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Recurrent mutations, expression analysis and functional characterization of cohesin subunits in myelodysplastic syndromes and acute myeloid leukemia

Abolfathi, Mohsen

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Myelodysplastic syndromes (MDS) are common hematopoietic disorders that are associated with bone marrow failure and the possibility of developing leukemia (1). MDS cells often contain chromosomal abnormalities, which significantly impact on the prognosis of the disease as documented by the contribution of chromosomal aberrations to the International Prognostic Scoring System (IPSS) used to prognostically classify MDS cases. Our understanding of the molecular mechanism of MDS has been increasing especially due to the advancements in genomics and next generation sequencing. An increasing list of mutated genes is being described in MDS including hematopoietic transcription factors (ETV6, CEBPA, RUNX1, SPI1 (PU.1)) (2-5), epigenetic regulators (ASXL1, TET2, DNMT3A, IDH1, IDH2, EZH2, SUZ12) (3, 6, 7) and microRNAs (8, 9), RNA splicing factors (SF3B1, SRSF2, U2AF1, ZRSR2) (10), cell cycle regulators (CDKN1A,TP53,BCL2, AURKA, AURKB, CDC20, MAD2L1, TUBG1) (10-14), members of the cohesin complex (STAG2, RAD21, SMC1, SMC3A) (10, 15, 16), members of other signaling pathways (JAK2, IRAK1, CTNNB1, NOTCH1, NPM1, SMAD7, TGFB1, NF-κB) (9, 16-20), proteins involved in immunological processes (TLR2, STAT3) (21, 22), and others factors (CBL, CALR, BCOR, BCORL1, SETBP1, GNAS, CDKN2B, Nup98, HoxD13) (2, 3, 10, 23-25). Identification of mutations in these genes has increased our understanding of the disease but there is a lot to be done in order to gain insights into the mechanisms of MDS pathogenesis. Mutations in the members of the cohesin complex including RAD21 and STAG2 have been found in MDS and other types of cancer such as bladder (26-28), breast (29), and colorectal cancer (30). These mutations are associated with chromosomal instability and aneuploidy in some cancer types such as bladder cancer (31) but this finding remains controversial in other cancer types (32) and requires further mechanistic studies and patient data analysis in order to be validated. Genome engineering has been improved greatly over the past couple of years with the recent introduction of the CRISPR/Cas9 system by the Zhang group (33), making functional validation of the genomic data obtained from massively parallel sequencing studies feasible. In this study, we characterized a panel of 63 genes that has been reported to be frequently mutated in MDS, in 90 patient samples from MDS patients with and without chromosomal aberrations in their bone marrow mononuclear cells using a targeted re-sequencing approach to assess the frequency of mutations in these genes. This approach allowed us to determine whether the mutation spectrum is different in MDS cases with and without chromosomal aberrations, especially with regard to the occurrence of cohesin complex subunit mutations. In addition, we functionally characterized the cohesin complex subunit STAG2 that has been reported to be mutated in several cancers including MDS and reported to be 8 associated with chromosomal instability at least in some studies. To do this, we used CRISPR/Cas9 genome engineering to knock out this gene and analyzed for signs of chromosomal instability. We have used HCT116-p53+/+ and HCT116-p53-/- cell lines for these functional analyses. Finally, we also analyzed the expression of STAG2 in AML samples using immunofluorescence microscopy and Western blotting. TP53 somatic SNVs were found in 7/90 (7.8%) of the cases and were mainly associated with complex karyotypes, which is in accordance with previous reports. STAG2 was mutated in only 4/90 (4.4%) of samples. No mutations in other cohesin components were found. On the other hand, STAG2 expression was lost in 18 out of 74 (24.3%) AML samples due to STAG2 mutations in 20% (2/10) and promoter methylation in 58.3% (7/12) of cases. In addition, we used CRISPR/Cas9 genome editing to knock out STAG2 in diploid, chromosomally stable HCT116-p53+/+ and HCT116-p53-/- cells. Whereas loss of STAG2 led to alterations in gene expression profiles in both cell lines, chromosome aberrations were only induced in the HCT116-p53-/- background. We conclude that the expression of STAG2 is lost in about one quarter of AML cases, frequently as a consequence of promoter methylation. Depending on the genetic background, both disturbed gene expression and chromosomal aberrations are associated with loss of STAG2.

Item Type: Dissertation
Supervisor: Krämer, Prof. Dr. Alwin
Place of Publication: Heidelberg, Germany
Date of thesis defense: 30 May 2017
Date Deposited: 13 Jun 2017 09:02
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
Controlled Keywords: Cohesin complex, MDS, AML
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