Supporting data for Functional study and therapeutic potential of CDK11 in liver cancer

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 in vivo and in vitro, 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 in vitro and in vivo.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（HCC）是一种致命性疾病，其治疗手段有限。因此，迫切需要了解HCC发展的分子机制以及识别新的治疗靶点。周期蛋白依赖性激酶（CDKs）属于丝氨酸/苏氨酸激酶家族，参与基因转录调控和细胞周期控制。针对CDKs是癌症患者的一种有前景的治疗途径，因为几种CDK4/CDK6抑制剂已被FDA批准用于乳腺癌治疗。在此，我们采用全基因组CRISPR文库筛选，鉴定出CDK11作为HCC中一个新的关键参与者。通过遗传扰动、shRNA介导的敲低和CDK11的选择性抑制，有效抑制了体内和体外HCC细胞生长，表明CDK11是HCC的一个潜在治疗靶点。从机制上讲，CDK11通过直接磷酸化HCC细胞中的丝氨酸62和丝氨酸293来维持c-Myc的稳定性，这表明CDK11除了其在基因转录、RNA剪接和细胞周期控制中的作用之外，还具有新的功能。此外，c-Myc的丝氨酸62磷酸化在HCC中高度表达，并与不良预后相关。c-Myc因其调节包括糖酵解、DNA修复和自噬在内的多种信号通路而闻名，事实上，我们发现CDK11通过c-Myc调节糖酵解、DNA修复和自噬。同时，我们发现CDK11的失活抑制了同源重组（HR）修复相关基因，而激活了非同源末端连接（NHEJ）修复，如PARP1。因此，OTS964与PARP1抑制剂奥拉帕利的联合使用，通过增加DNA损伤，对HCC肿瘤生长具有协同抑制作用。更重要的是，我们的研究揭示了CDK11如何通过调节c-Myc的稳定性来调节自噬、DNA修复和糖酵解的机制。全基因组CRISPR文库筛选发现，针对V-ATPases如ATP6V0C和ATP6V0D1可提高HCC细胞对OTS964的敏感性，而ATP6V0C是自噬和溶酶体抑制剂Baf A1的靶点，这表明靶向溶酶体和自噬途径可以使HCC细胞对OTS964治疗敏感。这一发现与靶向CDK11诱导自噬的结果一致。因此，联合使用OTS964和自噬抑制剂在体外和体内显著抑制了HCC细胞的生长。此外，自噬和溶酶体相关基因，如P62，在HCC患者中过表达，这使我们能够开发一种名为AUTOTAC的蛋白质降解技术，利用P62配体并与CDK11抑制剂结合，以获得一种新的自噬介导的蛋白降解剂，诱导CDK11降解。这种新的CDK11降解剂，称为CDK11-AUT，是一种异双功能小分子，它促进P62寡聚化，使CDK11能够通过自噬-溶酶体途径降解。此外，CDK11-AUT显示对CDK11降解的选择性能力，并有效地诱导HCC细胞凋亡和抑制HCC细胞生长。此外，CDK11-AUT在mouse HCC肿瘤模型中表现出优异的抗肿瘤能力和低毒性，这表明靶向CDK11降解也具有HCC治疗的潜力。总之，我们的研究证实了CDK11是HCC的一个新型潜在治疗靶点，并阐明了CDK11如何导致HCC发展的机制。同时，我们发现了一种新的CDK11降解剂，这为靶向CDK11治疗HCC提供了一种创新策略。