STAG Mutations in Cancer

TRECAN 00367 No. of Pages 15

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Review

STAG Mutations in Cancer Laura Romero-Pérez,1 Didier Surdez,2 Erika Brunet,3 Olivier Delattre,2 and Thomas G.P. Grünewald1,4,5,6,7,*,@ Stromal Antigen 1 and 2 (STAG1/2) are key subunits of the cohesin complex that mediate sister chromatid cohesion, DNA repair, transcriptional regulation, and genome topology. Genetic alterations comprising any of the 11 cohesin-associated genes possibly occur in up to 26% of patients included in The Cancer Genome Atlas (TCGA) studies. STAG2 shows the highest number of putative driver truncating mutations. We provide a comprehensive review of the function of STAG1/2 in human physiology and disease and an integrative analysis of available omics data on STAG alterations in a wide array of cancers, comprising 53 691 patients and 1067 cell lines. Lastly, we discuss opportunities for therapeutic intervention.

Highlights STAG1/2 are subunits of the cohesin complex required for sister chromatid cohesion, chromosome segregation, DNA repair, genome organization, and gene expression. Mutational rates for STAG1/2 vary across cancer entities, with STAG2 being the cohesin gene harboring most putatively pathogenic mutations. Integration of current literature and genomic datasets point to differential contributions of STAG1 and STAG2 alterations to tumorigenesis and emphasize the importance of STAG2 mutations.

STAG1 and STAG2 Human Stromal Antigen-1 and -2 (STAG1 and STAG2; also known as SA1 and SA2, and referred to in this review as STAG1/2) are paralog genes (see Glossary), located on chromosome 3 and the X chromosome, respectively. The protein products of STAG1/2 share 71% of their amino acid identity (Ensembl)i. STAG1/2 encode cohesin subunits with overlapping functions, which enables reciprocal compensation of the loss of either paralog to ensure survival in most somatic cells (Box 1). STAG1 or STAG2 containing cohesin (hereafter referred as cohesin-STAG1 and cohesinSTAG2, respectively) are found along chromatin, mostly at CTCF binding sites. Both cohesin complexes also have unique functions and preferential localizations (Box 1). Cohesin-STAG2 is found preferentially at centromeres [1,2]. Recent data from single molecule imaging approaches have demonstrated that STAG2 binds in a sequence-independent manner but with higher affinity than cohesin-STAG1 to DNA ends, single-stranded gap DNA, and DNA intermediate structures associated with DNA repair or replication [3]. In fact, cohesin-STAG2 has been recently implicated in replication fork progression by directly interacting with the replication machinery, therefore protecting cells against various types of DNA damages [4]. Moreover, there is evidence that STAG2 may associate with DNA near double-strand breaks in order to repress transcription of broken genes and enable proper repair, possibly preventing chromosomal rearrangements [5]. Eventually, lack of STAG2 is correlated with an increase in DNA damage, which activates the cGAS-STING pathway and interferon response [6] (Box 1). By contrast, STAG1 preferentially participates in cohesion at telomeres [2,7,8] and specifically recognizes AT-rich DNA sequences through its N-terminal-specific AT-hook domain, which is absent in STAG2 [9]. Owing to this specific feature, STAG1 can bind DNA independently of the cohesin ring, as demonstrated by silencing different subunits of the cohesin ring without altering the STAG1 specific function [9]. Additionally, STAG1 is involved in the cohesion and separation of sister telomeres bound to TRF1 and TIN2 proteins during mitosis [7]. It has also been shown that STAG2 can regulate telomere homeostasis by preventing inappropriate/unsuitable sister chromatid recombination [10] (Box 1). Downregulation of STAG1 and STAG2 results in different effects in terms of gene expression [8, 11–13] and genome architecture, including 3D chromosome organization [12,14]. Both cohesinTrends in Cancer, Month 2019, Vol. xx, No. xx

Phenotypic effects of STAG2 mutations depend on the cellular context, type of mutation, and type of induced genomic instability, which has impact on clinical outcomes. STAG1/2 are paralog genes that can functionally compensate each other. LOF mutations in either gene confer synthetic lethality to cancer cells upon targeting of the nonmutated STAG gene, which can be exploited therapeutically.

1

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU, Munich, Germany 2 INSERM U830, Équipe Labellisé LNCC “Genetics and Biology of Pediatric Cancers”, fhna PSL Université, SIREDO Oncology Centre, Institut Curie, Paris, France 3 Institut Imagine, INSERM UMR1163, Équipe Labellisé LNCC, Dynamics of the Genome and Immune System Lab, Paris, France 4 Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany 5 German Cancer Consortium (DKTK), partner site Munich, Munich, Germany

https://doi.org/10.1016/j.trecan.2019.07.001 © 2019 Elsevier Inc. All rights reserved.

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Box 1. The Cohesin Complex and the Physiological Roles of STAG1/2 Cohesin is a highly conserved protein complex that has diverse roles during development and physiology, including sister chromatid cohesion, chromosome segregation, and proper DNA repair, genome organization, and gene expression. It is composed of four core subunits: (SMC1A, SMC3, RAD21, and either STAG1 or STAG2); different regulatory factors, including WAPL, PDS5A/B, Sororin (CDCA5), NIPBL, and MAU2 [33]; and factors controlling this regulation, including CDK1, AURKB, PLK1, PP2A, SGO1, and separase [63]. Its ring-like structure is proteolytically cleaved during cell division to allow separation of the replicated chromosomes [32]. To date, different models of the cohesin complex exist; the prevailing model poses that each cohesin complex is formed by a single cohesin-ring that contains in a mutually exclusive manner, either STAG1 or STAG2 (the ‘simple-embrace’ model) [64]. However, more recent models such as the ‘handcuff’ and ‘double embrace’ models pose that the cohesin complex consists of two paired cohesin-rings [65,66]. These models conceptually enable the pairing of STAG1- and STAG2-cohesin and thus offer the possibility that a given cohesin complex can contain both STAG1 and STAG2 in a non-mutually exclusive manner [65,66]. STAG1 and STAG2 have partially overlapping functions (Figure I). They share activity in sister chromatid cohesion to support proper chromosome segregation. While STAG1 is preferentially located at telomeres where it ensures cohesion and replication [9–11], STAG2 is located preferentially at centromeres. STAG2 depletion leads to an increase in telomere recombination (telomere sister chromatid exchange, T-SCE), possibly due to increased and persistent telomere cohesion through STAG1 from G2 into the M-phase [1,2,10]. Both STAG1 and STAG2 strongly participate in 3D chromosome compartmentalization; however, STAG2 has a unique role in that it is able to bind to sites that are devoid of CTCF [14], directly or indirectly contributing to transcriptional regulation. Moreover, STAG2 is involved in DNA damage pathways: it binds in vitro to replication and homologous recombination repair intermediates, reduces homologous recombination efficiency in cell lines, induces fork progression, and aids in repressing transcription at sites of double-strand breaks to prevent instability [3,4]. In parallel, depletion of STAG2 leads to STING pathway activation and interferon response (presumably through an increase in spontaneous DNA damages), making the STAG2-deficient cells resistant to virus infections [6]. Each of these proteins plays a different role during development. While loss of Stag1 function in mice leads to accumulation of telomere defects and transcriptional abnormalities, resulting in developmental delay and prenatal death [11,12], loss of functional Stag2 is not embryonically lethal in murine models but causes developmental delays and congenital defects in humans [17,18]. Similarly, LOF of either protein and gain-of-function mutations of STAG2 have been shown to be involved in so-called cohesinopathies associated with developmental delay, dysmorphia, and intellectual and/or behavioral problems [17–19,67–69].

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Figure I. Physiological Roles of STAG1/2. Abbreviations: DSB, double-strand break; T-SCE, telomere sister chromatid exchange.

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*Correspondence: [email protected] (T.G.P. Grünewald). @ Twitter: @GrunewaldLab

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STAG1 and cohesin-STAG2 can localize at CTCF binding sites; however, recent evidence has indicated that only cohesin-STAG2 seems to be able to bind to enhancers that lack CTCF sites and that are usually enriched by cis-regulatory elements and co-occupied by transcriptional (co-)factors [14,15]. This particular feature of cohesin-STAG2 is associated with tissue-specific transcription and cannot be compensated by cohesin-STAG1. For instance, in breast and liver carcinoma cells, the cohesin complex colocalizes with tissue-specific transcriptional master regulators at sites that are not bound by CTCF [15,16], which is possibly mediated by the CTCF-independent function of cohesin-STAG2. Recent evidence has suggested that cohesin-STAG1 may play a prominent role in the stabilization of the TAD boundaries with CTCF, while cohesin-STAG2 could mediate physical contacts between enhancers and promoters specific to the given cell type and independent of CTCF [14]. Indeed, STAG1 can compensate the absence of STAG2 in TADs, but it cannot restore specific chromatin conformations, such as those required for hematopoietic lineage specifications [13]. In addition, cohesin-STAG1, and to a lesser extent, cohesin-STAG2, are involved in embryonal development [8,11,17–19] (Box 1). Given the plethora of essential cellular processes in which STAG1/2 are involved and the reported frequencies of STAG alterations in cancer, we focus in this review on the role of STAG1/2 in cancer pathogenesis by integrating recently published articles with a pan-cancer analysis that includes more than 53 000 tumor samples from 193 studies. In addition, we discuss controversial implications of STAG1/2 alterations in cancer-related processes and how these alterations may be relevant for various therapeutic approaches.

STAG1/2 mRNA in Normal Tissues and Cancer Available transcriptome data in normal tissues [The Human Protein Atlas (GTex project)]ii have demonstrated that although both genes show similar gene expression patterns across 31 different normal tissue types, STAG1 is less expressed than STAG2. Both genes are highly expressed in gynecological organs, the urinary bladder, and the thyroid gland (Figure 1A). Prior reports have shown that STAG1/2 expression is variable in cells undergoing cell cycle [20] (Figure 1A). In The Cancer Genome Atlas (TCGA) datasetsiii available through cBioPortal for Cancer Genomicsiv [21,22], STAG1/2 are relatively similarly expressed across cancer types (Figure 1B,C). However, there is substantial variability regarding STAG1/2 expression within each cancer subtype, particularly for STAG2 in bladder and uterine carcinoma. This heterogeneity appears to be correlated with the specific type of STAG1/2 mutation. While missense mutations typically do not globally affect gene expression, truncating mutations of STAG2 tend to be associated with lower expression levels [23] (Figure 1D). Changes in STAG2 mRNA expression have been correlated with specific clinical outcomes. In bladder carcinoma, contradictory outcomes were reported in cases of STAG2 loss [24]. While some studies described a correlation between reduced STAG2 mRNA levels and low-stage and -grade bladder carcinomas associated with better clinical outcomes [25,26], others have found an association between STAG2 mutations and worse outcomes in either only muscle-invasive bladder carcinomas (more lymph node metastases, increased risk of recurrence and mortality) [27], or both muscle-invasive or superficial bladder carcinomas [28]. Further analyses in larger series and/or specifying the mechanism of the loss of STAG2 mRNA expression (type of mutations, transcriptional regulators, etc.) may be required to determine the precise role of STAG2 mutation and/or expression in the outcomes of patients with bladder carcinomas.

Glossary Aneuploidy: any deviation from the normal number of chromosomes frequently observed in different cancer cells. Centromere: structure that holds together the two chromatids in a chromosome. It is also the point of attachment of the kinetochore to which the microtubules of the mitotic spindle become anchored. cGAS-STING pathway: cyclic GMPAMP synthase (cGAS)–endoplasmicreticulum (ER)-membrane adaptor STING pathway is a component of the innate immune system. It detects the presence of cytosolic DNA, associated with tumorigenesis or viral infection, and triggers the expression of inflammatory genes leading to cell death or to activation of defense mechanisms. CTCF: CCCTC-binding factor. It is involved in transcriptional regulation. Depending on the context, it can bind a histone acetyltransferase (HAT)containing complex and function as a transcriptional activator or bind a histone deacetylase (HDAC)-containing complex and function as a transcriptional repressor. If the protein is bound to a transcriptional insulator element, it can block communication between enhancers and upstream promoters. GATA1s: short isoform caused by Nterminal truncation of GATA1, characteristic of DS-AMKL patients and causative of abnormal megakaryocyte progenitor cell hyperproliferation. Genomic instability: large number of genetic alterations frequent in cancer cells, such as mutations, losses and gains of whole/part of chromosomes, and mitotic recombination. It involves specific forms: chromosome instability (gains, losses and rearrangements of chromosomes); microsatellite instability (caused by inactivation of the DNA mismatch repair system); and epigenetic instability (leading to a CpG island methylator phenotype, by which the promoters of tumor suppressor genes become permanently silenced by methylation in that lineage). Paralog gene: in respect to one gene, another gene at a different chromosomal location in the same organism that has structural similarities, indicating that both genes were derived from a common ancestral gene and share some common but also unique functions.

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In Ewing sarcoma, low STAG2 mRNA expression is associated with trends of higher rates of relapse [29] and metastatic disease [30]. Accordingly, truncating STAG2 mutations have exhibited significant association with poor patient outcomes [31].

STAG1/2 Alterations in Human Cancer STAG2 is the subunit of the cohesin complex that harbors more putative driver mutations in adult and pediatric cancers [32], and it is considered one of the 12 genes that are significantly mutated in four or more types of cancer [33]. According to genomics datasets of 53 691 patients (cBioPortal), the frequency of somatic STAG2 and STAG1 mutations (considering missense, truncating, and in-frame mutations) across all cancer types is 2% and 0.9%, respectively (Figure 2A,B, Key Figure). While 41% of STAG2 mutations in cancer are truncating mutations (which are considered as putative pathogenic mutations), only 18% of STAG1 mutations are truncating mutations and 80% are missense mutations. It should be noted that STAG1 and STAG2 show hotspot mutations (Figure 2A,B). Hotspot mutation A116T in STAG1 is a missense variant for which the prediction of consequences (Ensemble)i seems to be inconclusive. For most of the analyzed STAG1 transcripts, some tools have predicted this variant to be likely benign (PolyPhenv, CADDvi, Metal Rvii), whereas others have estimated that it has deleterious or likely pathogenic effects (SIFTviii, REVELix). The Catalogue of Somatic Mutations in Cancer (COSMIC)x uses the FATHMM prediction algorithm to describe A116T in STAG1 as a pathogenic somatic mutation that has been detected in one case of endometrioid carcinoma (TCGA-AP-A051-01), one lung adenocarcinoma (LUAD-RT-S01702) [34], one esophageal adenocarcinoma (ESO 717) [35], and several colorectal adenocarcinomas and cell lines [36,37]. This variant has not been reported in ClinVarxi and it has been observed at very low frequencies even in healthy individuals, according to the ExAC databasexii (allele frequency 1.513–05 in Europeans), which further spurs a debate on whether it is pathogenic. Since this variant has been detected with higher frequency in colorectal cancers and in one case of endometrioid carcinoma, it is tempting to speculate that it could be related to common genetic characteristics of these diseases (e.g., microsatellite instability). However, the A116T (c.346GNA) variant is not located in a repetitive or microsatellite sequence, making this possibility unlikely. Thus, more (experimental) research is required to elucidate whether the phenotypic effect of this hotspot variant is relevant to cancer. By contrast, the R216* hotspot mutation in STAG2 (c.646CNT) is a nonsense mutation that results in a premature stop codon truncating the STAG domain of the protein (Figure 2B). It is by all means predicted to be damaging (MutationTasterxiii, FATHMMxiv) and consequently has been shown to abolish the expression of STAG2, as evidenced by Western blot and immunohistochemistry in cancer cell lines and tumors [27,31,38]. Recently, the role of this mutation has been experimentally tested in the osteosarcoma cell line U2OS through CRISPR-Cas9-mediated deletion of exon 8 of STAG2, which mimics the effect of the R216* mutation, and which was accompanied with loss of STAG2 expression [38]. On the phenotypic level, STAG2 loss reduced cell growth and proliferation, but increased migration and drug resistance of U2OS cells [38]. In line with this, the STAG2 R216* mutation has been found in several cancers, such as leukemias, Ewing sarcoma, bladder carcinoma, and endometrial carcinosarcoma [27,29–31,39–41], in which inactivation of STAG2 has been associated with poor patient outcome [29–31,39]. Considering all types of mutations, STAG1 and STAG2 accumulate diverse variants at similar frequencies (2.3% and 2.6%, respectively; Figure 2C). In the case of STAG1, a large part of reported alterations may constitute ‘amplifications’, differing from STAG2, which shows more missense 4

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PRC2: polycomb repressive complex 2. PRC2 is required for initial targeting of the genomic region to be silenced. PRC1 is required to stabilize this silencing and underlies cellular memory after cellular differentiation. Both are required for long-term epigenetic silencing of chromatin and participate in stem cell differentiation and early embryonic development. TAD: topologically associated domain is a self-interacting genomic region. DNA sequences within this 3D chromosome structure physically interact with each other more frequently than with sequences outside the TAD. Telomere: structure at the end of a chromosome involved in the replication and stability of DNA molecules. A telomere is a length of DNA that is made up of a repeating sequence of six nucleotide bases (TTAGGG) in humans. The main function is to protect the ends of chromosomes from sticking and to protect genetic information during cell division.

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(A)

(C)

(B)

(D)

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Figure 1. STAG1/2 Expression in Normal Tissues and Cancer. (A) RNA-Seq analysis of STAG1 and STAG2 from the Genotype-Tissue Expression (GTEx) project. Mean mRNA expression values represent RPKM (reads per kilobase million). Data from The Human Protein Atlas. (B) and (C) Combined RNA-Seq V2 and mutational data showing gene expression levels and gene mutations of STAG1 and STAG2, respectively, across 10 953 patients included in the TCGA PanCancer study including 32 tumor types. Dataset from cBioPortal. (D) STAG2 expression in association with STAG2 mutations found in bladder and uterine carcinomas from TCGA PanCancer study. Y-axis values correspond to Z-scores from RNA-Seq by expectation maximization (RSEM) Batch normalized from Illumina HiSeq RNASeq V2. Abbreviation: CA, carcinoma.

and truncating mutations. In bladder carcinoma, STAG2 was mutated in 13.3% of the samples, with 73.8% (164/222) truncating mutations, across 1475 samples (1429 patients) from ten different studies (cBioPortal). However, STAG1 was mutated in 3.9% of samples and only 6% (4/67) were truncating mutations. Similarly, across 219 samples (215 patients) from two different Ewing sarcoma studies [30,31], STAG2 showed a somatic mutation frequency (SMF) of 13.7%, of which 90.9% (30/33) were truncating mutations. By stark contrast, STAG1 was Trends in Cancer, Month 2019, Vol. xx, No. xx

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Key Figure

STAG1/2 Alterations in Cancer

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(See figure legend at the bottom of the next page.)

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found to be mutated in only a single case [31] among the 219 Ewing sarcoma samples (cBioPortal) (SMF 0.5%); however, this mutation (D329V) was classified as a missense somatic mutation and predicted as being pathogenic in COSMIC and PolyPhen-2. These data provide suggestive evidence for differential contributions of STAG1 and STAG2 alterations, with STAG2 likely being more relevant to cancer (see Box 2). In addition, these mutational profiles further suggest that the functions of STAG1 and STAG2 do not entirely overlap, as mutational inactivation of STAG2 leads to consequences that STAG1 apparently cannot fully compensate. This further evokes our interest in understanding these nonredundant functions and their roles in cancer (see Outstanding Questions). Copy-number variations of STAG1 and STAG2 may also play a role in cancer. Apart from truncating or frameshift (point) mutations, deletions could also cause a loss-of-function (LOF), which may have clinical relevance in cancer. For example, STAG2 losses are correlated with the relapse of patients affected by B cell precursor acute lymphoblastic leukemia [42]. In addition, ‘amplifications’ were described for both genes in cBioPortal: for instance, it has been intriguingly observed that in available data from the study of the Molecular Basis of Neuroendocrine Prostate Cancer (Trento/Cornell/Broad 2016) [43] provided in cBioPortal, N28% of neuroendocrine carcinomas (NECs) of the prostate might harbor a STAG2 ‘amplification’ without any other STAG2 mutation (Figure 2E), and that N20% of prostatic NECs might have a STAG1 ‘amplification’ (Figure 2D). However, without the certainty that these alterations constitute true focal gene amplifications (rather than mere whole chromosome gains), and without (experimental) evidence that these ‘amplifications’ lead to gain-of-function of STAG1/2 that contributes to tumorigenesis, these findings should be interpreted with caution. Furthermore, STAG2 gene fusions have been reported in human T cell acute lymphoblastic leukemia/lymphoma cell lines [44]. However, there are neither reports from patients nor entries of such fusions in the COSMIC database, which is why the role of these fusions currently remains elusive.

Epigenetic Regulation of STAG1/2 in Cancer Shen et al. described a reduction in STAG2 protein levels in melanoma samples; however, they could not detect any mutation in coding exons to explain this decrease in protein expression. Hence, the authors suggested a downregulation mediated by epigenetic mechanisms [45]. Public methylation data regarding STAG1/2 promoters in 1067 different cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE)xv have shown that STAG2, whose promoter includes 21 CpG islands, is strongly hypermethylated (30%–35%) in cancer cell lines of Ewing sarcoma as well as endometrial and renal carcinoma, and moderately hypermethylated (~25%) in soft-tissue tumors, pancreatic, mammary, and ovarian carcinoma, as well as in hematological and lymphoid neoplasms (Figure 3A). However, in this context it should be taken into account that STAG2 is located on the X chromosome, one of which is

Figure 2. (A) and (B) Lollipop representation of the type and frequency of somatic mutations found in STAG1 and STAG2, respectively, in a set of 53 691 patients (from cBioPortal). A116T is a variant with uncertain pathogenic relevance. R216* is a loss-of-function mutation. (C) Type and frequency of gene alterations, including indels, fusions, and point mutations found in STAG1 and STAG2 among 193 different cancer studies (53 691 patients; from cBioPortal). Amplifications may comprise focal gene amplifications or larger chromosome parts/whole chromosome gains. (D,E) Alteration frequencies and types for STAG1 and STAG2 among different tumor types. Cancer types for which at least five cases with STAG gene alterations were reported and with a minimum alteration frequency of 3% are represented. Amplifications may comprise focal gene amplifications or larger chromosome parts/whole chromosome gains. Abbreviations: CA, carcinoma. CS, carcinosarcoma; SMF, somatic mutation frequency.

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Box 2. The Cohesin Complex in Cancer Cohesin complex subunits and regulators are frequently affected by LOF alterations in cancer [28,56,58,70]. These genes have been described as tumor suppressors in leukemia and solid tumors such as Ewing sarcoma, glioblastoma, melanoma, and bladder carcinoma [14,23,45,58]. While heterozygous germline mutations in cohesin genes are sufficient to promote tumorigenesis in mice [49], cohesin germline alterations in humans cannot induce cancer on their own [71,72]. Indeed, despite the large fraction of patients with Cornelia de Lange syndrome who display heterozygous mutations in the cohesin genes NIPBL, SMC1A, SMC3, and RAD21, cancer incidence does not appears to be affected in these patients [73–76]. However, on the level of somatic mutations, multiple lines of evidence have suggested that cohesin mutations contribute directly or indirectly to tumorigenesis via diverse mechanisms. First, cohesin genes are somatically mutated in a wide range of cancers, with high mutation frequencies in bladder carcinomas (11–26%) [25,26,28], Ewing sarcomas (15–22%) [29–31], and leukemias (~13–50%) [40,53,77]. However, it should be noted that not all cohesin genes are affected similarly: since STAG2 maps to chromosome X, a single genetic event may be sufficient to achieve LOF, whereas for other autosomal cohesin genes, two events may be required. For STAG2, this is because males (usually) only have one X chromosome and in females one of the two X chromosomes is inactivated. Somatic cohesin gene mutations are particularly frequent (~50%) in acute megakaryoblastic leukemia (AMKL) in children with trisomy 21 (Down syndrome) (DS-AMKL) [40]. The reason for this extremely high frequency is currently unknown, but it is hypothesized that cohesin mutations may promote leukemia by affecting the regulation of the expression of genes on chromosome 21 such as RUNX1 [a regulator of hematopoietic stem cells (HSCs) and megakaryocytes] or by affecting the chromatin binding of GATA1 and GATA1s, which may play a causal role in DS-AMKL [71,72]. Second, regarding myeloid neoplasms and Ewing sarcoma, somatic mutations in cohesin genes represent early events that define the dominant clone [30,31,40,53,58,78,79], which could hint at a potential cancer-driver role in these cancer types. In support of this notion, cohesin gene mutations, especially those in STAG2, are correlated with enhanced self-renewal and impaired differentiation of HSCs, possibly mediated via changes in the cellular transcriptome and aberrant targeting of epigenetic complexes such as polycomb PRC2 [13,72,80,81]. In fact, models of dose-specific loss of cohesin have shown that defective differentiation and increased proliferation can be associated with modifications in chromatin accessibility and altered gene expression [71,82]. Third, cohesin mutations (except for those in RAD21) may rewire the transcriptome by bringing otherwise silent protooncogenes such as c-Myc [83] and protocadherins [84] under the control of new regulatory elements [14,85,86]. Also, copy number gains of the SMC1A locus, combined with SMC1A mutations that affect DNA binding, contribute to the tumorigenesis of aggressive colorectal carcinoma in mice through the dysregulation of genes such as ATG12 or STEAP4 [87]. Fourth, STAG2 blocks sister chromatid recombination at telomeres, thus reducing the lifespan of normal cells. Hence, inactivation of STAG2 (e.g., due to LOF mutations) may enable mutated cells to live longer and thus increases the possibility that these cells will acquire additional tumor-driving mutations and genomic instability [10].

inactivated in females. Thus, the findings on STAG2 promoter methylation need to be interpreted with caution. By contrast, STAG1 is not strongly methylated (median b25%) in any cancer entity represented in the CCLE (Figure 3B). Regarding the variability of STAG2 promoter methylation across different cell lines derived from the same cancer type, a heterogeneous intertumoral epigenetic regulation has become evident. This regulation may suggest the activation of different epigenetic programs and gene regulations within the same tumor type, leading to variability in cellular responses to therapy [46]. Yet, so far, only one study in cancer, namely in testicular embryonal carcinoma, correlated STAG2 promoter hypermethylation with downregulation of the mRNA; however, no correlation with clinical outcome was observed [47]. Guo et al. examined the methylation status of the STAG2 promoter in transitional cell carcinoma (TCC) of the bladder and found STAG2 promoter hypermethylation in 7/30 tumors (23%) [28]. Although this STAG2 hypermethylation was not compared with any clinical characteristics, the authors describe bladder cancer as the first type of cancer with predominant alterations in genes involved in the process of sister chromatid cohesion and segregation, as well as the specific loss of STAG2, likely to contribute to TCC pathogenesis [28]. 8

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Figure 3. DNA Methylation of STAG1/2 in Cancer Cell Lines. DNA methylation percentages of STAG2 (A) and STAG1 (B) among 1067 different cancer cell lines. Graphs showing those cells with positive methylation values.

In sum, STAG1 and especially STAG2 appear to be differentially regulated by promoter hypermethylation across and within cancer entities; however, the causes and (clinical) consequences remain to be determined.

STAG Alterations, Chromosome Instability, and Aneuploidy Despite the extensive amount of data on the role of STAG1/2 in normal cellular processes, the contribution of STAG alterations to cancer pathogenesis through the induction of chromosome instability and aneuploidy is still controversial. Disruption of the cohesin complex seems to be directly involved in defective sister chromatid cohesion, which may represent a major cause of chromosome instability in human cancers [48]. Indeed, cohesin gene mutations have been described as a cause of aneuploidy and increased genomic instability in vitro through different mechanisms [48–51]. For example, STAG2 mutations in glioblastoma and colorectal carcinoma cell lines induced defective cell division leading to aneuploidy due to a premature separation of sister chromatids in metaphase [50]. Additionally, inactivation of Trends in Cancer, Month 2019, Vol. xx, No. xx

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STAG1 can generate aneuploidy through impaired telomere replication [49]. Similarly, SMC1A mutations have been postulated as promoters of aneuploidy in colorectal carcinoma due to their induction of abnormal anaphases and, consequently, chromosome missegregation [48,51]. Despite these in vitro observations, in situ findings are controversial. On the one hand, STAG2 mutations are not associated with chromosomal instability in bladder carcinoma [26]. On the other hand, mutations in different cohesin genes such as SMC1A, SMC3, RAD21, and STAG2, have been detected in acute myeloid leukemia and Ewing sarcoma, which usually do not display high degrees of aneuploidy [29–31,52,53]. These controversial observations may be related to the cellular context (e.g., colorectal carcinoma versus Ewing sarcoma), the type of gene mutation, and the type of induced genomic instability. Indeed, Kim et al. introduced nine tumor-derived mutations into the endogenous allele of STAG2 in cultured colorectal carcinoma cells (HCT116) and observed that all nonsense mutations led to altered sister chromatid cohesion, whereas STAG2 missense mutations did not. A subset of these nonsense mutations and one missense mutation induced anaphase defects, including lagging chromosomes, which are usually associated with chromosome instability. However, only one of these nonsense mutations led to aneuploidy [32]. Together, these findings indicate that not all STAG2 mutations found in cancer confer defects in chromatid cohesion that affect chromosome instability or chromosome count, but they may affect other STAG2 functions. According to genomic data across 193 cancer studies representing the major tumor types available through cBioPortal, 56.23% (717/1275) of STAG2 mutations are missense mutations and 40.7% (519/1275) are nonsense mutations (Figure 2B). The latter may be more damaging. Interestingly, cohesin gene mutations can induce genome instability without creating overt aneuploidy [5,14]. A recently identified STAG2 mutation in bladder carcinoma was found to be associated with intact sister chromatid cohesion, but linked to a defect in the ability of STAG2 to repress transcription at double-strand breaks. This specific STAG2 mutation class may lead to an increase in genome rearrangements in the absence of chromosome missegregation [5]. In summary, the consequences of STAG1/2 mutations on chromosome instability, aneuploidy, cellular differentiation, gene regulation, and cell survival, appear to depend on the specific type of mutation, the dose-specific loss, as well as on the cellular context and the developmental stage.

Synthetic Lethality (SL) of STAG1 and STAG2 SL is defined as an ‘interaction where the concomitant alteration of two non-essential genes leads to cell death, while the alteration of either gene individually is viable’ [23]. Since STAG proteins can at least, in part, functionally compensate for each other, and as only one of both STAG proteins can be incorporated into a given cohesin-ring [according to the prevailing structural models (Box 1)], LOF mutations of either gene in cancer could conceptually confer SL to such cancer cells, which may be exploited therapeutically [54] (Table 1). Indeed, despite the fact that computational methods (SynLethDB)xvi have not predicted SL between STAG1 and STAG2 [55], simultaneous depletion of both genes through RNA interference or genetic knockouts in glioblastoma, bladder carcinoma, Ewing sarcoma, and colon carcinoma cell lines provide a convincing proof-of-concept that STAG1 and STAG2 10

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Table 1. Consequences of STAG1/2 Mutations and Therapeutic Opportunities Condition Outcome in

Wild-type (WT)

STAG1 mutated (LOF)

STAG2 mutated (LOF)

STAG1/2 mutated (LOF)

Mouse models

Viable

(–/–) Embryonic lethality (+/–) Shorter lifespan and tumor prone

Developmental delay and congenital anomalies Hematological problems

Unknown Predicted synthetic lethality

Cell models

Viable

Viable

Viable

Synthetic lethality

Limited effects on normal cells and for WT parental residual cancer cells

Target STAG2 specific function (or STAG1/2 shared)

Target STAG1 specific function (or STAG1/2 shared)

Limited/depending on the deficiency level

Therapeutic opportunity

display the required functional interaction necessary for SL [4,23,54,56]. The combined loss of both genes in colon carcinoma cell line HCT116 affected cell division, confirming that the cohesin complex is not able to hold sister chromatids in the absence of both STAG subunits [56]. Similarly, the inhibition of STAG1 in the Ewing sarcoma cell line SK-ES1, which harbors a somatic homozygous LOF point mutation in STAG2, results in decreased cell viability [23]. There is also evidence of Stag1 and Stag2 SL in vivo. Viny et al. recently generated Stag2 knockout mice (hemizygous male) with hematopoietic cells that retained one or no copies of Stag1. The authors observed thrombocytopenia and impaired survival when one copy of Stag1 was present and rapid lethality in the absence of both alleles [13]. Although SL approaches may be attractive in the context of STAG1/2-deficient tumors, it remains uncertain whether this approach would also be suitable for STAG1/2 missense mutated tumors since many of these variants do not affect the capability of STAG proteins to interact with the cohesin complex [5,52] (Table 1). Therapeutic Applications of SL The concept of SL is especially relevant in cancer and is based on the inhibition of the SL paralog of a mutated tumor suppressor. Conceptually, the mutated cancer-specific gene can be used as a biomarker, and targeting the nonmutated paralog gene is supposed to preferentially eliminate cancer cells while normal cells are expected to remain largely unaffected [55,57]. In addition to the demonstrated in vitro and in vivo SL interactions between STAG1/2 [4,23,54, 56], it has been reported that STAG2 mutated cells become more susceptible to DNA damage (especially double-strand breaks) upon shRNA-mediated depletion of STAG1, due to an impairment in their capability to repair damaged DNA [54]. DNA repair capacity is based on the stability and integrity of the replication fork, which is preserved by an intact and functional cohesin complex. Accordingly, loss of cohesin function, mainly loss of STAG2, induces a stalled replication fork that sensitizes for the inhibition of proteins involved in DNA damage repair such as ATR or poly-ADP-ribose protein 1 (PARP1) [4,58,59], which is the main SL partner of STAG1 predicted by SynLethDB (0.855 confidence score). In fact, sensitization to PARP inhibition was demonstrated by the inhibition of STAG1 in STAG2-mutated cells of bladder carcinoma (UC3) and Ewing sarcoma (TC32), resulting in mitotic catastrophe and cell death [54]. Moreover in glioblastoma cells (H4), ATR and PARP inhibition lead to decreased cell survival, suggesting AZD6738 and olaparib, respectively, as candidates for SL clinical inhibition in a STAG2 mutated background [4,60]. Trends in Cancer, Month 2019, Vol. xx, No. xx

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Mondal et al. discussed other therapeutic opportunities, including ionizing radiation (gamma radiation) and more cytotoxic chemotherapeutic agents other than from PARP and ATR inhibitors. On the one hand, in synergism with the disruptive effect of STAG2 inactivation on the replication fork, ionizing radiation provokes cell death through the accumulation of DNA double-strand breaks in glioblastoma cells (H4). On the other hand, treatment with DNA alkylating agents, DNA crosslinking agents, or topoisomerase II inhibitors together with the STAG2 deficiency in glioblastoma (H4 and 42MGBA) and Ewing sarcoma (TC-106) cell lines, leads to reduced survival and colony formation when treated with cyclophosphamide [4]. However, there are some limitations that may lead to low specificity and minimal SL effects on cancer cells. These include the following. (i) Tumor heterogeneity: due to tumor heterogeneity, the LOF mutation in either STAG gene may only be present in particular (sub)clones of the tumor and, as such, the SL therapy may not be effective against all tumor clones. Thus, clonal and subclonal mutation frequencies have to be considered and eventually multitarget strategies need to be employed to improve the effectiveness of a targeted therapy approach [57]. Future studies should include patient-derived xenograft models, as these may model tumor heterogeneity and may enable the evaluation of the therapeutic potential through multitarget approaches. (ii) STAG1 ligandability: STAG1 presents a ligandability score of 75% for cancer, according to canSARxvii network prediction [61,62]; however, no ligandable cavities have been identified and no drugs have been proposed as potentially usable in clinics thus far. Since targeting the STAG1 protein specifically and directly is pharmacologically quite challenging, particularly due to its high sequence homology with STAG2, the preferred option would be to identify targetable candidates by performing genome-wide shRNA/sgRNA and/or high-throughput drug screening in STAG2 proficient and deficient contexts [57]. This approach could also be investigated in the STAG1 deficient context. (iii) Targeted delivery: it is necessary to design a method of delivering SL drugs and ionizing radiation specifically to STAG mutated cells, to avoid adverse effects in normal cells in which critical cellular functions may depend on nonredundant properties of STAG1/2. In addition, different indirect therapeutic approaches have been proposed that target other cohesin factors, including transcriptional inactivation of regulatory proteins such as NIPBL, activation of downstream genes, and using hypomethylating agents to re-establish the expression of genes methylated in certain tumors, such as RAD21 in 25% of colorectal cancers [63]. However, for all these approaches, the above-mentioned limitations apply. They especially apply to hypomethylating agents, which tend to rarely act specifically only on tumor cells. Finally, STAG2 alterations have been associated with resistance to anticancer treatments, such as BRAF inhibition (BRAFi) and cisplatin-mediated cell apoptosis [38,45]. In melanoma cells, loss of STAG2 inhibits CTCF-mediated expression of DUSP6, inducing reactivation of ERK signaling and therefore reducing the cell’s sensitivity to BRAF pathway inhibition [45]. In osteosarcoma cells, loss of STAG2 induces chemo-resistance through the inhibition of apoptosis [38]. Thus, targeting SL in STAG2 mutated tumors may constitute a promising approach to overcome acquired resistance toward BRAFi and/or chemo-resistance.

Concluding Remarks Functional cohesin complexes, including intact STAG1 and STAG2, are crucial for cell survival and the proliferation of normal cells. Whether these factors are present in a mutually exclusive manner in the cohesin complex remains unclear according to the current models discussed here. Elucidating this concept could help to establish conclusions based on exclusivity or duality within the cohesin complex. 12

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Outstanding Questions Given that different models of the cohesin complex are currently considered, what is the exact stoichiometry of the cohesin complex in vivo and how are STAG1 and STAG2 embedded in this complex? Which are the underlying mechanisms explaining the altered genome topology upon mutation of either cohesin-STAG1 or cohesin-STAG2? In addition to the blockade of sister chromatid recombination at telomeres, which unique functions of STAG2 are involved in cancer and why can STAG1 not compensate for them? Why do some cancer entities almost exclusively exhibit mutations in one STAG gene but not the other? What are the cause(s) and consequence(s) of inter- and intratumoral heterogeneity of STAG1/2 mutations and differential promoter methylation? How can drugs that specifically target STAG1 or STAG2, or only redundant functions of both proteins, be designed?

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The overlapping but also unique functions of STAG1 and STAG2 in processes associated with specific chromosomal regions, transcriptional regulation, 3D chromosome organization, and DNA repair are complex. Inactivation of STAG factors in the context of these genomeassociated functions is related to tumorigenesis and while their effects appear to depend on gene dosage, developmental state, cellular context, and the specific type of mutation present, the underlying mechanisms are still largely unknown. According to the most recent literature, STAG2 mutations appear to have more cancer-related consequences than STAG1 mutations, which can be partially explained by the type of mutations and the prominent role of STAG2 LOF in both DNA repair and the blockade of sister chromatid recombination at the telomeres. This STAG2 LOF may enable an extension of the replicative lifespan of cells, thus increasing the probability that a given cell will acquire additional mutations. However, to our knowledge, a striking increase in mutational burden has not been reported in all tumor types affected by STAG2 mutations, alternative unique functions of STAG2 that cannot be compensated by STAG1 may be involved in cancer pathogenesis. These STAG2 specific functions seem to be crucial for some tumor types. In addition to the unique functions carried out by both STAG1 and STAG2, their functional redundancy may offer a therapeutic vulnerability in cancer through the use of SL approaches. Since STAG2 is the most frequently mutated gene, drugs that inhibit either its SL partner STAG1, or DNA damage repair factors, may constitute promising treatment options. To design effective multitarget treatments, these drugs must overcome different limitations; for instance, they must achieve a specific ligandability to STAG1 or alternative candidates and thus be specifically delivered only to STAG2-mutated cells. Also, to be effective despite the complex clonal and subclonal genetic architecture of the given tumor, a mutational characterization of STAG1/2 at both the clonal and subclonal level would be required. Finally, we encourage the continuation of the experimental research to shed light on the questions that remained open throughout this review. Such questions include the inconclusive clinical association of the STAG2 mutations in bladder cancer; the types, causes, and consequences of ‘amplifications’ that affect STAG1/2 in various cancers; elucidation of the role of STAG2 fusions in cancer; clarification of the causes of differential STAG2 methylation in cancer; and the effectiveness of SL in those tumors where most STAG1/2 mutations are missense mutations. We conclude that future research should be directed toward these aspects to fully illuminate the pleiotropic roles of STAG1/2 in normal and cancer cells and to develop specific targeted and effective therapeutics (see Outstanding Questions). Acknowledgments L.R-P. is supported by the Dr Leopold and Carmen Ellinger Foundation and the Gert and Susanna Mayer foundation. The laboratory of T.G.P.G. is further supported by the ‘Verein zur Förderung von Wissenschaft und Forschung an der Medizinischen Fakultät der LMU München’ (WiFoMed), by the ‘Mehr LEBEN für krebskranke Kinder - Bettina-Bräu-Stiftung’, the Matthias-Lackas Foundation, the Friedrich-Baur foundation, the Dr Rolf M. Schwiete foundation, the Barbara and Hubertus Trettner foundation, the Wilhelm-Sander-Foundation (2016.167.1), the Deutsche Forschungsgemeinschaft (DFG-391665916), and the German Cancer Aid (DKH-70112257).

Resources i

www.ensembl.org/Homo_sapiens/Info/Index

ii

www.proteinatlas.org/about

iii

www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga

iv

www.cbioportal.org/

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v

http://genetics.bwh.harvard.edu/pph2/

vi

https://cadd.gs.washington.edu/snv

vii

https://sites.google.com/site/jpopgen/dbNSFP

viii

https://sift.bii.a-star.edu.sg/www/SIFT_seq_submit2.html

ix

https://sites.google.com/site/revelgenomics/about

x

https://cancer.sanger.ac.uk/cosmic

xi

www.ncbi.nlm.nih.gov/clinvar/

xii

http://exac.broadinstitute.org

xiii

www.mutationtaster.org/

xiv

http://fathmm.biocompute.org.uk/fathmm-xf/

xv

https://portals.broadinstitute.org/ccle

xvi

http://histone.sce.ntu.edu.sg/SynLethDB/

xvii

https://cansarblack.icr.ac.uk/

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