-L1治疗的疗效与肿瘤细胞上膜PD-L1（mPD-L1）的表达呈正相关。PD-L1抗体的主要机制是与mPD-L1结合，从而恢复T细胞的正常肿瘤杀伤功能。然而，单药抑制PD-L1的应答率很少超过30%，这表明单纯抑制mPD-L1不足以取得满意的临床疗效。此外，最近的研究表明，PD-L1在肿瘤细胞中的空间异质性表达似乎是导致抗PD-L1抗体疗效不理想的主要原因。mPD-L1通过HIP1R进入细胞膜，而细胞质中的PD-L1（cPD-L1）则通过TRAPPC4循环至细胞膜。同时，外泌体PD-L1（exoPD-L1）可从原发肿瘤分泌至其他组织，全面抑制肿瘤杀伤作用。由于其表达水平的异质性，有必要了解 PD-L1 在肿瘤细胞中的亚细胞分布及调控其表达的内在机制，从而帮助制定更具针对性的临床治疗策略。图1 PD-L1“空间异质性表达”示意图在这篇综述中，旨在全面了解 PD-L1 的空间异质性表达，包括其亚细胞分布、结构、功能和调控机制。通过探索 PD-L1 在癌症进展中的多方面作用，包括其在免疫/非免疫功能中的参与，希望能促进更有效的肿瘤治疗方法的发展。合理利用小分子调制剂和传统抗体疗法将为癌症治疗带来更大的希望。二、PD-L1的结构及其功能PD-L1 在不同的亚细胞位置表现出不同的构象，由于其空间异质性表达，PD-L1 在特定亚细胞结构中也发挥着特殊作用。典型的 PD-L1，即 mPD-L1，通常以跨膜蛋白的形式存在，其功能是作为 PD-1 配体，与细胞毒性 T 淋巴细胞膜上的 PD-1 结合，抑制肿瘤免疫反应。mPD-L1 主要以 PD-1 依赖性方式调节肿瘤免疫活性。非典型 PD-L1，特别是 cPD-L1、nPD-L1 和 exoPD-L1，表现出不同的生物学功能，与肿瘤细胞增殖、免疫反应、DNA 损伤反应和基因表达控制密切相关。这些效应包括 PD-1 依赖途径和 PD-L1 独立途径。图2 PD-L1在癌细胞中的细胞内功能2.1 mPD-L1的结构和功能mPD-L1中 Ig 样 V 型结构域是一个重要的治疗靶点。在功能方面，肿瘤细胞中表达的 mPD-L1 可与 T 细胞膜或肿瘤细胞中表达的 PD-1 特异性结合，诱导下游 PD-1 信号通路，导致肿瘤免疫逃逸（图 3）。除了能与 T 细胞上的 PD-1 结合，mPD-L1 还能与肿瘤细胞上的 PD-1 结合。此外，mPD-L1 还能非 PD-1 蛋白结合，从而对肿瘤生长产生不同的影响（图 3）。最近的研究表明，mPD-L1 的细胞质 C 尾可以通过阻止细胞凋亡和增加 mPD-L1 本身的稳定性来提高肿瘤细胞的存活率。mPD-L1的细胞质结构域是调节mPD-L1稳定性和介导肿瘤细胞免疫逃逸的关键。此外，mPD-L1 胞质尾部的碱性残基与细胞膜上的酸性磷脂相互作用，从而调节 mPD-L1 的构象并增强其稳定性。图3 PD-1 或其他受体识别 mPD-L1 会触发细胞信号通路，激活邻近细胞的共同信号通路2.2 cPD-L1 的结构和功能cPD-L1 存在于肿瘤细胞的内部，cPD-L1 的 Ig-V 和 Ig-C 结构域参与独立于 PD-1 的其他信号转导机制。到目前为止，cPD-L1 的主要功能与免疫系统无关，可调节肿瘤细胞的增殖、凋亡以及对化疗和放疗的耐受性。从机理上讲，cPD-L1 可与 DNA 损伤相关的 mRNA结合，阻止这些 RNA 降解，从而减少 DNA 损伤诱导的细胞死亡。此外，cPD-L1 还能维持 cGAS-STING 通路的组成性激活和低水平 IFN-β 的表达，从而诱导促生存 IRDS 的表达，进而导致耐药性。cPD-L1 除了在保护肿瘤细胞免受化疗和放疗影响方面发挥作用外，还参与了由乳腺癌易感蛋白-1（BRCA1）引发的肿瘤耐药过程。cPD-L1 不仅能帮助肿瘤细胞逃避化疗和放疗的细胞毒性作用，还能调节多种炎症反应，包括 COX2 和 STAT3，从而促进肿瘤的生存和生长。综上所述，cPD-L1 的非免疫功能在 DNA 损伤修复和肿瘤进展中也起着至关重要的作用。2.3 nPD-L1 的结构和功能通过核易位，cPD-L1 从细胞质穿梭到细胞核中，从而形成 nPD-L1，在前列腺癌、乳腺癌、结直肠癌以及骨肉瘤患者的 CTC 中观察到了 nPD-L1。nPD-L1可增强肿瘤细胞中免疫相关基因和促炎相关基因的表达，导致肿瘤进展。PD-L1 依赖性基因的表达是通过 nPD-L1 与 DNA 或与 DNA 结合的转录因子相互作用介导的，它作为转录因子调节免疫功能相关基因的表达。nPD-L1 也可以作为一种关键的核蛋白，在不与 PD-1 结合的情况下促进免疫依赖性细胞增殖。进一步的机理研究发现，cPD-L1 通过 p-ERK 激活转位到细胞核中，然后通过与甲状腺激素受体相关蛋白 3（THRAP3）结合，触发 BUB1（一种细胞周期蛋白控制蛋白）的表达，从而促进细胞周期的进展，促进细胞增殖。nPD-L1 的表达与肿瘤进展和预后不良密切相关。2.4 细胞外 PD-L1 的结构和功能在细胞培养物上清液和人血清等细胞外部位已发现几种不同形式的细胞外 PD-L1，包括外 PD-L1、微囊 PD-L1（mvPD-L1）和可溶性 PD-L1（sPD-L1）（图 1）。就结构而言，细胞外 PD-L1 有全长和短两种形式。exoPD-L1 的膜拓扑结构与 mPD-L1 相同，其细胞外结构域暴露在外泌体表面。至于其功能，细胞外 PD-L1 与抗肿瘤免疫反应有关。目前的研究表明，exoPD-L1 不仅在诱导免疫逃避方面起着关键作用，而且还可作为潜在的生物标志物预测抗 PD-1/PD-L1 免疫疗法的临床反应。一方面，exoPD-L1 由癌细胞分泌到远端组织，附着于 T 细胞表面的 PD-1，进而抑制和暴虐免疫系统。另一方面，研究发现exoPD-L1 能有效阻碍 T 细胞的活化和增殖，并在一定程度上对抗抗 PD-L1 抗体的阻断作用。除了抑制 T 细胞功能外，exoPD-L1 还可能影响巨噬细胞的极化。它能使肿瘤相关巨噬细胞（TAMs）极化为促进肿瘤生长的 M2 型巨噬细胞，并上调 TAMs 中 PD-L1 的表达。ExoPD-L1 还可导致对化疗药物的耐药性，并可作为临床预后的指标。exoPD-L1可作为预测肿瘤进展和免疫检查点抑制剂反应的生物标记物。除exoPD-L1外，sPD-L1也会损害T细胞的活力，使其对免疫疗法产生抗药性。三、mPD-L1 表达调控机制与肿瘤治疗策略3.1 mPD-L1 的表达调控mPD-L1的乙酰化修饰可影响mPD-L1的稳定性或亚细胞定位。乙酰化修饰能稳定 mPD-L1，乙酰化的 mPD-L1 可阻止自身向细胞质和细胞核扩散。棕榈酰化修饰可增强 mPD-L1 对靶蛋白的锚定作用并影响其稳定性。此外，mPD-L1 通过其 N 端结构域与酸性磷脂结合，增加了其稳定性。此外，mPD-L1 还能与其他膜蛋白结合以保护自身免受降解。下调后的mPD-L1可以减弱肿瘤免疫逃避，抑制癌症进展。在 mPD-L1 的重塑过程中，泛素化是一个不容忽视的关键过程。泛素化是一个酶解过程，包括泛素的附着以及蛋白酶体和溶酶体机制对标记细胞蛋白的降解（图 4）。除了泛素介导的降解外，mPD-L1 还可被 HIP1R、潜在的表面金属蛋白酶和自噬因子降解。图4 mPD-L1 的调节机制和治疗策略3.2 以 mPD-L1 为靶点加强肿瘤治疗的治疗策略目前针对 mPD-L1 的疗法可分为两大类：一类是直接灭活或降解 mPD-L1 的直接疗法，另一类是针对相关信号通路间接调节 mPD-L1 的间接疗法。通过抑制或降解 mPD-L1，这些方法可有效激发抗肿瘤免疫反应。四、cPD-L1 表达调控机制与肿瘤治疗策略图5 cPD-L1表达调控机制和治疗策略4.1 cPD-L1 表达的调控cPD-L1 可被 E3 连接酶定向降解，但泛素化的 cPD-L1 也可被去泛素化酶或去泛素化酶（DUBs）逆转，DUBs 可裂解并去除 cPD-L1 上的泛素链，保护其不被降解。此外，一些翻译后修饰（包括糖基化和磷酸化）和其他途径也用于保护 cPD-L1 免受溶酶体降解。4.2 以 cPD-L1 为靶点加强肿瘤治疗的策略cPD-L1主要有两种来源：一种来源于回收的mPD-L1，另一种来源于细胞质中新合成的PD-L1。因此，cPD-L1 可以成为更有效的治疗靶点，有望在更大范围内产生更强的抑制作用，从而促进 T 细胞的活化。通过调节相关信号通路来调节 cPD-L1 水平，在实现治疗目的方面具有巨大潜力。五、exoPD-L1 表达调控机制与肿瘤治疗策略5.1 exoPD-L1 表达调控一方面，exoPD-L1 通过旁分泌信号作用于紧邻的癌细胞和巨噬细胞，促进相邻肿瘤细胞的 PD-L1 表达和相邻巨噬细胞的 M2 极化，最终诱导免疫逃避；另一方面，它们可进入血液循环，远距离扩散至淋巴结，对新生成的可表达 PD-1 的 T 细胞产生抑制作用。exoPD-L1的表达水平主要由两个因素决定：参与外泌体形成的酶和外泌体内exoPD-L1的反馈调节机制。图6 肿瘤治疗的exoPD-L1表达调控机制和治疗策略5.2 以exoPD-L1为靶点加强肿瘤治疗的治疗策略肿瘤细胞向细胞外环境释放exoPD-L1，并将其转运至远处的淋巴结，从而抑制 T 细胞并促进免疫逃逸。抑制外泌体分泌有可能消除对 T 细胞的抑制，从而恢复抗肿瘤 T 细胞反应。越来越多的研究开始关注exoPD-L1，因此它有望成为未来癌症治疗的新靶点。然而，针对 exoPD-L1 的有效治疗药物的开发仍处于起步阶段，目前还没有直接针对 exoPD-L1 的治疗策略。现有的治疗药物几乎都使用外泌体抑制剂来抑制外泌体的产生，进而抑制exoPD-L1 的产生。exoPD-L1表达的调控是一个复杂的过程，涉及多种因素和信号通路。目前以 exoPD-L1 为靶点的治疗策略在临床前研究中显示出良好的效果，有望成为癌症治疗的新方向。六、结论与未来展望抗 PD-L1 抗体只能阻断 mPD-L1，而非典型 PD-L1 形式则无法解决，导致疗效不理想。PD-L1 的全貌更为复杂，它在细胞质、细胞核和外泌体中都有表达，这一特性被视为 "空间异质性表达"，它允许 PD-L1 通过 PD-1 独立途径介导其他肿瘤侵袭行为。这一发现将重塑 PD-L1 靶向疗法，并预示着未来一个新领域的开始。PD-L1 的这种异质性给强效调节剂的开发带来了严峻的挑战，而错综复杂的调控机制又给上游和下游调节剂的开发带来了额外的挑战。此外，PD-L1 被分为 mPD-L1、cPD-L1、nPD-L1 和 exoPD-L1，但它们之间的关系是动态的。PD-L1 靶向癌症疗法的未来将涉及多方面的方法，包括更深入地了解 PD-L1 的空间异质性表达、生物标志物鉴定、免疫系统调节、联合疗法、抗药性机制、Al 应用和改进的给药方式。通过探索这些途径，研究人员旨在开发更有效的癌症免疫疗法，改善各种癌症类型患者的预后。综述涵盖了各类PD-L1 的结构与功能，阐释了相关调控机制及其针对 PD-L1 的替代治疗策略的最新信息，为我们未来的研究提供了学术参考和研究思路，值得大家仔细研读。参考来源：Wang Y, Zhou Y, Yang L, Lei L, He B, Cao J et al. Challenges Coexist with Opportunities: Spatial Heterogeneity Expression of PD‐L1 in Cancer Therapy. Advanced Science 2023.识别微信二维码，添加生物制品圈小编，即可获取报告完整版！！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

NEWTOWN, Pa., March 15, 2023 (GLOBE NEWSWIRE) -- Onconova Therapeutics Inc. (NASDAQ: ONTX), (“Onconova”), a clinical-stage biopharmaceutical company focused on discovering and developing novel products for patients with cancer, today announced the publication of two abstracts that have been accepted for poster presentations at the American Association for Cancer Research (AACR) Annual Meeting, which is taking place at the Orange County Convention Center in Orlando, Florida from April 14 – 19, 2023. The full texts of the published abstracts can be found on the AACR Annual Meeting website. The corresponding posters will be presented during the “Cyclin-dependent Kinases and Cyclin-dependent Kinase Inhibitors” poster session, which is taking place from 9:00 a.m. – 12:30 p.m. ET on April 19, 2023. Additional information on the posters is shown below. Poster Title: Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma Abstract Number: 5974 This poster will describe studies evaluating narazaciclib in preclinical models of mantle cell lymphoma (MCL). Results from these studies demonstrated narazaciclib’s single-agent antitumor activity in MCL cell lines independent of their sensitivity to ibrutinib, which is a Bruton’s tyrosine kinase inhibitor (BTKi) approved by the U.S. Food and Drug Administration (FDA) for the treatment of MCL. When combining narazaciclib with ibrutinib, synergistic increases in antitumor activity against both BTKi-resistant and BTKi-sensitive MCL cell lines were observed. Additional analyses showed that narazaciclib’s antitumor activity against the evaluated MCL cell lines was superior to that of the FDA-approved CDK 4/6 inhibitors palbociclib and ribociclib, and similar to that of the FDA-approved CDK 4/6 inhibitor abemaciclib. Treatment with narazaciclib also led to tumor growth inhibition without detectable toxicity in a chicken embryo chorioallantoic membrane (CAM) xenograft model of MCL. Poster Title: Differential targets engaged by narazaciclib in comparison to the approved CDK4/6 inhibitors contribute to enhanced inhibition of tumor cell growth Abstract Number: 5987 This poster will describe cell-based, in vitro, and bioinformatic analyses comparing narazaciclib and palbociclib. Results from a cell-based murine mammary carcinoma model showed a stronger induction of programmed cell death with narazaciclib compared to palbociclib. In vitro and cell-based analyses revealed multiple targets that are engaged by narazaciclib but not by palbociclib. These targets included BUB1, the overexpression of which is correlated with poor survival in triple negative breast cancer. The results of additional cell-based assays that will be described in the poster suggest that narazaciclib may promote antitumor immunity and show that combining narazaciclib with autophagy inhibitors sensitizes breast cancer cells to cell death. About Onconova Therapeutics, Inc. Onconova Therapeutics is a clinical-stage biopharmaceutical company focused on discovering and developing novel products for patients with cancer. The Company has proprietary targeted anti-cancer agents designed to disrupt specific cellular pathways that are important for cancer cell proliferation. Onconova’s novel, proprietary multi-kinase inhibitor narazaciclib (formerly ON 123300) is being evaluated in two separate and complementary Phase 1 dose escalation and expansion studies. These trials are currently underway in the United States and China. Based on preclinical and clinical studies of CDK 4/6 inhibitors, Onconova is also planning a combination trial of narazaciclib with estrogen blockade in advanced endometrial cancer, as well as its clinical study in additional indications. Onconova’s product candidate rigosertib is being studied in multiple investigator-sponsored studies, including a dose-escalation and expansion Phase 1/2a study of oral rigosertib in combination with nivolumab in patients with KRAS+ non-small cell lung cancer, and a Phase 2 program evaluating rigosertib monotherapy in advanced squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa. For more information, please visit . Forward-Looking Statements Some of the statements in this release are forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended, Section 21E of the Securities Exchange Act of 1934, as amended, and the Private Securities Litigation Reform Act of 1995, and involve risks and uncertainties. These statements relate to Onconova’s expectations regarding its clinical development and trials, its product candidates, its business and financial position. Onconova has attempted to identify forward-looking statements by terminology including "believes," "estimates," "anticipates," "expects," "plans," "intends," "may," "could," "might," "will," "should," “preliminary,” “encouraging,” "approximately" or other words that convey uncertainty of future events or outcomes. Although Onconova believes that the expectations reflected in such forward-looking statements are reasonable as of the date made, expectations may prove to have been materially different from the results expressed or implied by such forward-looking statements. These statements are only predictions and involve known and unknown risks, uncertainties, and other factors, including the success and timing of Onconova's clinical trials, investigator-initiated trials and regulatory agency and institutional review board approvals of protocols, Onconova’s collaborations, market conditions and those discussed under the heading "Risk Factors" in Onconova's most recent Annual Report on Form 10-K and quarterly reports on Form 10-Q. Any forward-looking statements contained in this release speak only as of its date. Onconova undertakes no obligation to update any forward-looking statements contained in this release to reflect events or circumstances occurring after its date or to reflect the occurrence of unanticipated events. Company Contact: Mark Guerin Onconova Therapeutics, Inc. 267-759-3680 ir@onconova.us Investor Contact: Bruce Mackle LifeSci Advisors, LLC 646-889-1200 bmackle@lifesciadvisors.com

Medical researchers find an answer to the mystery of why most human embryos die young. New research by a scientist at the Milner Centre for Evolution at the University of Bath suggests that "selfish chromosomes" explain why most human embryos die very early on. The study, published in PLoS, Biology, explaining why fish embryos are fine but sadly humans' embryos often don't survive, has implications for the treatment of infertility. About half of fertilised eggs die very early on, before a mother even knows she is pregnant. Tragically, many of those that survive to become a recognised pregnancy will be spontaneously aborted after a few weeks. Such miscarriages are both remarkably common and highly distressing. Professor Laurence Hurst, Director of the Milner Centre for Evolution, investigated why, despite hundreds of thousands of years of evolution, it's still so comparatively hard for humans to have a baby. The immediate cause of much of these early deaths is that the embryos have the wrong number of chromosomes. Fertilised eggs should have 46 chromosomes, 23 from mum in the eggs, 23 from dad in the sperm. Professor Hurst said: "Very many embryos have the wrong number of chromosomes, often 45 or 47, and nearly all of these die in the womb. Even in cases like Down syndrome with three copies of chromosome 21, about 80% sadly will not make it to term." Why then should gain or loss of one chromosome be so very common when it is also so lethal? There are number of clues that Hurst put together. Firstly, when the embryo has the wrong number of chromosomes it is usually due to mistakes that occur when the eggs are made in the mother, not when the sperm is made in the father. In fact, over 70% of eggs made have the wrong number of chromosomes. Secondly, the mistakes happen in the first of two steps in the manufacture of eggs. This first step, it had been noticed before, is vulnerable to mutations that interfere with the process, such that the mutation can "selfishly" sneak into more than 50% of the eggs, forcing the partner chromosome to be destroyed, a process known as centromeric drive. This is well studied in mice, long suspected in humans and previously suggested to somehow relate to the problem of chromosome loss or gain. What Hurst noticed was that, in mammals, a selfish mutation that tries to do this but fails, resulting in an egg with one too many or one too few chromosomes, can still be evolutionarily better off. In mammals, because the mother continuously feeds the developing fetus in the womb, it is evolutionarily beneficial for embryos developing from faulty eggs to be lost earlier rather than be carried to full term. This means that the surviving offspring do better than the average. Hurst explained: "This first step of making eggs is odd. One chromosome of a pair will go to the egg the other will be destroyed. But if a chromosome 'knows' it is going to be destroyed it has nothing to lose, so to speak. Remarkable recent molecular evidence has found that when some chromosomes detect that they are about to be destroyed during this first step, they change what they do to prevent being destroyed, potentially causing chromosome loss or gain, and the death of the embryo. "What is remarkable, is that if the death of the embryo benefits the other offspring of that mother, as the selfish chromosome will often be in the brothers and sisters that get the extra food, the mutation is better off because it kills embryos." "Fish and amphibians don't have this problem," Hurst commented. "In over 2000 fish embryos not one was found with chromosomal errors from mum." Rates in birds are also very low, about 1/25th the rate in mammals. This, Hurst notes, is as predicted as there is some competition between nestlings after they hatch, but not before. By contrast, chromosome loss or gain is a problem for every mammal that has been looked at. Hurst commented, "It is a downside of feeding our offspring in the womb. If they die early on, the survivors benefit. It leaves us vulnerable to this sort of mutation." Hurst suspects that humans may indeed be especially vulnerable. In mice the death of an embryo gives resources to the survivors in the same brood. This is gives about a 10% increase in survival chance of the others. Humans, however, usually just have one baby at a time and the death of an embryo early on enables a mother to rapidly reproduce again -- she probably never even knew her egg had been fertilised. Preliminary data shows mammals such as cows, with one embryo at a time seem to have especially high embryo death rates owing to chromosomal errors, while those with many embryos in a brood, like mice and pigs, seem to have somewhat lower rates. Hurst's research also suggests that low levels of a protein called Bub1 could cause loss or gain of a chromosome in humans as well as mice. Hurst said: "The levels of Bub1 go down as mothers get older and as the rate of embryonic chromosomal problems goes up. Identifying these suppressor proteins and increasing their level in older mothers could restore fertility. "I would hope too that these insights will be one step to helping those women who experience difficulties getting pregnant, or suffer recurrent miscarriage."