Fusion Sarcoma Research Articles

Epithelioid Hemangioendothelioma as a Model of YAP/TAZ-Driven Cancer: Insights from a Rare Fusion Sarcoma.

Epithelioid hemangioendothelioma (EHE) is a rare soft-tissue sarcoma involving cells with histologic markers that suggest an endothelial origin. Around 90% of EHEs are caused by the fusion of Transcriptional Co-activator with a PDZ-motif (TAZ) with Calmodulin Binding Transcription Activator 1 (CAMTA1), a central nervous system-specific transcription activator. The 10% of EHEs that lack the TAZ–CAMTA1 fusion instead have a fusion of Yes-associated Protein (YAP) and Transcription Factor E3 (TFE3) genes (YAP-TFE3). YAP and TAZ are well-defined downstream effectors in the Hippo pathway that promote cell growth when translocated to the nucleus. The TAZ–CAMTA1 fusion transcript is insensitive to the Hippo inhibitory signals that normally prevent this process and thus constitutively activates the TAZ transcriptome. In EHE, this causes tumors to form in a variety of organs and tissue types, most commonly the liver, lung, and bone. Its clinical course is unpredictable and highly variable. TAZ activation is known to contribute to key aspects of the cancer phenotype, including metastasis and fibrosis, and increased expression of TAZ is thought to be causally related to the progression of many cancers, including breast, lung, and liver. Therefore, understanding TAZ biology and the molecular mechanisms by which it promotes unregulated cell proliferation will yield insights and possibly improved treatments for both EHE as well as much more common cancers.

Abstract B24: Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma

Abstract CIC-DUX4 soft-tissue tumors are an aggressive subset of undifferentiated round cell sarcomas that arise in children and young adults. The cytogenetic hallmark, (t(4;19) or t(10;19)), of this tumor is a chromosomal translocation that fuses the transcription factor, Capicua (CIC), with the double homeobox 4 protein, DUX4. Despite morphologic similarities to Ewing sarcoma, CIC-DUX4 sarcomas are clinically distinct with rapid development of lethal metastatic disease and chemoinsensitivity. The unique cytogenetic and clinical features that distinguish CIC-DUX4 sarcomas from other small round cell tumors provide a unique opportunity to identify and rationally target this population to improve clinical outcomes. To meet this need, we are taking a systematic functional approach to identify critical CIC-DUX4 targets that promote tumor progression and metastasis. Leveraging a comparative transcriptional analysis between cytogenetically distinct small round cell sarcomas, we reveal that the CIC-DUX4 fusion activates highly conserved molecular networks to control tumor growth and metastatic capacity. Using an orthotopic soft-tissue sarcoma model, we show that CIC-DUX4 induces ETV4 expression to drive invasion and metastasis. Genetic silencing of ETV4 in CIC-DUX4 expressing tumors limits tumor dissemination without a profound impact on tumor cell growth. These findings suggest that CIC-DUX4 enhances metastasis and tumor growth through distinct transcriptional repertoires. Consistent with this, we identify multiple components of the cell cycle machinery that are transcriptionally regulated by CIC-DUX4 expression. Specifically, the CIC-DUX4 fusion co-opts native CIC-binding specificity to transcriptionally activate these cell cycle target genes. Altogether, our molecular and functional dissection of the CIC-DUX4 fusion reveals highly conserved CIC-regulated transcriptional targets that promote tumor growth and metastasis. We aim to pharmacologically exploit these key CIC-DUX4-dependent transcriptional nodes to improve outcomes for this population with few therapeutic options. Citation Format: Ross A. Okimoto, Wei Wu, Trever G. Bivona. Molecular and functional dissection of the CIC-DUX4 fusion in undifferentiated round cell sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B24.

Clear cell sarcomas of the kidney are characterised by BCOR gene abnormalities, including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion.

Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically. By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression. The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.

Novel exon–exon breakpoint in CIC-DUX4 fusion sarcoma identified by anchored multiplex PCR (Archer FusionPlex Sarcoma Panel)

AimsWe describe the clinical and pathological features and novel genetic findings of a case of CIC-DUX4 sarcoma occurring in the thigh of a 35-year-old man.MethodsFusion gene detection using a next-generation...

Abstract 2916: TAF15 as an important mediator of RNA polymerase II-dependent drug tolerance

Abstract Background: Cancer relapse after chemotherapy is thought to originate from drug-tolerant cancer cell subpopulations. We studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Previous DTC formation-based compound screening identified an RNA polymerase (RNAP) II inhibitor, α-amanitin (α-AMA), suggesting the existence of an RNAPII-dependent drug tolerance in cancer cells. However, the mechanism underlying this regulation is unclear. Materials and Methods: DTCs derived from the gastric cancer cell line MKN45 were used as an in vitro model for gastrointestinal tumor recurrence. For the generation of the DTCs and those of untreated colonies (i.e., control colonies), cells were sparsely disseminated (100 cells/cm2) into the presence or absence of cisplatin at 50% colony inhibitory concentration. To identify the DTC formation responsible genes, a DNA microarray analysis was performed followed by gene ontology analysis and quantitative RT-PCR (qRT-PCR) validation. For gene knockdown experiments with those that have been identified as DTC-formation responsible genes, cells were treated with small interference RNAs (siRNAs) for 48 hrs and then sparsely disseminated into the microplates in the presence of cisplatin for the DTC formation. Results: In terms of the RNAPII-dependence of the DTC formation, we focused on RNAPII-binding protein-coding genes in the list of the top 2.5% of specifically induced genes in DTCs. The transcriptional screening identified only one gene, TATA-binding protein-associated factor 15 (TAF15). The level of TAF15 mRNA was 2.1-fold higher in DTCs than in the control colonies. A protein family consisting of fusion in sarcoma (FUS), Ewing's sarcoma (EWS), and TAF15 is called the FET (FUS/EWS/TAF15) family, and they play various roles in transcription as well as RNA processing. Among the FET family proteins, the TAF15 protein level was specifically increased in DTCs, suggesting that TAF15-specific function may contribute to DTC formation. Next, we examined whether TAF15 protein depletion by siRNA affects DTC formation. The TAF15 depletion preferentially inhibited DTC formation despite the fact that the degree of inhibition to the control colony formation was limited. With an anti-TAF15 siRNA treatment, the original small, round-shaped MKN45 cells exhibited spindle-like morphological changes within 48 hrs. Interestingly, α-AMA treatment also induced similar morphological changes, suggesting that RNAPII inhibition caused TAF15 depletion. Consistent with the morphological changes, both TAF15 mRNA and protein levels decreased in response to α-AMA treatment. Conclusion: These results suggest that TAF15 is an important mediator of RNAPII-dependent drug tolerance and one of the potential molecular therapeutic targets of α-AMA in the context of cancer relapse after chemotherapy. Citation Format: Kohei Kume, Kohei Ito, Takeshi Iwaya, Satoshi S. Nishizuka. TAF15 as an important mediator of RNA polymerase II-dependent drug tolerance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2916.

Abstract 2315: Reversible disruption of mSWI/SNF-like BAF complexes by the SS18-SSX oncogenic fusion in synovial sarcoma.

Abstract Recent exon sequencing studies have revealed frequent mutations in subunits of SWI/SNF or BAF complexes and implicated them as tumor suppressors. Indeed, we estimate that they are mutated in 15.8 percent of sequenced human cancers. Interestingly, the subunits most commonly mutated are not required for chromatin remodeling in vitro and thus may contribute to chromatin regulation in a novel way. One difficultly in understanding the role of these complexes in cancer is distinguishing primary driving mutations from passenger mutations. To investigate the underlying mechanism we studied human synovial sarcoma (SS), in which transformation results from a remarkably precise and uniform translocation of exactly 78 amino acids of SSX to the SS18 protein. In a proteomic study we discovered that SS18 is a dedicated, non-exchangeable subunit of BAF complexes. We demonstrate that the hallmark SS18-SSX fusion incorporates into BAF complexes, evicting both the wild-type SS18 protein and the tumor suppressor, hSNF5 (BAF47). The altered complex binds the Sox2 locus, reversing polycomb-mediated repression and results in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for their proliferation. Increasing the concentration of wild-type SS18 leads to reassembly of the wild-type complex, retargeting of BAF complexes away from the Sox2 locus, polycomb-mediated repression of Sox2 and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX and hence provides a potential foundation for therapeutic intervention. Citation Format: Cigall Kadoch, Gerald R. Crabtree. Reversible disruption of mSWI/SNF-like BAF complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2315. doi:10.1158/1538-7445.AM2013-2315

Reversible Disruption of mSWI/SNF (BAF) Complexes by the SS18-SSX Oncogenic Fusion in Synovial Sarcoma

Recent exon sequencing studies have revealed that over 20% of human tumors have mutations in subunits of mSWI/SNF (BAF) complexes. To investigate the underlying mechanism, we studied human synovial sarcoma (SS), in which transformation results from the translocation of exactly 78 amino acids of SSX to the SS18 subunit of BAF complexes. We demonstrate that the SS18-SSX fusion protein competes for assembly with wild-type SS18, forming an altered complex lacking the tumor suppressor BAF47 (hSNF5). The altered complex binds the Sox2 locus and reverses polycomb-mediated repression, resulting in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for proliferation. Increasing the concentration of wild-type SS18 leads to reassembly of wild-type complexes retargeted away from the Sox2 locus, polycomb-mediated repression of Sox2, and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX, providing a potential foundation for therapeutic intervention.

Microsatellites with Macro-Influence in Ewing Sarcoma

Numerous molecular abnormalities contribute to the genetic derangements involved in tumorigenesis. Chromosomal translocations are a frequent source of these derangements, producing unique fusion proteins with novel oncogenic properties. EWS/ETS fusions in Ewing sarcoma are a prime example of this, resulting in potent chimeric oncoproteins with novel biological properties and a unique transcriptional signature essential for oncogenesis. Recent evidence demonstrates that EWS/FLI, the most common EWS/ETS fusion in Ewing sarcoma, upregulates gene expression using a GGAA microsatellite response element dispersed throughout the human genome. These GGAA microsatellites function as enhancer elements, are sites of epigenetic regulation and are necessary for EWS/FLI DNA binding and upregulation of principal oncogenic targets. An increasing number of GGAA motifs appear to substantially enhance EWS/FLI-mediated gene expression, which has compelling biological implications as these GGAA microsatellites are highly polymorphic within and between ethnically distinct populations. Historically regarded as junk DNA, this emerging evidence clearly demonstrates that microsatellite DNA plays an instrumental role in EWS/FLI-mediated transcriptional regulation and oncogenesis in Ewing sarcoma. This unprecedented role of GGAA microsatellite DNA in Ewing sarcoma provides a unique opportunity to expand our mechanistic understanding of how EWS/ETS fusions influence cancer susceptibility, prognosis and transcriptional regulation.

Ubiquitinated, p62 immunopositive cerebellar cortical neuronal inclusions are evident across the spectrum of TDP-43 proteinopathies but are only rarely additionally immunopositive for phosphorylation-dependent TDP-43

Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), frontotemporal lobar degeneration with motor neuron disease/amyotrophic lateral sclerosis (FTLD-MND/ALS) and MND/ALS are thought to represent a clinicopathological spectrum of TDP-43 proteinopathies. The cerebellum has been little studied in these conditions, probably because of the lack of cerebellar signs in most cases. We examined p62 immunohistochemistry on cerebellar sections from 43 TDP-43 proteinopathies (including cases of FTLD-TDP, FTLD-MND/ALS and MND/ALS) together with 72 cases of other neurodegenerative diseases, seven controls and three other disease conditions. In 11 of the TDP-43 proteinopathies (26%) there were numerous p62-positive cerebellar inclusions, predominantly within the granular layer, but also the molecular and Purkinje cell layer. Furthermore, only one of the remaining 82 cases (a familial tauopathy) showed similar p62 positivity. Immunohistochemistry for ubiquitin was positive in the granular layer inclusions. The immunohistochemistry for phosphorylation-independent TDP-43, hyperphosphorylated tau, α-synuclein, fusion sarcoma protein (FUS), and neurofilament was negative. In only one case (a case of FTLD-TDP) were the inclusions positive for phosphorylation-dependent TDP43 (p-TDP-43). Those TDP-43 proteinopathy cases that showed the cerebellar inclusions also tended to display other common features, such as a notable excess of p62 pathology when compared to TDP-43 pathology, especially within the pyramidal neurones of the hippocampus but also in some cases within the neocortex. The results suggest that p62-positive inclusions within the cerebellum are seen in a proportion of cases across the range of the TDP-43 proteinopathy spectrum and they appear to be relatively specific for this group of diseases. The question as to whether these cerebellar-positive cases represent a distinct subgroup remains to be answered. Furthermore, the relationship of the p62 positivity in the cerebellum to the underlying pathological processes awaits to be established.

Abstract 3907: Emergent properties of EWS/FLI regulation via GGAA-microsatellites in Ewing's sarcoma

Abstract ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing's sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing's sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. In addition to EWS/FLI, we found that other Ewing's sarcoma associated EWS/ETS fusions are capable of mediating gene regulation via the NR0B1 microsatellite. Furthermore, regulation via the GGAA microsatellite was specific to Ewing's sarcoma fusion proteins as two other ETS family members, ETS1 and ELF1, could not cause transcriptional activation, highlighting a neomorphic function of the Ewing's sarcoma fusion proteins. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for ETS family member binding to DNA at cancer-relevant genetic loci, and highlight emergent properties of EWS/FLI that are required for the development of Ewing's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3907.

Emergent Properties of EWS/FLI Regulation via GGAA Microsatellites in Ewing's Sarcoma

ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing's sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing's sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. We found that the ability to bind GGAA microsatellites is shared by multiple ETS family members from distinct phylogenetic subfamilies. Importantly, however, only EWS/ETS-containing fusions are capable of mediating transcriptional activation via these elements, highlighting a neomorphic function of the Ewing's sarcoma fusion proteins. Additional analysis revealed that the GGAA microsatellite binds EWS/FLI with an affinity that is 2 to 3 orders of magnitude lower than previously identified high-affinity consensus/redundant binding sites. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for an ETS family member binding to DNA at cancer-relevant genetic loci and highlight emergent properties of EWS/FLI that are required for the development of Ewing's sarcoma.

Abstract A70: Oncogenic potential of multiple splicing isoforms of the prostate-cancer specific TMPRSS2-ERG gene fusion product

Abstract Genetic rearrangements are key events in cancer development, as seen by the recurrent chromosomal translocations characteristic of some hematological and mesenchymal tumors. It has been recently reported, however, that a majority of prostate cancer tumors appear to carry specific translocation, in which a transcription factor of the ETS (E26 transformation-specific) family (namely ERG, ETV1, ETV4, ETV5) is expressed under the control of TMPRSS2, a prostate-specific, androgen-dependent protease. In particular, the fusion of TMPRSS2 exon 1 to ERG exon 4 has been observed in approximately 50% of human prostate cases, suggesting a causative role in PC development, but its specific functions remain unclear. This is further complicated by the presence of ERG splicing variants, and by the use of alternative promoters or polyadenylation sites in both the normal and fusion contexts, which could generate over 50 different isoforms. Interestingly, the presence of specific isoforms seems to correlate with tumor aggressiveness. ERG is already known to be involved in oncogenic fusion, such as EWS-ERG fusion in Ewing sarcoma, which activates targets involved in proliferation, metastasis and angiogenesis, suggesting that the androgen-dependent over-expression of ERG following the TMPRSS2 fusion could be a driving mechanism of prostate carcinogenesis. We have identified multiple novel splicing isoforms of both the wild-type ERG gene and the TMPRSS2:ERG fusion gene in prostate tumors and we are currently investigating their respective biological roles and mechanism of action in order to determine their function and oncogenic potential in vitro and in vivo. Citation Information: Cancer Res 2009;69(23 Suppl):A70.

The genomic breakpoint and chimeric transcripts in the EWSR1–ETV4/E1AF gene fusion in Ewing sarcoma

Chromosome translocation creates a fusion between the EWSR1 gene and an ETS family gene. The fusion between these two genes is a characteristic feature of Ewing sarcoma. We previously identified a fourth translocation, t(17;22)(q12;q12), in genomic DNA isolated from cells of patients affected with Ewing sarcoma. The discovery of this translocation suggested that there might be a novel EWSR1–ETV4 fusion gene. In the present study, we determined the genomic breakpoint and characterized the chimeric transcript of the EWSR1–ETV4 fusion gene in two t(17;22) Ewing sarcomas. Reverse transcriptase-PCR assay showed an in-frame fusion between the 5′-terminal region of EWSR1 and the 3′ end of ETV4 (alias E1AF, PEA3); the chimeric transcript could thus serve as a template for expression of a protein composed of the N-terminal portion of EWSR1 fused to the DNA-binding domain of ETV4. Long PCR and sequence analysis of genomic DNA revealed that either exon 8 or intron 7 of EWSR1 is fused to the same intron of ETV4 in both tumors. Several palindromic oligomer sequences were found close to the breakpoints in both genes. The 159-bp Alu-like sequence was repeated in the breakpoint region of the ETV4 gene. These observations suggest a mechanism of EWSR1–ETV4 gene fusion.