BCOR as a Novel Fusion Partner of Retinoic Acid Receptor Alpha In a t(X;17)(p11;q12) Variant of Acute Promyelocytic Leukemia

Abstract 1703

The majority of acute promyelocytic leukemia (APL) cases are characterized by the presence of a PML-RARA fusion gene. In a small subset, RARA is fused to a different partner including PLZF, NPM1, NuMA, STAT5b, PRKAR1A and FIP1L1. Here we identified a novel RARA fusion transcript, BCOR-RARA in a t(X;17)(p11;q12) variant of APL. The patient was a 45-year-old man. Although the patient was clinically responsive to ATRA, repeated standard chemotherapy with ATRA did not effect a cure. The bone marrow promyelocytes had unique morphologic features, including rectangular and round cytoplasmic inclusion bodies. They were more granular than those of AML M2 but less granular than the classical t(15;17) APL. Flow cytometric analysis revealed strong expression of CD13, CD33, CD56, weak expression of CD11c and lack of HLA-DR and CD7. The karyotype analysis detected a novel chromosomal translocation described as 45,-Y, t(X;17)(p11.2;q12)[19]/ 46,XY[1]. FISH analysis indicated one intact and two split signals of RARA and two intact signals of PML. To amplify unknown chimeric fusion transcripts, we performed 5'-RACE. The sequence revealed that BCL6 co-repressor, BCOR cDNA from exons 9 to 12 to be fused to RARA exon 3. By RT-PCR, we confirmed full length chimeric fusion transcripts spanning from the start codon to 4,948 nt of BCOR cDNA (NM_001123384) fused to RARA cDNA from exon 3 to the stop codon. The chimeric cDNA had an in-frame codon from BCOR through RARA, creating a 1,931 amino acid fusion protein. One of the consistent features in all known RARA fusion partners is self-association. To determine whether BCOR-RARA self-associate, we performed co-immunoprecipitation assays. These results showed that BCOR-RARA is able to self-associate both through the region of BCOR-S and the ankyrin repeat domain of BCOR. In addition, BCOR-RARA associated with BCL6. RXR recruitment is a critical determinant of transforming potential of oligomeric RARA fusion proteins. To investigate how BCOR-RARA associates with RARE in vitro, we performed EMSA. These results showed that BCOR-RARA/RXRA complex associates with RARE in an alternative manner compared to RARA and PML-RARA. Deregulation of RARA transcriptional activations has a central role in pathogenesis of APL. Therefore, we evaluated ATRA-induced transcriptional activation of 4× RAREs with a reporter assay in HepG2 cells. Without ATRA, BCOR-RARA repressed the reporter activity. With addition of ATRA, BCOR-RARA induced transcriptional activation very weakly. Subsequently, we evaluated dominant-negative effects of the samples in the RARA/RXRA pathway. In contrast to BCOR, BCOR-RARA clearly inhibited ATRA-induced RARA transcriptional activation as well as PML-RARA. Furthermore, we asked which domains are sufficient for the dominant-negative effects with the deletion mutants. The results indicated that the region spanning from 999 to 1,409 aa of BCOR-RARA has pivotal roles in the dominant-negative effects. Correct protein function is highly dependent on intracellular localization. To investigate subcellular localization of BCOR-RARA, we performed immunofluorescence analysis in 293T cells. In BCOR-RARA-expressing cells, BCOR-RARA localized as two patterns; (I) diffusely in the nucleus as well as PML-RARA in 82% of the cells, (II) diffusely in the nucleus and aggregately in the cytoplasm in 18% of the cells. The subcellular localization of BCOR-RARA was clearly distinguishable from the punctuate pattern as shown in the nucleus of BCOR-expressing cells. Moreover, co-immunofluorescence analysis between BCOR-RARA and BCL6 indicated that the subcellular localization of BCOR-RARA/BCL6 is distinct from BCOR/BCL6. BCOR-RARA was found to possess common features with other RARA fusion proteins. These included: (I) the same break point in RARA cDNA; (II) self-association; (III) RXRA is necessary for BCOR-RARA to associate with the RARA responsive element; (IV) action in a dominant-negative manner on RARA transcriptional activation; (V) aberrant subcellular relocalization. It should be noted that there was no intact BCOR found in the 45,-Y,t(X;17)(p11;q12) APL cells because they featured only a rearranged × chromosome. These results highlight essential features of pathogenesis in APL in more detail. BCOR appears to be involved not only in human congenital diseases but also in a human cancer.