Functional Classification of Fusion Proteins in Sarcoma.

Sarcomas comprise a heterogeneous group of malignant tumors of mesenchymal origin. More than 80 entities are associated with different mesenchymal lineages. Sarcomas with fibroblastic, muscle, bone, vascular, adipocytic, and other characteristics are distinguished. Nearly half of all entities contain specific chromosomal translocations that give rise to fusion proteins. These are mostly pathognomonic, and their detection by various molecular techniques supports histopathologic classification. Moreover, the fusion proteins act as oncogenic drivers, and their blockade represents a promising therapeutic approach. This review summarizes the current knowledge on fusion proteins in sarcoma. We categorize the different fusion proteins into functional classes, including kinases, epigenetic regulators, and transcription factors, and describe their mechanisms of action. Interestingly, while fusion proteins acting as transcription factors are found in all mesenchymal lineages, the others have a more restricted pattern. Most kinase-driven sarcomas belong to the fibroblastic/myofibroblastic lineage. Fusion proteins with an epigenetic function are mainly associated with sarcomas of unclear differentiation, suggesting that epigenetic dysregulation leads to a major change in cell identity. Comparison of mechanisms of action reveals recurrent functional modes, including antagonism of Polycomb activity by fusion proteins with epigenetic activity and recruitment of histone acetyltransferases by fusion transcription factors of the myogenic lineage. Finally, based on their biology, we describe potential approaches to block the activity of fusion proteins for therapeutic intervention. Overall, our work highlights differences as well as similarities in the biology of fusion proteins from different sarcomas and provides the basis for a functional classification.

Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer.

Rhabdomyosarcoma (RMS) is a highly aggressive pediatric cancer with features of skeletal muscle differentiation. More than 80% of the high-risk patients ultimately fail to respond to chemotherapy treatment, leading to limited therapeutic options and dismal prognostic rates. The lack of response and subsequent tumor recurrence is driven in part by stem cell-like cells, the tumor subpopulation that is enriched after treatment, and characterized by expression of the AXL receptor tyrosine kinase (AXL). AXL mediates survival, migration, and therapy resistance in several cancer types; however, its function in RMS remains unclear. In this study, we investigated the role of AXL in RMS tumorigenesis, migration, and chemotherapy response, and whether targeting of AXL with small-molecule inhibitors could potentiate the efficacy of chemotherapy. We show that AXL is expressed in a heterogeneous manner in patient-derived xenografts (PDX), primary cultures and cell line models of RMS, consistent with its stem cell-state selectivity. By generating a CRISPR/Cas9 AXL knock-out and overexpressing models, we show that AXL contributes to the migratory phenotype of RMS, but not to chemotherapy resistance. Instead, pharmacologic blockade with the AXL inhibitors bemcentinib (BGB324), cabozantinib and NPS-1034 rapidly killed RMS cells in an AXL-independent manner and augmented the efficacy of the chemotherapeutics vincristine and cyclophosphamide. In vivo administration of the combination of bemcentinib and vincristine exerted strong antitumoral activity in a rapidly progressing PDX mouse model, significantly reducing tumor burden compared with single-agent treatment. Collectively, our data identify bemcentinib as a promising drug to improve chemotherapy efficacy in patients with RMS.

The Chromatin Remodeler CHD4 Sustains Ewing Sarcoma Cell Survival by Controlling Global Chromatin Architecture.

Ewing sarcoma is an aggressive cancer with a defective response to DNA damage leading to an enhanced sensitivity to genotoxic agents. Mechanistically, Ewing sarcoma is driven by the fusion transcription factor EWS-FLI1, which reprograms the tumor cell epigenome. The nucleosome remodeling and deacetylase (NuRD) complex is an important regulator of chromatin function, controlling both gene expression and DNA damage repair, and has been associated with EWS-FLI1 activity. Here, a NuRD-focused CRISPR/Cas9 inactivation screen identified the helicase CHD4 as essential for Ewing sarcoma cell proliferation. CHD4 silencing induced tumor cell death by apoptosis and abolished colony formation. Although CHD4 and NuRD colocalized with EWS-FLI1 at enhancers and super-enhancers, CHD4 promoted Ewing sarcoma cell survival not by modulating EWS-FLI1 activity and its oncogenic gene expression program but by regulating chromatin structure. CHD4 depletion led to a global increase in DNA accessibility and induction of spontaneous DNA damage, resulting in an increased susceptibility to DNA-damaging agents. CHD4 loss delayed tumor growth in vivo, increased overall survival, and combination with PARP inhibition by olaparib treatment further suppressed tumor growth. Collectively, these findings highlight the NuRD subunit CHD4 as a therapeutic target in Ewing sarcoma that can potentiate the antitumor activity of genotoxic agents. SIGNIFICANCE: CRISPR/Cas9 screening in Ewing sarcoma identifies a dependency on CHD4, which is crucial for the maintenance of chromatin architecture to suppress DNA damage and a promising therapeutic target for DNA damage repair-deficient malignancies.

Elimusertib has Antitumor Activity in Preclinical Patient-Derived Pediatric Solid Tumor Models.

The small-molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical antitumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger antitumor effects than some standard-of-care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical antitumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients.

PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution.

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting.

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.

Genomic and Epigenetic Changes Drive Aberrant Skeletal Muscle Differentiation in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

CRISPR activation screen identifies TGFß-associated PEG10 as a crucial tumor suppressor in Ewing sarcoma.

As the second most common pediatric bone and soft tissue tumor, Ewing sarcoma (ES) is an aggressive disease with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an "undruggable" fusion protein acting as transcriptional modulator. EWS-FLI1 rewires the protein expression in cancer cells by activating and repressing a multitude of genes. The role and contribution of most repressed genes remains unknown to date. To address this, we established a CRISPR activation system in clonal SKNMC cell lines and interrogated a custom focused library covering 871 genes repressed by EWS-FLI1. Among the hits several members of the TGFß pathway were identified, where PEG10 emerged as prime candidate due to its strong antiproliferative effect. Mechanistic investigations revealed that PEG10 overexpression caused cellular dropout via induction of cell death. Furthermore, non-canonical TGFß pathways such as RAF/MEK/ERK, MKK/JNK, MKK/P38, known to lead to apoptosis or autophagy, were highly activated upon PEG10 overexpression. Our study sheds new light onto the contribution of TGFß signalling pathway repression to ES tumorigenesis and suggest that its re-activation might constitute a novel therapeutic strategy.

Inhibition of HDACs reduces Ewing sarcoma tumor growth through EWS-FLI1 protein destabilization.

Oncogenic transcription factors lacking enzymatic activity or targetable binding pockets are typically considered "undruggable". An example is provided by the EWS-FLI1 oncoprotein, whose continuous expression and activity as transcription factor are critically required for Ewing sarcoma tumor formation, maintenance, and proliferation. Because neither upstream nor downstream targets have so far disabled its oncogenic potential, we performed a high-throughput drug screen (HTS), enriched for FDA-approved drugs, coupled to a Global Protein Stability (GPS) approach to identify novel compounds capable to destabilize EWS-FLI1 protein by enhancing its degradation through the ubiquitin-proteasome system. The protein stability screen revealed the dual histone deacetylase (HDAC) and phosphatidylinositol-3-kinase (PI3K) inhibitor called fimepinostat (CUDC-907) as top candidate to modulate EWS-FLI1 stability. Fimepinostat strongly reduced EWS-FLI1 protein abundance, reduced viability of several Ewing sarcoma cell lines and PDX-derived primary cells and delayed tumor growth in a xenograft mouse model, whereas it did not significantly affect healthy cells. Mechanistically, we demonstrated that EWS-FLI1 protein levels were mainly regulated by fimepinostat's HDAC activity. Our study demonstrates that HTS combined to GPS is a reliable approach to identify drug candidates able to modulate stability of EWS-FLI1 and lays new ground for the development of novel therapeutic strategies aimed to reduce Ewing sarcoma tumor progression.

BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.

Fenretinide Acts as Potent Radiosensitizer for Treatment of Rhabdomyosarcoma Cells.

Fusion-positive rhabdomyosarcoma (FP-RMS) is a highly aggressive childhood malignancy which is mainly treated by conventional chemotherapy, surgery and radiation therapy. Since radiotherapy is associated with a high burden of late side effects in pediatric patients, addition of radiosensitizers would be beneficial. Here, we thought to assess the role of fenretinide, a potential agent for FP-RMS treatment, as radiosensitizer. Survival of human FP-RMS cells was assessed after combination therapy with fenretinide and ionizing radiation (IR) by cell viability and clonogenicity assays. Indeed, this was found to significantly reduce cell viability compared to single treatments. Mechanistically, this was accompanied by enhanced production of reactive oxygen species, initiation of cell cycle arrest and induction of apoptosis. Interestingly, the combination treatment also triggered a new form of dynamin-dependent macropinocytosis, which was previously described in fenretinide-only treated cells. Our data suggest that fenretinide acts in combination with IR to induce cell death in FP-RMS cells and therefore might represent a novel radiosensitizer for the treatment of this disease.

A combinatorial drug screen in PDX-derived primary rhabdomyosarcoma cells identifies the NOXA - BCL-XL/MCL-1 balance as target for re-sensitization to first-line therapy in recurrent tumors.

First-line therapy for most pediatric sarcoma is based on chemotherapy in combination with radiotherapy and surgery. A significant number of patients experience drug resistance and development of relapsed tumors. Drugs that have the potential to re-sensitize relapsed tumor cells toward chemotherapy treatment are therefore of great clinical interest. Here, we used a drug profiling platform with PDX-derived primary rhabdomyosarcoma cells to screen a large drug library for compounds re-sensitizing relapse tumor cells toward standard chemotherapeutics used in rhabdomyosarcoma therapy. We identified ABT-263 (navitoclax) as most potent compound enhancing general chemosensitivity and used different pharmacologic and genetic approaches in vitro and in vivo to detect the NOXA-BCL-XL/MCL-1 balance to be involved in modulating drug response. Our data therefore suggests that players of the intrinsic mitochondrial apoptotic cascade are major targets for stimulation of response toward first-line therapies in rhabdomyosarcoma.

Negative correlation of single-cell PAX3:FOXO1 expression with tumorigenicity in rhabdomyosarcoma.

Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage Myf6Cre,Pax3:Foxo1,p53 mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor Tp53 (Myf6Cre,Pax3:Foxo1,p53), exhibit marked heterogeneity in PAX3:FOXO1 (P3F) expression at the single cell level. In mouse RMS cells, P3F expression is directed by the Pax3 promoter and coupled to eYFP YFPlow/P3Flow mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFPlow/P3Flow compared with YFPhigh/P3Fhigh cells. Both YFPlow/P3Flow and YFPhigh/P3Fhigh cells gave rise to mixed clones in vitro, consistent with fluctuations in P3F expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFPlow/P3Flow RMS cells. Heterogeneous expression of PAX3:FOXO1 at the single cell level may provide a critical advantage during tumor progression.

Clinicopathologic and molecular analysis of embryonal rhabdomyosarcoma of the genitourinary tract: evidence for a distinct DICER1-associated subgroup.

Embryonal rhabdomyosarcoma (ERMS) of the uterus has recently been shown to frequently harbor DICER1 mutations. Interestingly, only rare cases of extrauterine DICER1-associated ERMS, mostly located in the genitourinary tract, have been reported to date. Our goal was to study clinicopathologic and molecular profiles of DICER1-mutant (DICER1-mut) and DICER1-wild type (DICER1-wt) ERMS in a cohort of genitourinary tumors. We collected a cohort of 17 ERMS including nine uterine (four uterine corpus and five cervix), one vaginal, and seven urinary tract tumors. DNA sequencing revealed mutations of DICER1 in 9/9 uterine ERMS. All other ERMS of our cohort were DICER1-wt. The median age at diagnosis of patients with DICER1-mut and DICER1-wt ERMS was 36 years and 5 years, respectively. Limited follow-up data (available for 15/17 patients) suggested that DICER1-mut ERMS might show a less aggressive clinical course than DICER1-wt ERMS. Histological features only observed in DICER1-mut ERMS were cartilaginous nodules (6/9 DICER1-mut ERMS), in one case accompanied by foci of ossification. Recurrent mutations identified in both DICER1-mut and DICER1-wt ERMS affected KRAS, NRAS, and TP53. Copy number analysis revealed similar structural variations with frequent gains on chromosomes 2, 3, and 8, independent of DICER1 mutation status. Unsupervised hierarchical clustering of array-based whole-genome DNA methylation data of our study cohort together with an extended methylation data set including different RMS subtypes from genitourinary and extra-genitourinary locations (n = 102), revealed a distinct cluster for DICER1-mut ERMS. Such tumors clearly segregated from the clusters of DICER1-wt ERMS, alveolar RMS, and MYOD1-mutant spindle cell and sclerosing RMS. Only one tumor, previously diagnosed as ERMS arising in the maxilla of a 6-year-old boy clustered with DICER1-mut ERMS of the uterus. Subsequent sequencing analysis identified two DICER1 mutations in the latter case. Our results suggest that DICER1-mut ERMS might qualify as a distinct subtype in future classifications of RMS.

Paracrine Placental Growth Factor Signaling in Response to Ionizing Radiation Is p53-Dependent and Contributes to Radioresistance.

Placental growth factor (PlGF) is a pro-angiogenic, N-glycosylated growth factor, which is secreted under pathologic situations. Here, we investigated the regulation of PlGF in response to ionizing radiation (IR) and its role for tumor angiogenesis and radiosensitivity. Secretion and expression of PlGF was induced in multiple tumor cell lines (medulloblastoma, colon and lung adenocarcinoma) in response to irradiation in a dose- and time-dependent manner. Early upregulation of PlGF expression and secretion in response to irradiation was primarily observed in p53 wild-type tumor cells, whereas tumor cells with mutated p53 only showed a minimal or delayed response. Mechanistic investigations with genetic and pharmacologic targeting of p53 corroborated regulation of PlGF by the tumor suppressor p53 in response to irradiation under normoxic and hypoxic conditions, but with so far unresolved mechanisms relevant for its minimal and delayed expression in tumor cells with a p53-mutated genetic background. Probing a paracrine role of IR-induced PlGF secretion in vitro, migration of endothelial cells was specifically increased towards irradiated PlGF wild type but not towards irradiated PlGF-knockout (PIGF-ko) medulloblastoma cells. Tumors derived from these PlGF-ko cells displayed a reduced growth rate, but similar tumor vasculature formation as in their wild-type counterparts. Interestingly though, high-dose irradiation strongly reduced microvessel density with a concomitant high rate of complete tumor regression only in the PlGF-ko tumors. IMPLICATIONS: Our study shows a strong paracrine vasculature-protective role of PlGF as part of a p53-regulated IR-induced resistance mechanism and suggest PlGF as a promising target for a combined treatment modality with RT.