The RASGRP2 gene encodes the Ca2+ and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which plays a key role in integrin activation in platelets and neutrophils. We here report two new RASGRP2 variants associated with platelet dysfunction and bleeding in patients. The homozygous patients had normal platelet and neutrophil counts and morphology. Platelet phenotyping showed: prolonged PFA-100 closure times; normal expression of major glycoprotein receptors; severely reduced platelet aggregation response to ADP and collagen (both patients); aggregation response to PAR1 and arachidonic acid markedly impaired in one patient; PMA-induced aggregation unaffected; platelet secretion, clot retraction, and spreading minimally affected. Genetic analysis identified two new homozygous variants in RASGRP2: c.706C>T (p.Q236X) and c.887G>A (p.C296Y). In both patients, CalDAG-GEFI protein was not detectable in platelet lysates, and platelet αIIbβ3 activation, as assessed by fibrinogen binding, was greatly impaired in response to all agonists except PMA. Patient neutrophils showed normal integrin expression, but impaired Mn2+-induced fibrinogen binding. In summary, we have identified two new RASGRP2 mutations that can be added to this rapidly growing form of inherited platelet function disorder.
We have characterized the molecular changes underlying the transformation of a JAK2V617F+-myelofibrosis with trisomy 8, into a JAK2V617F-negative leukemia. Leukemic clone did not carry JAK2V617F mutation, but showed ASXL1 mutation (R693X). This mutation was identified in a low percentage at diagnosis by next-generation sequencing. Using this technology in serial specimens during the follow-up, we observed a progressive expansion of the ASXL1-mutated minor clone, whereas the JAK2V617F+-clone carrying trisomy 8 decreased. Hematologic progression occurred simultaneously with an ASXL1-R693X-negative lung-cancer. This is the first report showing a clear association between the expansion of an ASXL1-mutated clone and the leukemic transformation of myelofibrosis.
Despite the absence of mutations in the DNA repair machinery in myeloid malignancies, the advent of high-throughput sequencing and discovery of splicing and epigenetics defects in chronic myelomonocytic leukaemia (CMML) prompted us to revisit a pathogenic role for genes involved in DNA damage response. We screened for misregulated DNA repair genes by enhanced RNA-sequencing on bone marrow from a discovery cohort of 27 CMML patients and 9 controls. We validated 4 differentially expressed candidates in CMML CD34 bone marrow selected cells and in an independent cohort of 74 CMML patients, mutationally contextualized by targeted sequencing, and assessed their transcriptional behavior in 70 myelodysplastic syndrome, 66 acute myeloid leukaemia and 25 chronic myeloid leukaemia cases. We found BAP1 and PARP1 down-regulation to be specific to CMML compared with other related disorders. Chromatin-regulator mutated cases showed decreased BAP1 dosage. We validated a significant over-expression of the double strand break-fidelity genes CDKN1A and ERCC1, independent of promoter methylation and associated with chemorefractoriness. In addition, patients bearing mutations in the splicing component SRSF2 displayed numerous aberrant splicing events in DNA repair genes, with a quantitative predominance in the single strand break pathway. Our results highlight potential targets in this disease, which currently has few therapeutic options.
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. Disclosures Díez-Campelo: Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Janssen: Research Funding.
Background: De-ubiquitinating enzyme BAP1, a fundamental deubiquitinase in the epigenetic regulation of transcription factors and functionally related to ASXL1, is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. In a recent murine study, absolute BAP1 depletion generated specimens with similar characteristics to myelodysplastic / myeloproliferative syndromes in humans (ineffective hematopoiesis and myeloproliferation), mainly to chronic myelomonocytic leukemia (CMML) (Dey, et al. Science 2012). Aim: The aim of this study was to quantify BAP1 gene expression in patients diagnosed with a variety of myeloid neoplasms, and compared it with healthy donors. We furthermore explored the possible association of BAP1 low expression level and the presence of ASXL1 mutations or BRCA1 protein levels. In addition, a regression analysis to determine the possible correlation of peripheral blood and bone marrow expression levels was performed. Methods: We included patients diagnosed between 2008-2014 of CMML, myelodysplastic sydrome (MDS) chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), of whom bone marrow DNA and RNA were available at diagnosis. As controls, 6 healthy bone marrow donors were used. BAP1 and BRCA1 expressions levels were quantified by RT-qPCR, using the same healthy bone marrow donor sample as an inter-assay normalizing- calibrator. The study of somatic ASXL1 mutations was carried out by the Sanger method. For statistical studies, the T-Student, Pearson correlation and/or U Mann-Whitney test, were used when needed. For survival analysis COX regression and the ROC curves were used. A two-side P<0.05 was used as statistical significance threshold. Results: Samples of 116 patients were included in the study: CMML=26; MDS=15; AML=50; CML=25 and 6 controls. This study shows that levels of BAP1 expressions are decreased when compared to controls along the spectrum of myeloid diseases. In the comparison among entities, CMML shows the lowest values (percentage respect to the calibrator), 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; LMMC vs CML, p=0.346. No differences were found between CMML patients with dysplastic and myeloproliferative variant, WHO types 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. Conclusion:In summary; this study shows that BAP1 decreased expression is a common mechanism among the myeloid malignances, being CMML mainly affected. This mechanism is independent of the presence of ASXL1 mutations, and it could constitute a new therapeutic target in chronic myelomonocytic leukemia. Disclosures No relevant conflicts of interest to declare.
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