Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma

Abstract

More than 70% of Epstein–Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

Inhibitory feedback control of NF-κB signalling in health and disease

“…Progress is also now being made upstream of the core IKK complex with the development of potent and selective inhibitors of TAK1 and NIK. Takinib is a potent (9 nM) and highly selective inhibitor of TAK1 [ 365 ] that exhibits activity in arthritis models [ 366 ] and in a Hodgkin's Lymphoma model with mutation of the A20 DUB [ 367 ]. In addition, potent and selective inhibitors of NIK are now becoming available [ 368 ] including XT2, which is potent (9 nM) and effective at inhibiting NIK-induced inflammatory signalling [ 369 ].…”

High-Throughput CRISPR Screening in Hematological Neoplasms

“…A comprehensive understanding of how the ubiquitination pathway regulates HL pathogenesis in deficient-A20 HL has identified the essential role of TAK1 kinase by the use of a custom unique ubiquitin regulator-focused CRISPR library. Interestingly, the TAK1 inhibitor takinib has shown promising activity against HL in vitro and in vivo models [ 89 ].…”

Do Epstein–Barr Virus Mutations and Natural Genome Sequence Variations Contribute to Disease?

“…The Reed–Sternberg cell is thought to be derived from a B cell which has undergone a defective B cell receptor mutation or rearrangement that would be expected to result in cell apoptosis. In most cases of the EBV negative disease, these cells survive because of a mutation in the A20 apoptosis regulator, which affects signaling to NF-kB [ 42 ]. In the EBV-positive Reed–Sternberg cells, the viral LMP2A protein can produce survival signals including those provided by the BCR [ 43 ], whereas LMP1 is thought to provide signals that substitute for T cell help, preventing cell death and allowing the disease to develop.…”