(P 235) ELUCIDATING THE ONCOGENIC EFFECT OF BCOR-ITD IN SARCOMA

Objective: To establish the structural / functional effect of internal tandem duplication within the BCOR gene on non-canonical Polycomb Repressive Complex 1 (ncPRC/PRC1.1) in sarcomagenesis.

Methods: The PUFD domain of BCOR combines with the RAWUL domain of second protein PCGF1 to bind to a lysine methylase KDM2B as part of a complex in this non-canonical polycomb pathway that contributes to epigenetic regulation via chromatin structure. Using isogenic cell lines engineered to express FLAG-tagged BCOR wild-type, versus 3 distinct FLAG-tagged BCOR-ITDs, we have carried out mass spectrometry with immunoprecipitation and co-immunoprecipitations of various PRC1.1 protein subunits, along with gel filtration with western blotting of the subunits of PRC1.1. By whole genome sequencing, the ITDs from 132 patient samples duplicate a sequence encoding a specific beta strand plus alpha helix (b5-a5) super-secondary motif in BCOR that is a key part of the PUFD-RAWUL interface. [Variation in the ITDs arises because they also include sequences upstream or downstream of the duplicated b5-a5 ] Based on ‘insertion logic” for these 3 various BCOR ITD sequences, molecular dynamic analysis of the key protein domains within the core polycomb subunits of PRC1.1 was carried out, generating ‘movies’ for each run simulating these key subunit interactions.

Results:We have shown by the various experimental means detailed above including initially mass spec-IP and then also gel filtration with Western blotting that the PRC1.1. ‘disintegrates’ in the context of BCOR-ITD, with separation of the enzymatic subcomplex from the DNA-binding subcomplex.

Conclusion: BCOR-ITD results in a structural disintegration/non-integration of the PRC1.1 with oncogenic effect.