(P 424) INTERNAL TANDEM DUPLICATION OF BCOR (BCOR-ITD) ALTERS THE TUMOR EPIGENOME

Objective: In this work we aim to integrate RNA expression, DNA methylation, ChIP Seq, size exclusion chromatography and IP-mass spec data from our cell line models to elucidate the oncogenic mechanism in BCOR family tumors.

Methods: We generated an inducible cell line model of YWHAE::NUTM2 fusion tumors, and further cell line models stably expressing BCOR-wild type versus three of the internal tandem duplications previously described by our group in a large series of clear cell sarcomas of kidney to include the commonest ITD and an observed triplication event also. IP-mass spectrometry was carried out comparing ITD versus wild type-BCOR-expressing constructs along with size exclusion chromatography studies also. DNA methylation analyses were similarly carried out on BCOR ITD versus wild-type cell lines as well as and ChIPSeq. RNA sequencing of all cell lines including BCOR and YWHAE::NUTM2 construct-bearing lines was completed also.

Results: Reassuringly, the cell line models show gene expression changes including many previously reported in BCOR-family tumor samples in the literature. Three lines of evidence, including IP-mass spectrometry, molecular dynamic studies and size exclusion chromatography all support the hypothesis that BCOR-ITD leads to non-assembly/disaggregation of the PRC1.1, NOT through failed interaction of BCOR-ITD with PCGF1, its direct interaction partner subunit, as has been reported in leukemias in which there is typically a truncating mutation of BCOR, rather than ITD, and resulting in loss of BCOR's PUFD domain required for the interaction with PCGF1. Instead, this BCOR-PCGF1 interaction and indeed subsequent heterotrimerisation with KDM2B persist in the context of BCOR-ITD, but with separation of the enzymatic RING1B/RYBP subunits from the DNA-binding BCOR/PCGF1/KDM2B subcomplex. We propose a novel and intriguing mechanism for the 'disintegration/non-assembly' of the complex in this way, which relies on a further PRC1.1 protein undergoing conformational change in this context. We show by ChIPSeq and RNA profiling how the histone modification by PRC1.1 is altered in the ITD context with both direct and indirect consequences on gene expression in these cell lines.

Conclusion: A novel mechanism of oncogenesis is revealed, which differs importantly from the loss of function truncating mutations identified in BCOR in the leukemia setting. Further exploration is expected to pave the way for novel therapeutic approaches.