<b>Supporting data for </b><b>Functional study and therapeutic potential of CDK11 in liver cancer</b>

Hepatocellular carcinoma (HCC) is a lethal disease with limited treatment. Therefore, understanding the molecular mechanism of HCC development and identifying novel therapeutic targets are urgently needed. Cyclin-dependent kinases (CDKs) belong to the serine / threonine kinase family, which is involved in gene transcription regulation and cell cycle control. Targeting CDKs is a promising therapeutic avenue for cancer patients as several CDK4/CDK6 inhibitors have already been approved by FDA for breast cancer therapy. Here, we employed a genome-wide CRISPR library screening and identified CDK11 as a novel key player in HCC. Genetic perturbation, shRNA-mediated knockdown, and selective inhibition of CDK11 effectively suppressed HCC cell growth both <i>in vivo</i> and <i>in vitro</i>, suggesting that CDK11 is a potential therapeutic target for HCC. Mechanistically, CDK11 maintains c-Myc stability through directly phosphorylating serine 62 and serine293 in HCC cells, suggesting that a novel function of CDK11 beyond its role in gene transcription, RNA splicing and cell cycle control. Moreover, Serine62 phosphorylation of c-Myc is highly expressed and associated with poor survival in HCC.c-Myc is well-known for regulating multiple signal pathways including glycolysis, DNA repair and autophagy, indeed, we found that CDK11 regulates glycolysis, DNA repair and autophagy through c-Myc. Meanwhile, we found that CDK11 inactivation suppressed homologous recombination (HR) repair related genes while activated non homogenous end join (NHEJ) repair like PARP1. Therefore, a combination of OTS964 with PARP1 inhibitor, Olaparib, has a synergistic effect on suppression of HCC tumor growth through augment DNA damage. More importantly, our study shed light on the mechanism of how CDK11 regulates autophagy, DNA repair and glycolysis through regulating c-Myc stability.A genome-wide CRISPR library screening identified that targeting V-ATPases like ATP6V0C and ATP6V0D1 sensitizes HCC cells to OTS964, and ATP6V0C is a target of Baf A1 that is an inhibitor of autophagy and lysosome, which suggests that targeting lysosome and autophagy pathways allow HCC cells to sensitize OTS964 treatment. This finding is consistent with that targeting CDK11 induces autophagy. Thus, Co-treatment with OTS964 and autophagy inhibitors significantly suppressed HCC cells growth both <i>in vitro</i> and <i>in vivo</i>.Furthermore, autophagy and lysosome related genes such as P62 were overexpressed in HCC patients, which enable us to develop a protein degradation technology, termed AUTOTAC, to utilize P62 ligand and link with CDK11 inhibitor to gain a novel autophagy-mediated protein degrader to induce CDK11 degradation. This novel CDK11 degrader, called CDK11-AUT, is a heterobifunctional small-molecule, which facilitates P62 oligomerization to allow CDK11 to be degraded by autophagy-lysosome pathway. Moreover, CDK11-AUT displays selective ability for CDK11 degradation, and CDK11-AUT effectively induces HCC cell apoptosis and inhibits HCC cell growth. Furthermore, CDK11-AUT shows excellent anti-tumor ability with low toxicity in mouse HCC tumor models indicating that targeting CDK11 degradation also exhibits therapeutic potential for HCC.In summary, our study demonstrated that CDK11 is a novel potential therapeutic target for HCC and elucidated the mechanism of how CDK11 contributes to HCC development. Meanwhile, we discovered a novel CDK11 degrader, which provides an innovative strategy to target CDK11 for HCC treatment.

肝细胞癌（Hepatocellular carcinoma, HCC）是一种致死性疾病，现有治疗手段十分有限。因此，阐明HCC发生发展的分子机制并发掘全新治疗靶点，已成为当前亟待解决的关键问题。细胞周期蛋白依赖性激酶（Cyclin-dependent kinases, CDKs）属于丝氨酸/苏氨酸激酶家族，参与基因转录调控与细胞周期进程的调控。靶向CDKs是癌症治疗中颇具前景的策略，目前已有多款CDK4/CDK6抑制剂获美国食品药品监督管理局（FDA）批准用于乳腺癌的临床治疗。
本研究通过全基因组CRISPR文库筛选，鉴定出CDK11是HCC发生过程中的新型关键调控因子。通过遗传扰动、短发夹RNA（shRNA）介导的基因敲低以及选择性抑制CDK11，均可在体内和体外有效抑制肝癌细胞的增殖，提示CDK11是HCC潜在的治疗靶点。
从分子机制来看，CDK11可通过直接磷酸化肝癌细胞内c-Myc的丝氨酸62与丝氨酸293位点，维持c-Myc的蛋白稳定性，这表明CDK11具有超越其在基因转录、RNA剪接与细胞周期调控之外的全新生物学功能。此外，c-Myc的丝氨酸62磷酸化在HCC患者组织中呈高表达状态，且与患者不良预后显著相关。
c-Myc已知可调控糖酵解、DNA修复与自噬等多条信号通路，本研究证实CDK11可通过c-Myc介导调控上述通路。同时，我们发现CDK11失活会抑制同源重组（Homologous Recombination, HR）修复相关基因的表达，同时激活非同源末端连接（Non-homologous End Joining, NHEJ）修复通路相关因子如PARP1。因此，OTS964与PARP1抑制剂奥拉帕利（Olaparib）联合使用，可通过加剧DNA损伤协同抑制肝癌的生长。更重要的是，本研究阐明了CDK11通过调控c-Myc稳定性进而影响自噬、DNA修复与糖酵解的具体分子机制。
全基因组CRISPR文库筛选还发现，靶向V-ATPases家族成员如ATP6V0C与ATP6V0D1，可增强肝癌细胞对OTS964的敏感性；而ATP6V0C是自噬与溶酶体抑制剂巴弗洛霉素A1（Bafilomycin A1, Baf A1）的作用靶点，这表明靶向溶酶体与自噬通路可使肝癌细胞对OTS964治疗增敏。这一发现与CDK11靶向诱导自噬的结果相一致。因此，OTS964与自噬抑制剂联合使用，可在体内外显著抑制肝癌细胞的增殖。
进一步研究发现，HCC患者体内自噬与溶酶体相关基因（如P62）表达上调，据此我们开发了一种名为AUTOTAC的蛋白质降解技术，该技术利用P62配体与CDK11抑制剂结合，构建新型自噬介导的蛋白降解剂以诱导CDK11降解。这款新型CDK11降解剂命名为CDK11-AUT，属于双功能小分子化合物，可促进P62寡聚化，使CDK11通过自噬-溶酶体通路被降解。此外，CDK11-AUT可选择性降解CDK11，并有效诱导肝癌细胞凋亡、抑制细胞增殖。在小鼠肝癌模型中，CDK11-AUT展现出优异的抗肿瘤活性与低毒性，表明靶向降解CDK11同样具有治疗HCC的潜力。
综上，本研究证实CDK11是HCC新型潜在治疗靶点，并阐明了CDK11参与HCC发生发展的分子机制。同时，我们开发的新型CDK11降解剂，为HCC靶向CDK11的治疗提供了创新策略。

搜集汇总

背景与挑战

背景概述

该数据集支持肝细胞癌（HCC）中CDK11功能及治疗潜力的研究，基于全基因组CRISPR筛选发现CDK11通过磷酸化c-Myc调控糖酵解、DNA修复和自噬等过程，是HCC的潜在治疗靶点。同时，数据集涉及开发了一种新型自噬介导的CDK11降解剂CDK11-AUT，该降解剂通过靶向CDK11降解在小鼠模型中表现出抗肿瘤效果，为HCC治疗提供了创新策略。