Background/Objectives: Fusion-positive rhabdomyosarcoma (FP-RMS) is characterized by the presence of tumor-specific chromosomal translocation products, most commonly PAX3::FOXO1, and typically results in lower survival rates compared to fusion-negative RMS cases. PAX3::FOXO1 plays a critical role in FP-RMS oncogenesis in both tumor initiation and maintenance, making it an excellent target for therapeutic intervention in FP-RMS. Methods: We created Proteolysis Targeting Chimeras (PROTACs) by combining PAX3::FOXO1-binding small molecules with E3 ligase recruiters for cereblon (CRBN) or S-Phase Kinase Associated Protein 1 (SKP1). Results: The PROTACs achieved up to 70% degradation of the endogenous PAX3::FOXO1 protein in FP-RMS cell lines in a concentration-, time-, and proteasome-dependent manner. Moreover, the PROTAC-mediated targeted degradation of PAX3::FOXO1 in FP-RMS cells deregulated the endogenous PAX3::FOXO1 gene expression signature and induced myogenic differentiation. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs synergized with vincristine treatment and impaired >80% of anchorage-independent growth in soft agar. Conclusions: Taken together, we demonstrate the proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1 in FP-RMS cells. The PROTACs created in this study will not only be useful tools in studying PAX3::FOXO1 biology in laboratory models but could also serve as molecular scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs in FP-RMS patients.

Ezrin is a cytoplasmic protein that can exist in multiple conformations that are regulated by phosphorylation at Thr567. The phosphorylated, open form of ezrin generally has been considered the active form because it translocates to the plasma membrane. In contrast, the unphosphorylated, closed form of ezrin is sequestered in the cytoplasm and is considered inactive, although it directly interacts with cytoplasmic RNA binding proteins. Here, we found that the closed form of ezrin is itself an RNA binding protein with biological activity. The abundance of ezrin correlated with that of RBPs in human osteosarcoma samples. Purified recombinant ezrin protein engineered to maintain a closed conformation (rEZRIN-T567A) directly bound RNA, with greatest affinity for guanine-rich sequences and RNA G-quadruplexes (G4 RNAs). Expressing closed ezrin in ezrin-null osteosarcoma cells restored the transcriptomic and proteomic profiles. Closed ezrin bound to endogenous mRNAs associated with pathways related to RNA processing and splicing, DNA maintenance, and cellular metabolism. In zebrafish, expression of closed ezrin rescued the metastatic capability of ezrin-null osteosarcoma xenografts. Our findings demonstrate that the closed conformation of ezrin-previously thought to be inactive-can directly bind RNA, regulate transcription and translation, and contribute to a metastatic phenotype in osteosarcoma cells.

Diffuse large B-cell lymphoma (DLBCL) remains a challenging disease with limited therapeutic options beyond standard immunochemotherapy. ETS transcription factors, including SPIB and SPI1, are implicated in lymphoma pathogenesis and can be targeted by the small molecule TK216, which disrupts ETS-DHX9 interactions. To explore mechanisms of resistance, we generated stable TK216-resistant clones from the ABC-DLBCL line U2932. Resistant clones exhibited a 4-5-fold increase in IC50 values and lost the ability to undergo G2-M arrest upon treatment. Transcriptomic and mutational analyses revealed three resistance patterns: (i) MDR1/ABCB1 overexpression, leading to multidrug efflux; (ii) Cluster A, enriched for proliferation, Wnt, and transcriptional programs, with mutations in ESR2, USP24, and SFSWAP; and (iii) Cluster B, characterized by actin/microtubule remodeling, altered metabolism, and mutations in SRSF11 and PATJ. Pharmacologic screening revealed an increased sensitivity of resistant cells to BCL2, MCL1, and XPO1 inhibitors, while also showing reduced sensitivity to aurora kinase and microtubule-targeting agents. Venetoclax and selinexor retained activity in resistant models, supporting their potential for rational combinations with TK216. These findings demonstrate that multiple, heterogeneous mechanisms drive resistance to ETS inhibition in DLBCL, highlighting therapeutic strategies to overcome it.

Ewing sarcoma is a pediatric bone and soft tissue cancer thought to arise from mesenchymal stem cells (MSCs). It is characterized by fusion of the RNA-binding protein EWS to an ETS family transcription factor, most often FLI1. EWS::FLI1 induces changes in gene expression that have been well-studied and have been linked to its ability to bind to and alter chromatin state at GGAA repeat sequences within the genome. In addition to these effects, EWS::FLI1 alters mRNA splicing; however, the mechanism of this activity remains unclear. We curated a sarcoma-focused transcriptome from published long-read RNA sequencing of Ewing sarcoma, osteosarcoma, and synovial sarcoma patient tumors and matched adjacent normal tissue (215M reads). We supplemented this with published long-read RNA sequencing data from relevant normal cell types, osteosarcoma cell lines, and Ewing sarcoma lines with or without EWS::FLI1 knockdown (105M reads). We used this transcriptome as a reference to evaluate expression and splicing from short-read RNA-seq of Ewing sarcoma cell lines, MSCs, and MSC-based models of Ewing sarcoma and established Ewing-associated expression and splicing signatures. We generated scores based on these signatures in published knockdown-rescue experiments in which endogenous EWS::FLI1 was silenced by shRNA and replaced with exogenous shRNA-resistant mutants. Expression and splicing scores were highly correlated within these datasets (r2 = 0.89), suggesting that similar regions of the fusion protein might be required for both its functions. In contrast, the scores had limited correlation within an internal dataset of RNA-binding protein perturbations (r2 = 0.02). We next examined the effects of pharmacological agents thought to alter EWS::FLI1 activity. Interestingly, we found that lurbinectedin which disrupts EWS::FLI1’s DNA-binding could partially revert both expression and splicing scores. In contrast, YK-4-279 which disrupts the fusion’s protein-protein interactions and MS0621 which reverts a Ewing-associated chromatin signature had limited effects on these scores. To further interrogate the potential role of EWS::FLI1 DNA-binding in splicing regulation, we silenced EWS::FLI1 in TC-32 cells and replaced it with either the wildtype fusion or a DNA-binding null mutant. Unlike the wildtype construct, the DNA-binding null mutant could not rescue expression or splicing. Finally, we examined the effects of the previously published KRAB-ZFA7 repressor which silences chromatin at GGAA repeats while sparing single GGAA sites and found that it could revert both scores. Together, these data establish a potential connection between EWS::FLI1 activity at GGAA repeats and its effects on mRNA splicing. Additionally, we find that lurbinectedin, an FDA-approved agent currently in a phase 1/2 trial for Ewing sarcoma, suppresses both the splicing and expression effects of EWS::FLI1. Emerging compounds designed to target EWS::FLI1 DNA-binding directly will likely have similar effects. David V. Allegakoen, Jeffrey A. Toretsky. EWS::FLI1 activity at GGAA repeats is essential for maintenance of Ewing-like splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B013.

BACKGROUND:Ewing sarcoma (ES) is a rare tumor that affects children, adolescents, and young adults. ES is associated with high morbidity in all patients and high mortality for those who present with metastatic disease. A chromosomal translocation, either t(11;22)(q24;p12) or t(21;22)(q22;q12) leads to the fusion oncoproteins EWS::FLI1 or EWS::ERG in 95% of ES patients. We recognized a critical need for a stably sourced high-affinity antibody that recognizes EWS::FLI1 with maximal specificity. Understanding EWS::FLI1 protein complexes is a pivotal gap in ES knowledge that necessitates the development of antibodies capable of identifying native proteins in solution. Further, variable epitope sequencing of a monoclonal antibody enables the construction of degraders and nanobody identifiers. METHODS:Monoclonal antibodies were produced following informed peptide synthesis, injection, and hybridoma creation. Hybridoma antibodies were validated for specificity and function. RESULTS:Our results indicate that the FLI1 1.2 monoclonal antibody, which recognizes the EWS::FLI1 fusion oncoprotein, can be reliably applied to multiple molecular biology applications like immunoblot, immunoprecipitation, immunofluorescence, and immunohistochemistry. This FLI1 1.2 monoclonal antibody has a high affinity of 0.3 nM KD to EWS::FLI1. In terms of specificity, this antibody is highly specific to EWS::FLI1 and some cross reactivity with ERG. CONCLUSIONS:This reagent will provide the research community with valuable tools for further biochemical and genomic interrogation of the oncogenic activity of EWS::FLI1 in ES.

Despite many advances in cancer treatment, metastasis remains the major cause of mortality: over 90% of cancer-related deaths are due to metastasis. We chose Ewing sarcoma (ES) as a model to study cancer metastasis since less than 25% of ES patients with metastatic disease live beyond five years and there are no effective therapies available to specifically target metastatic disease. Through an in vivo genome-wide CRISPR/Cas9 transcriptional activation screen using human ES cells as xenografts, we identified the human INAFM2 gene as a strong driver of metastasis and therefore named the vertebrate gene and its protein product, ROME (Regulator of Metastasis). Up until now, INAFM2 has not been studied in-depth and INAFM2 protein expression profile or function is not known. We characterized aspects of ROME expression and function in vertebrate development, physiology, and pathology. Blocking ROME expression in zebrafish embryos resulted in severe developmental defects and early mortality. Single cell RNA sequencing of zebrafish embryos with rome knockdown and RNA sequencing of cancer cell lines with ROME modulation revealed that ROME negatively regulates calcium and canonical Wnt pathways. We also discovered that ROME directly interacts with CAV1, FLOT1, and Vimentin proteins. ROME overexpression increased metastasis of ES xenografts in zebrafish and immunodeficient mice while ROME knockout in ES xenografts reduced metastasis. Human tumor RNA sequencing data revealed a negative correlation between ROME expression and patient survival. In summary, we discovered a previously unstudied human glycoprotein located on the plasma membrane that can regulate cell motility and invasion, is essential for normal development in vertebrates, and increases the metastatic phenotype of human tumor cells. Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Üren. ROME, a novel membrane protein in vertebrates, is a key modulator of embryonal development and cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6439.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, and is classified as fusion-positive (FP) or fusion-negative (FN) based on the presence of the PAX3::FOXO1 or PAX7::FOXO1 fusions. These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. FP status is associated with worse event free survival for patients with RMS. Regardless of fusion status, most RMS patients receive a chemotherapy regimen consisting of some combination of vincristine, actinomycin D, and cyclophosphamide (VAC). Unfortunately, tumors may acquire resistance to these drugs over time, leading to relapse. Our work aimed to uncover genes that drive VAC resistance in FP-RMS using a two-pronged approach. To assess how FP-RMS cells gradually acquire resistance, we cultured FP-RMS cell lines (RH30, RH41, CW9019, U48484, U66788) in sublethal concentrations of vincristine, actinomycin D, or 4-hydroperoxy cyclophosphamide (4-HC, the active metabolite of cyclophosphamide) for multiple passages. We periodically assessed the IC50 of cells growing in drugs and increased drug doses as cells developed resistance. We generated four cell lines with a ∼5- to ∼20-fold increase in IC50. Using RNAseq, we identified differentially expressed genes between the parental and resistant cell lines that may act as drivers of chemotherapeutic resistance. To assess de novo resistance in FP-RMS, we used CRISPR inactivation and activation library screening. We generated FP-RMS cell lines (RH30, RH41, CW9019) stably expressing Cas9 or dCas9-VPR, an endonuclease-dead version of Cas9 fused to three transcriptional activators. We then transduced these cells with a CRISPR inactivation sgRNA library (18, 885 gene targets) or a CRISPR activation sgRNA library (19, 113 gene targets). Transduced cells were cultured in vincristine, actinomycin D, or 4-HC, and surviving cells were expanded and sequenced to determine which genes provided drug resistance when activated or deleted. We hope that the findings of these experiments will contribute to novel therapeutic targets for patients with VAC-resistant RMS tumors. Taryn E. Shaw, Foram Shah, Jeffrey Toretsky, Aykut Üren. Determinants of chemotherapeutic resistance in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6448.

Abstract In order to discover genes that can accelerate metastasis of Ewing sarcoma (ES), we performed a genome-wide CRISPR/Cas9 transcriptional activation screen in zebrafish using human ES cells as xenografts. We identified the human INAFM2 gene as a strong driver of metastatic activity of ES cells. INAFM2 is the human homolog of the Drosophila InaF gene and there were no publications about the protein product of this gene in any vertebrate animals. We named the new protein ROME (Regulator of metastasis) and characterized its expression and its role in ES metastasis using multiple in vitro and in vivo models. We established that the human ROME protein is indeed expressed at the plasma membrane. We demonstrated that the ROME protein is both phosphorylated and glycosylated. The functional significance of these posttranslational modifications remains to be determined. Lack of ROME expression in zebrafish embryos resulted in severe developmental defects and early mortality. We performed single cell RNA sequencing of zebrafish embryos that were injected with ROME targeting morpholino. RNA sequencing of ES cell lines that were either overexpressing ROME or lack ROME expression (by CRISPR). We determined that ROME expression regulates Ca++, Wnt and MAPK signaling pathways. Overexpression of ROME in four ES cell lines caused increased cell migration and chemotaxis in vitro, and increased intravasation in zebrafish xenografts. ROME knockout in two ES cell lines decreased chemotaxis. Rescue of ROME expression restored the cells’ ability to migrate in CRISPR knockout clones. In vivo studies by tail vein injection of ES cells in immunodeficient mice demonstrated significantly increased gross metastases with ROME overexpression and decreased gross metastases with ROME knockout compared to the respective control groups. Additional ES xenograft studies where the primary tumor was removed by amputating the affected leg, showed that overexpression of ROME results in enhanced lung metastasis. Analysis of patient tumor RNA sequencing data from many different types of human cancers revealed correlations between ROME expression and both an increased metastatic phenotype and poor patient survival. This provides strong evidence for a substantial role of ROME in ES and cancer metastasis in general. In summary, we present the first ever characterization of the ROME protein in zebrafish embryos and ES cancer cell lines. Our data suggest that ROME is a membrane protein that can regulate cell motility and invasion through different signaling pathways, which results in increased metastatic potential. Citation Format: Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Uren. ROME, a novel membrane protein in vertebrates, enhances metastatic phenotype of Ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B076.

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is commonly divided into subtypes based on histopathological appearance. The oncogenic fusion proteins PAX3::FOXO1 or PAX7::FOXO1 are present in the alveolar subtype of RMS (ARMS). These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. These fusion proteins have been shown to drive malignant transformation in ARMS, and patients with fusion-positive ARMS have a worse prognosis than patients with fusion-negative ARMS and embryonal RMS. We recently reported that piperacetazine, a phenothiazine derivative and first-generation antipsychotic, is capable of binding to and inhibiting the transcriptional activity of PAX3::FOXO1, reducing the expression of PAX3::FOXO1 target genes, and inhibiting the ability of fusion-positive RMS cells to grow in soft agar. We hypothesize that other phenothiazine derivatives may have stronger binding affinity for PAX3::FOXO1 and greater ability to reduce tumor growth in vivo. We employed a structure-activity relationship campaign to develop new phenothiazine derivatives that may be more potent inhibitors of PAX3::FOXO1 than piperacetazine. We synthesized and tested 10 new compounds, in addition to 9 existing FDA-approved phenothiazine derivatives, for their ability to bind to PAX3::FOXO1 and inhibit its transcriptional activity and target gene expression as well as inhibiting the growth of fusion-positive RMS cells in 3D culture. Seven derivatives inhibited PAX3::FOXO1 reporter activity better than piperacetazine, nine of them performed worse, and three of them showed comparable activity to piperacetazine. We demonstrated that piperacetazine does not alter PAX3::FOXO1 levels, subcellular localization, binding to a PAX3::FOXO1 target DNA sequence, or phosphorylation at Ser256 in the FOXO1 portion of the fusion protein. We therefore hypothesize that piperacetazine may be disrupting interactions between PAX3::FOXO1 and its partner proteins. We are employing a proximity-based biotinylation method to identify proteins that are competed away from PAX3::FOXO1 by piperacetazine. Our data suggest that it is possible to target PAX3::FOXO1 protein using small molecules that can directly bind to it and inhibit its activity in fusion-positive RMS cells. Citation Format: Taryn Shaw, Kay Nakazawa, Purushottam Tiwari, Eryn Nelson, Jeffrey Formen, Christian Wolf, Jeffrey Toretsky, Aykut Üren. Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1093.

PURPOSE Ewing Sarcoma (ES), a rare cancer with a pathognomonic translocation resulting in the Ewing sarcoma gene (EWS)::FLI1 oncoprotein, has a poor prognosis in the relapsed/refractory (R/R) setting. Tokalas (TK)216 was designed to bind EWS::FLI1 proteins directly, disrupt protein-protein interactions, and inhibit transcription factor function. TK216 plus vincristine showed synergistic activity in preclinical tumor models. To our knowledge, we report the results of a first-in-class, first-in-human phase I/II trial of TK216 in R/R ES. PATIENTS AND METHODS TK216 was administered intravenously as a continuous infusion to patients with R/R ES in 11 cohorts. The dosing duration of 7 days was later extended to 10, 14, and 28 days. Vincristine could be added on day 1 after cycle 2, per investigators’ choice. The trial used a 3 + 3 design with an expansion cohort at the recommended phase II dose (RP2D). RESULTS A total of 85 patients with a median age of 27 years (range, 11-77) were enrolled. The maximum tolerated dose for the 14-day infusion of TK216, 200 mg/m 2 once daily, was determined in cohort 9 and selected as the RP2D. The median previous number of systemic therapies regimens was three (range, 1-10). The most frequent-related adverse events in patients treated at the RP2D included neutropenia (44.7%), anemia (29.4%), leukopenia (29.4%), febrile neutropenia (15.3%), thrombocytopenia (11.8%), and infections (17.6%). In cohorts 9 and 10, two patients had a complete response, one had a partial response, and 14 had stable disease; the 6-month progression-free survival was 11.9%. There were no responses among the eight patients in cohort 11. CONCLUSION TK216 administered as 14-day continuous infusion with or without vincristine was well tolerated and showed limited activity at the RP2D in R/R ES.

Abstract Metastasis is the main cause of mortality in cancer patients and accounts for about 90% of cancer deaths, which has not changed in the past 50 years. This indicates a significant need for further study of metastasis pathways to find novel druggable targets. Ewing sarcoma (ES) is an aggressive childhood cancer that occurs in the bones or soft tissue. The five-year survival rate for Ewing sarcoma patients diagnosed with metastatic disease is still only 25%, making ES a good model for studying metastasis. We performed a genome-wide CRISPR/Cas9 transcriptional activation screen in a zebrafish xenograft model to discover genes whose activation leads to increased metastasis. The screen identified a number of genes that may accelerate ES metastasis; we chose INAFM2 for further study because multiple unique INAFM2-targeting gRNAs were enriched in the screen and we discovered a potential link to metastasis and patient survival in publicly available clinical data. Overexpression of INAFM2 in six different cancer cell lines (four ES, one hepatocellular carcinoma, and one stomach adenocarcinoma) caused increased cell migration and chemotaxis in vitro, and increased intravasation in zebrafish xenografts. INAFM2 knockdown in four cancer cell lines (two ES, one clear cell renal cell carcinoma, and one stomach adenocarcinoma) decreased chemotaxis and rescue of INAFM2 expression restored the cells’ ability to migrate. Analysis of patient tumor RNA sequencing data from many different types of cancers revealed correlations between INAFM2 expression and both an increased metastatic phenotype and poor patient survival. Further in vivo studies by tail vein injection of ES cells in immunodeficient mice demonstrated significantly increased gross metastases with INAFM2 overexpression and decreased gross metastases with INAFM2 knockdown compared to the respective control groups. This provides strong evidence for a substantial role of INAFM2 in cancer metastasis. Human INAFM2 is an unstudied gene and our preliminary work has revealed that the INAFM2 protein localizes to the cell membrane, is glycosylated, and may be involved in Wnt and/or MAPK pathway signaling. Inafm2 silencing by morpholino in zebrafish embryos lead to significant mortality, necrosis, and developmental delays/defects including severe cardiac edema. Current studies are focused on further discerning the molecular pathways that mediate the role of INAFM2 in promoting metastasis using in vitro and in vivo models as well as characterizing the functional significance of the direct protein binding partners and post-translational modifications for INAFM2. Citation Format: Anna Molotkova, Emre Deniz, Matthew Swift, Eric Glasgow, Jeffrey Toretsky, Aykut Üren. In vivo CRISPR/Cas9 transcriptional activation screen leads to discovery of INAFM2, a novel driver of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1560.

Reduction of PAX3::FOXO1 protein expression does not alter proliferation rates of FP-RMS cell lines but reduces anchorage-independent growth.

Piperacetazine inhibits endogenous PAX3::FOXO1 activity in multiple cell lines. A, The effect of piperacetazine treatment on cell viability was measured using the CellTiter-Blue assay (black lines). The effect of piperacetazine treatment on PAX3::FOXO1 activity was measured via luciferase assay (blue lines), using the PAX3::FOXO1-responsive pGL3-PDGFRA luciferase reporter (RH30, RH41, RH28, U66788, RD), and PGK reporter (SN12C, negative control). B, Left: Luciferase activity was measured in HEK293T cells transfected with PAX3::FOXO1-responsive ASS1P reporter and an empty or PAX3::FOXO1 expression vector. Cells were treated with DMSO or piperacetazine for 48 hours. Matching Western blots are shown under each bar. Right: The same experimental setup was repeated with EWS::FLI1-responsive NR0B1 luciferase reporter and an EWS::FLI1 expression vector or empty vector. The cells were treated with the same concentration of piperacetazine or DMSO for 48 hours, and the normalized luciferase readouts and accompanying Western blots are shown. (*, P < 0.0001; Student t test; ns, P > 0.05)

PDF - 65KB, Growth of SK-N-MC flank tumors in athymic nude mice treated with 12-15 Gy.

Piperacetazine alters the expression of PAX3::FOXO1 target genes. A, Protein expression of PAX3::FOXO1 target genes was evaluated by Western blot analysis in RH30 cells treated with 10 µmol/L piperacetazine for 6 days. B, RNA-seq was performed in fusion-positive RH30 cells treated with 30 µmol/L piperacetazine for 24 hours. The ranked gene expression list was compared with existing lists of genes using GSEA. Gene set descriptions: Top left: genes upregulated during human skeletal muscle myoblast differentiation. Top right: Hallmark genes during myogenesis. Bottom left: Genes downregulated in SAOS-2 (osteosarcoma) cells upon expression of PAX3::FOXO1. Bottom right: Genes downregulated in fusion-positive versus fusion-negative RMS cell lines. NES = normalized enrichment score, FDR = false discovery rate). C, Piperacetazine does not cause PAX3::FOXO1 to shift its intracellular localization. RH30 cells were treated with 10 µmol/L piperacetazine for 24 hours prior to cellular fractionation, which were analyzed via Western blot analysis. Lamin A/C and alpha-tubulin were used as positive controls for nuclear and cytoplasmic fractions, respectively. D, Piperacetazine does not alter PAX3::FOXO1 protein levels or phosphorylation of Ser256, as evaluated by Western blot analysis. RH30 cells were treated with 10 µmol/L piperacetazine or vehicle for 3 days. E, PAX3::FOXO1 protein was immobilized on a CM5 chip, and double-stranded PAX3::FOXO1 oligonucleotide (100 nmol/L), piperacetazine (10 µmol/L), or a combination of the two were injected over the chip surface. Piperacetazine did not inhibit DNA binding to PAX3::FOXO1.

Piperacetazine inhibits anchorage-independent growth of FP-RMS cells. A, Average 48 hour IC50 values for indicated cell lines. Sample size indicated above each bar. Red bars: FP-RMS, blue bars: FN-RMS, black bars: non-RMS. U48484, U66788, U37125, and U57810 are cell lines from transgenic RMS mouse models. B, RH30 and RD colonies in soft agar with DMSO or 15 µmol/L piperacetazine treatment. The top row images are whole well images taken with Gelcount and the bottom row images are microscope images of the colonies at 40X magnification. C, Gelcount quantification of colonies in B (*, P < 0.0001; Student t test; ns, P > 0.05).

Validation of PKC412 and BMS-754807 synergy in a panel of Ewing sarcoma cell lines. CI values are shown for each concentration point where the drugs were combined.

Supplementary Tables 1, 2, and 3

PDF - 46KB, Growth of HT1080 flank tumors in athymic nude mice treated with 8 Gy of radiation and olaparib