BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)
Authors: Dkhissi Fatima , Aggoune Djamel , Pontis Julien , Bonnet Marie Laure , Meunier Marie Claude , Sorel Nathalie , Bouneau Clémentine , Bennaceur-Griscelli Annelise , Guilhot François , Chomel Jean-Claude , Ait-Si-Ali Slimane , Turhan Ali G 
Published: October 2019

BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation.

Disclosures:

Guilhot: Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.