Recurrent Mutations of Multiple Components of Cohesin Complex in Myeloid Neoplasms

Abstract

782 Recent genetic studies have revealed a number of novel gene mutations in myeloid malignancies, unmasking an unexpected role of deregulated histone modification and DNA methylation in both acute and chronic myeloid neoplasms. However, our knowledge about the spectrum of gene mutations in myeloid neoplasms is still incomplete. In the previous study, we analyzed 29 paired tumor-normal samples with chronic myeloid neoplasms with myelodysplastic features using whole exome sequencing (Yoshida et al., Nature 2011). Although the major discovery was frequent spliceosome mutations tightly associated with myelodysplasia phenotypes, hundreds of unreported gene mutations were also identified, among which we identified recurrent mutations involving STAG2, a core cohesin component, and also two other cohesin components, including STAG1 and PDS5B. Cohesin is a multimeric protein complex conserved across species and is composed of four core subunits, i.e., SMC1, SMC3, RAD21 and STAG proteins, together with several regulatory proteins. Forming a ring-like structure, cohesin is engaged in cohesion of sister chromatids in mitosis, post-replicative DNA repair and regulation of gene expression. To investigate a possible role of cohesin mutations in myeloid leukemogenesis, an additional 534 primary specimens of various myeloid neoplasms was examined for mutations in a total of 9 components of the cohesin and related complexes, using high-throughput sequencing. Copy number alterations in cohesin loci were also interrogated by SNP arrays. In total, 58 mutations and 19 deletions were confirmed by Sanger sequencing in 73 out of 563 primary myeloid neoplasms (13%). Mutations/deletions were found in a variety of myeloid neoplasms, including AML (22/131), CMML (15/86), MDS (26/205) and CML (8/65), with much lower mutation frequencies in MPN (2/76), largely in a mutually exclusive manner. In MDS, mutations were more frequent in RCMD and RAEB (19.5%) but rare in RA, RARS, RCMD-RS and 5q- syndrome (3.4%). Cohesin mutations were significantly associated with poor prognosis in CMML, but not in MDS cases. Cohesin mutations frequently coexisted with other common mutations in myeloid neoplasms, significantly associated with spliceosome mutations. Deep sequencing of these mutant alleles was performed in 19 cases with cohesin mutations. Majority of the cohesin mutations (16/19) existed in the major tumor populations, indicating their early origin during leukemogenesis. Next, we investigated a possible impact of mutations on cohesin functions, where 17 myeloid leukemia cell lines with or without cohesin mutations were examined for expression of each cohesin component and their chromatin-bound fractions. Interestingly, the chromatin-bound fraction of one or more components of cohesin was substantially reduced in cell lines having mutated or defective cohesin components, suggesting substantial loss of cohesin-bound sites on chromatin. Finally, we examined the effect of forced expression of wild-type cohesin on cell proliferation of cohesin-defective cells. Introduction of the wild-type RAD21 and STAG2 suppressed the cell growth of RAD21- (Kasumi-1 and MOLM13) and STAG2-defective (MOLM13) cell lines, respectively, supporting a leukemogenic role of compromised cohesin functions. Less frequent mutations of cohesin components have been described in other cancers, where impaired cohesion and consequent aneuploidy were implicated in oncogenic action. However, 23 cohesin-mutated cases of our cohort had completely normal karyotypes, suggesting that cohesin-mutated cells were not clonally selected because of aneuploidy. Alternatively, a growing body of evidence suggests that cohesin regulate gene expression, arguing for the possibility that cohesin mutations might participate in leukemogenesis through deregulated gene expression. Of additional note, the number of non-silent mutations determined by our whole exome analysis was significantly higher in 6 cohesin-mutated cases compared to non-mutated cases. Since cohesin also participates in post-replicative DNA repair, this may suggest that compromised cohesin function could induce DNA hypermutability and contribute to leukemogenesis. In conclusion, we report a new class of common genetic targets in myeloid malignancies, the cohesin complex. Our findings highlight a possible role of compromised cohesin functions in myeloid leukemogenesis. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

The evolving role of next generation sequencing in myelodysplastic syndromes

“…Mutations in genes encoding components of the cohesion complex represent approximately 10% of MDS cases (Bejar et al , ; Kon et al , ; Haferlach et al , ). STAG2 , RAD21 , SMC1A and SMC3 are genes which encode proteins (having the same names as the genes) that make up the multimeric cohesion complex.…”

Thrombocytopenia and Cornelia de Lange syndrome: Still an enigma?

“…Moreover, it has also been shown that cohesins regulate expression of genes involved in hematopoiesis and megakaryocytic lineage commitment [Horsfield et al, ; Elagib et al, ; Panigrahi and Pati, ; Kon et al, ].…”

Genome stability: What we have learned from cohesinopathies

“…In addition, in recent years mutations in core cohesin genes and auxiliary factors have been associated with carcinogenesis (Table , Fig. ) [Barber et al, ; Solomon et al, ; Welch et al, ; Balbas‐Martinez et al, ; Kon et al, ; Brohl et al, ; Cucco et al, ; Viny et al, ]. GIN is a well‐established hallmark of cancer cell phenotypes and for some cancers, such as colorectal carcinoma, it is predictive of a poor prognosis [Carter et al, ].…”

“…Experimental evidence indicates that cohesin pathway is a convergent hub for many biological processes intrinsically connected: sister chromatid cohesion, genome organization, gene expression regulation, DNA repair, and genome safeguarding [Michaelis et al, ; Parelho et al, ; Bauerschmidt et al, ; Kagey et al, ; Caron et al, ; Cipressa et al, ; Gelot et al, ]. The identification of both germinal and somatic mutations in cohesin genes in human developmental disorders, collectively termed cohesinopathies [Krantz et al, ; Tonkin et al, ; Schule et al, ; Vega et al, ; Musio et al, ; Deardorff et al, , ,; van der Lelij et al, ; Chetaille et al, ; Kaiser et al, ], and cancers including colon and bladder cancer and leukemia [Barber et al, ; Solomon et al, ; Welch et al, ; Kon et al, ; Cucco et al, ], all characterized by GIN, indicates that cohesin may be a pivotal actor in preserving genome stability. Taking this information as guide, we review the ways that the study of cohesinopathies can contribute to our understanding of GIN mechanisms.…”