Downregulation of BAP1 in Chronic Myelomonocytic Leukemia: Correlated with a Post-Translational Reduction of BRCA1 Levels and Independent of Promoter Methylation

Background: De-ubiquitinating enzyme BAP1, functionally related to ASXL1, is mutated in various hereditary cancers and its deletion is associated with the appearance of myelodysplastic/myeloproliferative features in mice. BRCA1 is known to drive homologous recombination, playing a critical role in preserving genomic integrity. Finding an impaired DNA-damage via in chronic myelomonocytic leukemia (CMML) would open the synthetic lethality strategy to MDS/MPN diseases, moreover if the already targeted hypermethylation mechanism is not involved.

Methods: BAP1 and BRCA1 expressions levels were quantified by RT-qPCR. Methylation detection was performed on 57 CMML patients and 27 controls using Methylation Cancer Panel I from Illumina (San Diego, CA) interrogating 2 CpG sites at the 5´promoter region of BAP1.

We developed a sandwich-type ELISA assay to measure the grade of ubiquitination of BRCA1. Fifty micrograms of total protein from blood-sorted monocytes, granulocytes and lymphocytes lysate of 19 patients were used. Total BRCA1 was captured in precoated microtiter and an anti-mono and poly ubiquitin conjugates monoclonal antibody (HRP-linked FK2) was added. A second ELISA assay determines the nanogrames of BRCA1 protein quantity in those 50 µg of sample lysate. It allows to determine the ratio of ubiquitylated BRCA1 light units/total BRCA1 (uBRCA1/BRCA1), which measures in a quantitative manner the percentage of BRCA1that is ubiquitylated in a given sample.

Results: Samples of 175 patients were included in the study: CMML=61; MDS=34; AML=50; CML=30 and 10 controls. CMML showed the lowest values (65% compared with the controls), significantly lower than the other groups, except for CML patients: CMML vs MDS, p=0.001; CMML vs AML, p<0.001; CMML vs Controls, p<0.001; CMML vs CML, p=0.346. No significant differences in the expression of BRCA1 In the bone marrow could be found through the myeloid disease spectrum.

BRCA1 protein was significantly more ubiquitinated in the monocytes of patients than in their healthy controls counterparts: monocytic uBRCA1/BRCA1 in patients 10.7 vs 1.7 in controls, p=0.03). We found no significant differences in BRCA1 ubiquitination in the lymphocyte subsets. A correlation was found between low levels of BAP1 expression in bone marrow and lower levels of total BRCA1 (R²=0.7) and higher levels of the ubiquitination ratio (R²=0.75) in monocytes. In the granulocyte subset a statistical trend was found.

We compared overall average methylation β-values and the percentage of hypermethylated calls from the 2 BAP1 CpG sites in bone marrow-extracted DNA from 57 CMML patients and 27 controls. Average methylation and the number of hypermethylated calls from CpG sites were not significantly different in patients with CMML (average β-value 0.293; average number of hypermethylated CpG calls, 5.1%) compared with patients with healthy controls (average β-value 0.313, p=0.8; average number of hypermethylated CpG calls, 5.4%, p=0.9).

No differences were found between CMML patients with dysplastic and myeloproliferative variant, WHO subtypes I and II or according to the presence of ASXL1 mutations (33% CMML patients were mutated). Of potential clinical interest, BAP1 expression in bone marrow and peripheral blood showed a direct and significant correlation (R=0.884, p= 0.001).   BRCA1 expression were decreased uniformly through the different myeloid diseases, suggesting that the heterogeneous BAP1 expression could be responsible for different BRCA1 protein levels by posttranslational regulation.

Discussion: It has been shown that BAP1 is required for efficient assembly of the homologous recombination factor BRCA. We show here, with a newly developed assay, that BRCA1 is hyperubiquitylated in CMML patients. An imbalance that correlates with the degree of downregulation of BAP1 in the bone marrow. The latter, as well as the lack of hypermethylation in the promoter region of BAP1, prompts the study of synthetic lethality strategies in this disease.