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.