ABSTRACT
BACKGROUND & AIMS: Despite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Here, we aim to develop new therapeutic approaches for liver cancer. METHODS: A compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with cyclin-dependent kinase (CDK) 4/6 inhibitor. The combination effects of CDK4/6 inhibitor and exportin 1 (XPO1) inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. RESULTS: The combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic drug screen, cereblon (CRBN)-based proteolysis targeting chimera (PROTAC) ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Up-regulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that ubiquitin specific peptidase 2 (USP2) directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. CONCLUSIONS: Our study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.
Subject(s)
Adaptor Proteins, Signal Transducing , Cellular Senescence , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , Exportin 1 Protein , Karyopherins , Liver Neoplasms , Protein Kinase Inhibitors , Receptors, Cytoplasmic and Nuclear , Ubiquitin-Protein Ligases , Humans , Cellular Senescence/drug effects , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Karyopherins/antagonists & inhibitors , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Receptors, Cytoplasmic and Nuclear/metabolism , Ubiquitin-Protein Ligases/metabolism , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Liver Neoplasms/metabolism , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Retinoblastoma Binding Proteins/metabolism , Retinoblastoma Binding Proteins/genetics , Drug Synergism , Senotherapeutics/pharmacology , Xenograft Model Antitumor Assays , Signal Transduction/drug effects , Proteolysis/drug effects , Hydrazines/pharmacology , Hydrazines/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Hep G2 Cells , Mice , Piperazines , Pyridines , TriazolesABSTRACT
BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.