Therapeutic vulnerability to PARP1/2 inhibition in RB1-mutant osteosarcoma

ABSTRACTBackgroundLoss-of-function mutations of the retinoblastoma tumour suppressor RB1 are key drivers in cancer, with prominent involvement in the natural history of Osteosarcoma (OS). RB1 loss-of-function compromises genome maintenance in cells and hence could yield vulnerability to therapeutics targeting such processes.MethodWe assessed the response to Poly-ADP-Polymerase1/2 inhibitors (PARPi) in histiotype-matched cancer cell lines differing in RB1 status including an extended panel of OS lines, measuring viability, clonogenic activity and inhibition of xenograft growth in vivo. We used mutational signature analysis and RAD51 immunostaining to assess competence for homologous repair defect (HRd).ResultsWe report selective hypersensitivity to clinically-approved PARPi in OS lines with RB1 mutation, which extends to other cancer histiotypes and is induced in RB1-normal OS following engineered RB1 loss. PARPi treatment caused extensive cell death in RB1-mutated OS and extended survival of mice carrying human RB1-mutated OS grafts. Sensitivity in OS with natural or engineered RB1 loss surpassed that seen in BRCA-mutated backgrounds where PARPi are showing clinical benefit. PARPi sensitivity was not associated with loss of RAD51 recruitment and HRd-linked mutational signatures, which predict PARPi sensitivity in cancers with BRCA1/2 loss, but linked to rapid activation of replication checkpoint signalling with S phase transit critical for the death response observed.ConclusionOur work demonstrates that mutations in RB1 causes clinically relevant hypersensitivity to approved PARP1/2-targeting therapeutics and advocates PARP1/2 inhibition as a novel, genome lead strategy for RB1-mutated osteosarcoma.

Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1

The activity of Rb (retinoblastoma protein) is regulated by phosphorylation and acetylation events. Active Rb is hypophosphorylated and acetylated on multiple residues. Inactivation of Rb involves concerted hyper-phosphorylation by cyclin–CDK (cyclin-dependent kinase) complexes combined with deacetylation of appropriate lysine residues within Rb. In the present study, using in vivo co-immunoprecipitation experiments, we identified mammalian SIRT1 (sirtuin 1) as a binding partner for Rb and its family members p107 and p130. Formation of Rb–SIRT1 complexes required the pocket domain of Rb. p300 catalysed the acetylation of Rb, and SIRT1 was a potent deacetylase for Rb. The ability of SIRT1 to catalyse the deacetylation of Rb was dependent on NAD and was inhibited by the SIRT1 inhibitor nicotinamide. Deacetylated lysine residues within Rb formed a domain similar to the SIRT1-targeted domain of the p53 tumour suppressor protein. Cultures of arrested cells, via contact inhibition or DNA damage, exhibited decreased Rb phosphorylation and increased Rb acetylation. Overexpression of SIRT1 in either confluent or etoposide-treated cells resulted in a significant reduction in Rb acetylation, which was restored with nicotinamide. Gene knockdown of SIRT1 by siRNA (short interfering RNA) produced an accumulation of acetylated Rb. This increase was augmented further when siRNA against SIRT1 was used in conjunction with nicotinamide. In conclusion, our results demonstrate that SIRT1 is an in vitro and in vivo deacetylase for the Rb tumour suppressor protein.