ABSTRACT
Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.
Subject(s)
Gene Expression Profiling , Rhabdomyosarcoma , Single-Cell Analysis , Transcriptome , Humans , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma/metabolism , Single-Cell Analysis/methods , Animals , Gene Expression Profiling/methods , Cell Line, Tumor , Mice , Child , Drug Resistance, Neoplasm/genetics , Cell Differentiation , Muscle Development/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Minimal/measurable residual disease (MRD) diagnostics using real-time quantitative PCR analysis of rearranged immunoglobulin and T-cell receptor gene rearrangements are nowadays implemented in most treatment protocols for patients with acute lymphoblastic leukemia (ALL). Within the EuroMRD Consortium, we aim to provide comparable, high-quality MRD diagnostics, allowing appropriate risk-group classification for patients and inter-protocol comparisons. To this end, we set up a quality assessment scheme, that was gradually optimized and updated over the last 20 years, and that now includes participants from around 70 laboratories worldwide. We here describe the design and analysis of our quality assessment scheme. In addition, we here report revised data interpretation guidelines, based on our newly generated data and extensive discussions between experts. The main novelty is the partial re-definition of the "positive below quantitative range" category by two new categories, "MRD low positive, below quantitative range" and "MRD of uncertain significance". The quality assessment program and revised guidelines will ensure reproducible and accurate MRD data for ALL patients. Within the Consortium, similar programs and guidelines have been introduced for other lymphoid diseases (e.g., B-cell lymphoma), for new technological platforms (e.g., digital droplet PCR or Next-Generation Sequencing), and for other patient-specific MRD PCR-based targets (e.g., fusion genes).
Subject(s)
Neoplasm, Residual , Humans , Neoplasm, Residual/genetics , Neoplasm, Residual/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Gene Rearrangement , Quality Assurance, Health Care , Practice Guidelines as Topic/standards , Genes, Immunoglobulin , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standardsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Axl Receptor Tyrosine Kinase , Benzocycloheptenes , Proto-Oncogene Proteins , Receptor Protein-Tyrosine Kinases , Rhabdomyosarcoma , Humans , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma/genetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Mice , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/genetics , Benzocycloheptenes/pharmacology , Xenograft Model Antitumor Assays , Cell Line, Tumor , Child , Cell Proliferation/drug effects , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Cell Movement/drug effects , Protein Kinase Inhibitors/pharmacology , TriazolesABSTRACT
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.
Subject(s)
Mi-2 Nucleosome Remodeling and Deacetylase Complex , Sarcoma, Ewing , Humans , Cell Line, Tumor , Cell Survival , Chromatin/genetics , DNA , Gene Expression Regulation, Neoplastic , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , RNA-Binding Protein EWS , Sarcoma, Ewing/genetics , Sarcoma, Ewing/pathologyABSTRACT
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.
Subject(s)
Antineoplastic Agents , Neoplasms , Child , Humans , Xenograft Model Antitumor Assays , Neoplasms/drug therapy , Neoplasms/pathology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Biomarkers , Cell Line, TumorABSTRACT
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.
Subject(s)
RNA Polymerase II , Rhabdomyosarcoma, Alveolar , Humans , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Cysteine/metabolism , Transcription Factors/metabolism , PAX3 Transcription Factor/genetics , Rhabdomyosarcoma, Alveolar/genetics , RNA/metabolism , Transcriptional Activation , Protein Binding , Forkhead Box Protein O1/metabolismABSTRACT
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.
Subject(s)
Rhabdomyosarcoma , Humans , Carcinogenesis/genetics , Cell Line, Tumor , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Transcription Factors , Cell Transformation, Neoplastic , Cell DifferentiationABSTRACT
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.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents , Rhabdomyosarcoma, Alveolar , Rhabdomyosarcoma , Child , Humans , Rhabdomyosarcoma, Alveolar/drug therapy , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Muscle, Skeletal/metabolism , Cell Differentiation , Antineoplastic Agents/therapeutic use , Cell Line, TumorABSTRACT
Low hypodiploidy defines a rare subtype of B-cell acute lymphoblastic leukemia (B-ALL) with a dismal outcome. To investigate the genomic basis of low-hypodiploid ALL (LH-ALL) in adults, we analyzed copy-number aberrations, loss of heterozygosity, mutations, and cytogenetics data in a prospective cohort of Philadelphia (Ph)-negative B-ALL patients (n = 591, ages 18-84 years), allowing us to identify 80 LH-ALL cases (14%). Genomic analysis was critical for evidencing low hypodiploidy in many cases missed by cytogenetics. The proportion of LH-ALL within Ph-negative B-ALL dramatically increased with age, from 3% in the youngest patients (under 40 years old) to 32% in the oldest (over 55 years old). Somatic TP53 biallelic inactivation was the hallmark of adult LH-ALL, present in virtually all cases (98%). Strikingly, we detected TP53 mutations in posttreatment remission samples in 34% of patients. Single-cell proteogenomics of diagnosis and remission bone marrow samples evidenced a preleukemic, multilineage, TP53-mutant clone, reminiscent of age-related clonal hematopoiesis. SIGNIFICANCE: We show that low-hypodiploid ALL is a frequent entity within B-ALL in older adults, relying on somatic TP53 biallelic alteration. Our study unveils a link between aging and low-hypodiploid ALL, with TP53-mutant clonal hematopoiesis representing a preleukemic reservoir that can give rise to aneuploidy and B-ALL. See related commentary by Saiki and Ogawa, p. 102. This article is highlighted in the In This Issue feature, p. 101.
Subject(s)
Lymphoma, B-Cell , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Aged , Adult , Adolescent , Young Adult , Middle Aged , Aged, 80 and over , Clonal Hematopoiesis , Prospective Studies , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Mutation , Aneuploidy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted.
Subject(s)
Circulating Tumor DNA , Neoplasms , Rhabdomyosarcoma, Embryonal , Humans , Child , Mice , Animals , Circulating Tumor DNA/genetics , Feasibility Studies , Prospective Studies , Biomarkers, Tumor/genetics , MutationABSTRACT
Rhabdomyosarcomas (RMSs) are the most common soft tissue sarcomas in children/adolescents less than 18 years of age with an annual incidence of 1-2/million. Inter/intra-tumour heterogeneity raise challenges in clinical, pathological and biological research studies. Risk stratification in European and North American clinical trials previously relied on clinico-pathological features, but now, incorporates PAX3/7-FOXO1-fusion gene status in the place of alveolar histology. International working groups propose a coordinated approach through the INternational Soft Tissue SaRcoma ConsorTium to evaluate the specific genetic abnormalities and generate and integrate molecular and clinical data related to patients with RMS across different trial settings. We review relevant data and present a consensus view on what molecular features should be assessed. In particular, we recommend the assessment of the MYOD1-LR122R mutation for risk escalation, as it has been associated with poor outcomes in spindle/sclerosing RMS and rare RMS with classic embryonal histopathology. The prospective analyses of rare fusion genes beyond PAX3/7-FOXO1 will generate new data linked to outcomes and assessment of TP53 mutations and CDK4 amplification may confirm their prognostic value. Pathogenic/likely pathogenic germline variants in TP53 and other cancer predisposition genes should also be assessed. DNA/RNA profiling of tumours at diagnosis/relapse and serial analyses of plasma samples is recommended where possible to validate potential molecular biomarkers, identify new biomarkers and assess how liquid biopsy analyses can have the greatest benefit. Together with the development of new molecularly-derived therapeutic strategies that we review, a synchronised international approach is expected to enhance progress towards improved treatment assignment, management and outcomes for patients with RMS.
Subject(s)
Rhabdomyosarcoma, Embryonal , Rhabdomyosarcoma , Soft Tissue Neoplasms , Adolescent , Child , Consensus , Humans , Molecular Diagnostic Techniques , Neoplasm Recurrence, Local , Prospective Studies , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma/therapy , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/therapy , Risk Assessment , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/pathology , Soft Tissue Neoplasms/therapyABSTRACT
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.
Subject(s)
Apoptosis Regulatory Proteins , DNA-Binding Proteins , Neuroectodermal Tumors, Primitive, Peripheral , RNA-Binding Proteins , Sarcoma, Ewing , Apoptosis Regulatory Proteins/genetics , Cell Line, Tumor , Child , Clustered Regularly Interspaced Short Palindromic Repeats , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Humans , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/genetics , RNA-Binding Protein EWS/metabolism , RNA-Binding Proteins/genetics , Sarcoma, Ewing/pathology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
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.