Oncogenic Fusion Research Articles

Deciphering the role of FUS::DDIT3 expression and tumor microenvironment in myxoid liposarcoma development

BackgroundMyxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development.MethodsTo study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing.ResultsCharacterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS.ConclusionsOur experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.

Clinical, pathologic, and genomic characteristics of two pediatric glioneuronal tumors with a CLIP2::MET fusion

Integration of molecular data with histologic, radiologic, and clinical features is imperative for accurate diagnosis of pediatric central nervous system (CNS) tumors. Whole transcriptome RNA sequencing (RNAseq), a genome-wide and non-targeted approach, allows for the detection of novel or rare oncogenic fusion events that contribute to the tumorigenesis of a substantial portion of pediatric low- and high-grade glial and glioneuronal tumors. We present two cases of pediatric glioneuronal tumors occurring in the occipital region with a CLIP2::MET fusion detected by RNAseq. Chromosomal microarray studies revealed copy number alterations involving chromosomes 1, 7, and 22 in both tumors, with Case 2 having an interstitial deletion breakpoint in the CLIP2 gene. By methylation profiling, neither tumor had a match result, but both clustered with the low-grade glial/glioneuronal tumors in the UMAP. Histologically, in both instances, our cases displayed characteristics of a low-grade tumor, notably the absence of mitotic activity, low Ki-67 labeling index and the lack of necrosis and microvascular proliferation. Glial and neuronal markers were positive for both tumors. Clinically, both patients achieved clinical stability post-tumor resection and remain under regular surveillance imaging without adjuvant therapy at the last follow-up, 6 months and 3 years, respectively. This is the first case report demonstrating the presence of a CLIP2::MET fusion in two pediatric low-grade glioneuronal tumors (GNT). Conservative clinical management may be considered for patients with GNT and CLIP2:MET fusion in the context of histologically low-grade features.

Development of combination therapies with BTK inhibitors and dasatinib to treat CNS-infiltrating E2A-PBX1+/preBCR+ ALL

AbstractThe t(1;19) translocation, encoding the oncogenic fusion protein E2A (TCF3)-PBX1, is involved in acute lymphoblastic leukemia (ALL) and associated with a pre–B-cell receptor (preBCR+) phenotype. Relapse in patients with E2A-PBX1+ ALL frequently occurs in the central nervous system (CNS). Therefore, there is a medical need for the identification of CNS active regimens for the treatment of E2A-PBX1+/preBCR+ ALL. Using unbiased short hairpin RNA (shRNA) library screening approaches, we identified Bruton tyrosine kinase (BTK) as a key gene involved in both proliferation and dasatinib sensitivity of E2A-PBX1+/preBCR+ ALL. Depletion of BTK by shRNAs resulted in decreased proliferation of dasatinib-treated E2A-PBX1+/preBCR+ cells compared with control-transduced cells. Moreover, the combination of dasatinib with BTK inhibitors (BTKi; ibrutinib, acalabrutinib, or zanubrutinib) significantly decreased E2A-PBX1+/preBCR+ human and murine cell proliferation, reduced phospholipase C gamma 2 (PLCG2) and BTK phosphorylation and total protein levels and increased disease-free survival of mice in secondary transplantation assays, particularly reducing CNS-leukemic infiltration. Hence, dasatinib with ibrutinib reduced pPLCG2 and pBTK in primary ALL patient samples, including E2A-PBX1+ ALLs. In summary, genetic depletion and pharmacological inhibition of BTK increase dasatinib effects in human and mouse with E2A-PBX1+/preBCR+ ALL across most of performed assays, with the combination of dasatinib and BTKi proving effective in reducing CNS infiltration of E2A-PBX1+/preBCR+ ALL cells in vivo.

Abstract SY05-01: Targeting chromatin adaptor proteins in cancer

Abstract Menin interacts with oncogenic fusion proteins produced by MLL1 rearrangements, and small molecules capable of disrupting these interactions are currently under investigation in clinical trials for the treatment of leukemia. Our research, which combines chromatin-focused and genome-wide CRISPR screenings with a range of genetic, pharmacological, and biochemical strategies in both mouse and human models, has led to the discovery of a molecular mechanism that acts as a switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes, influencing the efficacy of Menin-MLL inhibitors. We demonstrate that the MLL1-Menin complex plays a critical role in maintaining leukemia cell survival by preventing the MLL3/4-UTX complex from binding to certain gene promoters. Interruption of the Menin-MLL1 interaction results in the UTX-dependent transcriptional activation of a tumor suppressor gene program, which significantly influences therapeutic outcomes in both murine and human leukemia models. Notably, the therapeutic activation of this gene program through CDK4/6 inhibitors can overcome resistance in leukemia cells that do not respond to Menin inhibitors. The identification of this molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin highlights the complex roles of these evolutionarily conserved epigenetic regulators and underscores their importance in understanding and addressing the molecular mechanisms underlying response and resistance in leukemia treatment trials. Citation Format: Yadira Soto-Feliciano. Targeting chromatin adaptor proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY05-01.

FusionVAC22_01: a phase I clinical trial evaluating a DNAJB1-PRKACA fusion transcript-based peptide vaccine combined with immune checkpoint inhibition for fibrolamellar hepatocellular carcinoma and other tumor entities carrying the oncogenic driver fusion.

EU CT Number: 2022-502869-17-01 and ClinicalTrials.gov Registry (NCT05937295).

Oncogenic ETS fusions promote DNA damage and proinflammatory responses via pericentromeric RNAs in extracellular vesicles.

Aberrant expression of the E26 transformation-specific (ETS) transcription factors characterizes numerous human malignancies. Many of these proteins, including EWS:FLI1 and EWS:ERG fusions in Ewing sarcoma (EwS) and TMPRSS2:ERG in prostate cancer (PCa), drive oncogenic programs via binding to GGAA repeats. We report here that both EWS:FLI1 and ERG bind and transcriptionally activate GGAA-rich pericentromeric heterochromatin. The respective pathogen-like HSAT2 and HSAT3 RNAs, together with LINE, SINE, ERV, and other repeat transcripts, are expressed in EwS and PCa tumors, secreted in extracellular vesicles (EVs), and are highly elevated in plasma of patients with EwS with metastatic disease. High human satellite 2 and 3 (HSAT2,3) levels in EWS:FLI1- or ERG-expressing cells and tumors were associated with induction of G2/M checkpoint, mitotic spindle, and DNA damage programs. These programs were also activated in EwS EV-treated fibroblasts, coincident with accumulation of HSAT2,3 RNAs, proinflammatory responses, mitotic defects, and senescence. Mechanistically, HSAT2,3-enriched cancer EVs induced cGAS-TBK1 innate immune signaling and formation of cytosolic granules positive for double-strand RNAs, RNA-DNA, and cGAS. Hence, aberrantly expressed ETS proteins derepress pericentromeric heterochromatin, yielding pathogenic RNAs that transmit genotoxic stress and inflammation to local and distant sites. Monitoring HSAT2,3 plasma levels and preventing their dissemination may thus improve therapeutic strategies and blood-based diagnostics.

Abstract 2853: Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions

Abstract Introduction High grade hemispheric gliomas in infancy (IHG) are characterized by a more favorable outcome compared to high grade gliomas in older children. We have shown in previous studies that these tumors are characterized by a stable genome. The occurrence of tyrosine kinase gene fusions in high-grade hemispheric gliomas of infancy may represent therapeutic targets. Methods: 33 high-grade hemispheric gliomas occurring in children younger than three years were retrieved from the archives of the Brain Tumor Reference Center, Institute of Neuropathology, University of Bonn. DNA and RNA were extracted from FFPE tumor samples. Methylation-based classification was done by hybridization to Illumina methylation arrays and subsequent use of the Heidelberg brain tumor classifier versions v11b4, v12.5 and v12.8. In addition UMAP plots were used to visualize similarities of methylation profiles. Gene fusions were identified on the DNA level by FISH using break-apart probes for ALK, NTRK1, -2, -3, ROS1 and MET and Molecular Inversion Probe (MIP) methodology. On the RNA level, fusion transcripts were detected by targeted RNA sequencing as well as Nanostring assay with fusion-specific probes. Results: 32 IHG occurred in the first year of life, one at the age of two years. 23/33 IHG were classified as such by methylation arrays; 6 further cases could be annotated to the IHG group by UMAP plots. 18/33 cases (54.5 %) showed fusions of ALK to different partners; all occurred in the first year of life. Fusions of ROS1 were found in 4, MET in 2, NTRK1 and -3 in 7 cases. 2 cases showed no fusions. The different methods for fusion detection led to comparable results. Only recurrent fusions with known fusion partners were detectable with Nanostring probes designed for the fusion sequence, and library construction for targeted RNA sequencing was not successful in a fraction of cases. Break-apart FISH led to reliable results on the next day, and MIP technology represented the most sensitive method for analysis of FFPE samples. Conclusions: Gene fusions involving the tyrosine kinase genes ALK, MET, ROS1 and NTRK1 and -3 occurred in 93 % of high grade hemispheric gliomas of infancy; the most frequent were ALK fusions occurring in children younger than 1 year. DNA-based MIP technology represented the most robust and sensitive assay. A combination of RNA- and DNA-based methods to detect these fusions with high reliability is recommended. Citation Format: Torsten Pietsch, Gerrit Gielen, Andreas Waha, Evelyn Dörner, Verena Dreschmann, Christof Kramm, Christian Vokuhl. Comparison of methods for detection of hemispheric gliomas of infancy and their characteristic oncogenic tyrosine kinase gene fusions [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 2853.

Abstract 1081: A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma

Abstract Background: Tumor necrosis factor receptor superfamily member 10b (TNFRSF10b, also known as DR5) agonists are attractive targeted agents for cancer cells because these agents activate cellular apoptosis. Ewing sarcoma (ES) is a bone and soft-tissue sarcoma occurring in adolescence defined by a t(11;22)(q24;q12) balanced chromosomal translocation that results in chimeric transcription factor and oncogenic driver protein, EWS-FLI1. Independent studies and unbiased cell line screens have reported ES to be sensitive to DR5 targeting. INBRX-109 is a tetravalent, DR5-targeted antibody. Here, we analyzed the sensitivity of ES models to DR5 targeting by INBRX-109 and explored how the EWS-FLI1 transcriptional program impacts the continuum of sensitivity-resistance to this targeting. Methods: In a panel of 11 ES lines, in vitro sensitivity was assessed to both single agent and INBRX-109 combinations. A screen of 1,363 FDA approved compounds identified cooperative combinations that were prioritized mechanistically. Differential sensitivity for single agent and in combination with irinotecan was confirmed in vivo in xenograft models. Resistant clones were isolated from in vivo experiments and evaluated for DR5 expression and sensitivity. Cellular factors associated with sensitivity and resistance were determined by siRNA knockdown and Cleavage Under Targets and Tagmentation assay (CUT and Tag). Results: ES models showed variable sensitivity to INBRX-109, with a GI90 as low as 10 picomolar in some models. All models showed robust expression of DR5, but absolute expression of DR5 did not correlate with sensitivity. In vitro sensitivity was replicated in vivo, with all 7 mice bearing RD-ES xenografts demonstrating initial complete tumor regression and 3 of 7 mice demonstrating no recurrence beyond 90 days. Importantly, cells isolated from recurrent tumors showed a novel mechanism of resistance: direct repression of DR5 by EWS-FLI1. INBRX-109 was ineffective as a single agent at 10 nM in resistant ES models. However, there was striking synergy in vitro with SN38, the active metabolite of irinotecan, and this effect translated into tumor regressions of a resistant TC32 xenograft in vivo. The FDA compound screen captured novel synergy with a panel of agents. Conclusion: INBRX-109 is an exciting candidate for ES as a single agent in subsets of tumors or in combination with irinotecan, which is currently being evaluated in the clinic. Investigation of the exact relationship between EWS-FLI1 and sensitivity to DR5 targeting is ongoing. Initial findings suggest EWS-FLI1, combined with other disease modifying mutations, is a key mediator of this sensitivity. Further, EWS-FLI1 plays a direct role repressing DR5 in the setting of therapeutic challenge in one model in vivo. These findings highlight the potential for INBRX-109 in combination with irinotecan or novel candidates as therapies for ES patients. Citation Format: Curtis C. Parker, Lauren M. Gaetano, Elissa A. Boguslawski, Seneca Kinn-Gurzo, Rebecca Kaufman, Matthew C. Stout, Chase Deveraux, Patrick J. Grohar. A role for the oncogenic driver fusion protein EWS-FLI1 in the targeting of DR5 by INBRX-109 in Ewing sarcoma [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 1081.

Abstract 7550: KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models

Abstract Adenoid Cystic Carcinoma (ACC) is an aggressive rare type of cancer of the secretory glands with no approved targeted therapy and high unmet clinical need. Deregulated transcription driven by MYB-NFIB or MYBL1-NFIB transcription factor (TF) fusions are a hallmark of ACC pathogenesis. More aggressive disease and resistance to therapy has been associated with co-mutation in NOTCH. Although direct targeting of oncogenic TF fusions has remained challenging, targeting of their activity via transcriptional cofactors has emerged as an attractive and clinically actionable strategy. Cyclin-dependent kinase 9 (CDK9) is a key potentiator of TF activity via its ability to act both as an upstream regulator of TF expression and a downstream cofactor. KB-0742 is a potent, selective, and orally bioavailable inhibitor of CDK9 with a long plasma half-life. KB-0742 is being studied in an ongoing Phase 1/2 study (NCT04718675) in advanced solid tumors including ACC. KB-0742 has demonstrated on-mechanism, single agent anti-tumor activity and a manageable safety profile in heavily pre-treated patients (0-11 prior lines of therapy) with transcriptionally addicted solid tumors. Here we present the preclinical rationale for targeting of oncogenic fusion TF activity in ACC. XenoSTART Patient-Derived Xenograft (XPDX) models were established from viable human tumor tissue for in vivo and ex vivo testing. Cell viability was assessed by CellTiter GLO reagent and phase imaging. Changes in MYB-fusion transcripts were assessed by RNA-Seq. Tumor Fragments (~70 mg) from ACCx5M1, ACCx6, ACCx9, ACCx11 models were implanted into athymic nude mice. Animals were treated with either vehicle (normal saline) or 60 mg/kg KB-0742 on a Q3D/week until tumors reached 2500 mm3 or up to 60 days post start of treatment. In a retrospective analysis using real world data, ACC has a low incidence of genomic instability and high rates of MYB fusions detectable by RNA-seq, making immunotherapy challenging but creating an opportunity for targeting MYB transcription. In primary MYB-fusion positive and NOTCH co-mutated patient-derived spheroid models, KB-0742 treatment induced strong antiproliferative activity and cytotoxicity. Importantly, KB-0742 demonstrated stronger cytotoxic effects when compared to historical control agents. In XPDXs, CDK9 inhibition with KB-0742 resulted in antiproliferative activity and stronger tumor growth inhibition in MYB-fusion positive and NOTCH co-mutated tumor models compared to MYB-fusion positive only models.These data demonstrate KB-0742 is effective in preclinical models of ACC suggesting KB-0742 may be a promising therapeutic option for ACC patients. KB-0742 is currently being evaluated in a phase 1/2 dose-escalation and cohort expansion trial in patients with ACC and other transcriptionally addicted tumors (NCT04718675). Citation Format: Michael R. McKeown, Luis A. Carvajal, Tressa R. Hood, Nicole S. Burr, Jeffrey Kaufman, Adam Boynton, Kameron Mori, Tessa DesRochers, Michael Wick, Charles Y. Lin, Jorge F. DiMartino. KB-0742, an oral highly selective CDK9 inhibitor, demonstrates preclinical activity in transcription factor fusion driven adenoid cystic carcinoma patient-derived models [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 7550.

Abstract 1089: Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens. Method: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR in FP-RMS cell lines RH4 and SCMC. Western blot was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, a 2,480 drug library with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. Mouse xenograft models of FP-RMS were used to investigate in vivo efficacy. Results: We validated HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody for P3F-HiBiT matched the genomic locations from ChIP-seq with P3F antibody in RH4 and SCMC. Using a cutoff value of > 90 (Area Under the Curve (AUC) of CellTiter-Glo minus AUC of Nano-Glo), in both RH4 and SCMC, we identified 182 compounds which downregulate P3F before cell death. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F including HDAC inhibitors (3), BRD4 inhibitors (3), and CDK inhibitors (8). FP-RMS was sensitive to CDK1/2, CDK4/6, CDK9, and multi-CDK inhibitors. A multi-CDK inhibitor TG02, currently in human trials, downregulated P3F and RNA-seq GSEA showed marked suppression of P3F targets. TG02 also significantly delayed tumor progression in mouse xenograft model of FP-RMS without weight loss. Interestingly, the commonly used chemotherapeutic Vincristine (VCR) also downregulated P3F in vitro. TG02 with VCR showed synergy by Loewe analysis. In vivo, testing showed significant delay in tumor progression by the combination compared to TG02 or VCR alone. Of note, tumor RNA-seq GSEA following treatment with TG02, or VCR alone, and in combination significantly downregulated P3F targets. Conclusion And Future Directions: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels of which TG02 was a top hit. TG02 showed in vivo efficacy indicating that FP-RMS is susceptible to CDK inhibition. We also found synergy between TG02 and VCR, resulting in a significant delay in FP-RMS tumor progression compared to single agents. Interestingly, we found that VCR alone can downregulate P3F and its targets. Combination therapy of TG02 with VCR shows promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine [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 1089.

Abstract 562: Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor that predominantly occurs in children, adolescents, and young adults. The treatment for OS that combines surgery with chemotherapy, which consists of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, was established in 1970s, and it is still used as a standard therapy. Oncogenic driver mutations and fusion genes common to OS have not been identified, which is one of the reasons for the lack of success in drug development for OS. Herein, to explore the characteristic vulnerability in OS, molecular targeted drugs were screened against OS cell lines and patient-derived cells; obatoclax, a pan-BH3 mimetic, and a concomitant drug that enhances obatoclax-induced apoptosis, OSI906, a dual inhibitor of IGF1R and IR, were found. Fluorescence in situ hybridization (FISH) analysis revealed that copy number gains of MCL1, an antiapoptotic Bcl-2 family protein, were observed in 46.3% of formalin-fixed paraffin-embedded (FFPE) specimens from patients with OS (N = 41) and in 45.5% of OS cell lines (N = 11), thereby causing vulnerability to BH3 mimetics. IHC staining also revealed high expression of Mcl-1 protein in specimens with MCL1-amplification in FISH analysis. Pharmacological or genetic inhibition of Mcl-1 induced potent apoptosis in MCL1-amplified OS cells, which was enhanced by the co-inhibition with Bcl-xL. Moreover, chromosome 1q21.2-3, where MCL1 is located, contains several IGF1R/PI3K pathway-related genes, including PIP5K1A, TARS2, OUTD7B, and ENSA, whose copy numbers were also increased in MCL1-amplified OS cells, suggesting that 1q21.2-3-amplified OS cells readily activate downstream pathways via IGF1R. IGF signaling activation in 1q21.2-3-amplified OS cells negated the obatoclax-induced apoptosis, which was overcome by OSI906 treatment. Combined obatoclax and OSI906 administration remarkably suppressed MCL1-amplified OS tumor growth in the xenograft mouse model and resulted in tumor regression; however, none of the treatments reduced tumor growth of OS without MCL1 amplification. These results suggest that combining obatoclax with OSI906 could be a potential therapeutic intervention for 1q21.2-3-amplified OS, and that prior stratification of OS based on the 1q21.2-3 amplification status would be important for the success of this combination therapy. Thus, we elucidated the mechanism of action of drugs and their potential as a novel therapeutic strategy for approximately 50% of patients with OS. Moreover, a FISH analysis that can detect copy number gains of MCL1 in FFPE specimens from patients with OS was established to stratify patients eligible for this therapy. Citation Format: Satoshi Takagi, Ryohei Katayama. Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma [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 562.

Abstract 613: Oral CHK1 inhibitor BBI-355 allows flexibility of dose and schedule with demonstration of monotherapy and combinational antitumor activity in extrachromosomal DNA (ecDNA) driven preclinical models

Abstract Focal, high copy number amplifications of prevalent oncogenes (e.g., MYC, EGFR) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular, non-chromosomal DNA. While use of targeted therapies is associated with improved survival in patients with cancers that have activating oncogene mutations or fusions, they have demonstrated limited activity for patients whose tumors harbor oncogene amplifications, including those mediated by ecDNA. We have previously identified Checkpoint Kinase 1 (CHK1) as a target essential for ecDNA function in cancer, as tumor cells with oncogene amplification on ecDNA exhibit increased DNA replication stress (RS) and are hypersensitive to inactivation of CHK1, cells’ master regulator of RS. Thus, we have developed BBI-355, a novel, orally bioavailable, and selective small molecule inhibitor of CHK1, which is currently being studied in the first-in-human Phase 1/2 POTENTIATE clinical trial for patients with cancer harboring oncogene amplifications, including on ecDNA (NCT05827614). In vitro antiproliferation activity of BBI-355 was observed in a panel of ecDNA+ oncogene amplified tumor lines and non-amplified lines, with heightened sensitivity and induction of RS observed in ecDNA+ cells. Oral administration of BBI-355 resulted in dose dependent on-target activity, with induced p-CHK1-S345 protein expression, in agreement with measured plasma and tumor concentrations. Subsequent pharmacokinetic/pharmacodynamic (PK/PD) modeling guided continuous and intermittent dose/schedules of single agent BBI-355 that demonstrated durable antitumor activity, including tumor regressions, in multiple ecDNA+ tumor models. Single agent targeted therapy treatment of cancers with oncogene amplification on ecDNA can result in acquired resistance via ecDNA-based mechanisms, further increasing reliance on CHK1. Enhanced antitumor activity of BBI-355 in combination with agents targeting the protein products of amplified oncogenes was demonstrated in multiple in vivo tumor models; these included combination with a pan-FGFR inhibitor in FGFR2 ecDNA+ amplified gastric cancer and combination with CDK4/6 inhibitors in CDK4 ecDNA+ amplified sarcoma models. The first ecDNA-directed therapy (ecDTx) in development, BBI-355 demonstrated significant antitumor activity as a single agent and in combination with targeted therapies in multiple ecDNA+ oncogene amplified tumor models. Oral dosing of BBI-355 provides dose/schedule flexibility to attempt to optimize antitumor activity with a tolerable safety profile in patients with highly aggressive cancers and significant unmet need. The POTENTIATE clinical trial is open and currently enrolling. Citation Format: Ryan J. Hansen, Auzon Steffy, Joshua Plum, Ardalan Ardeshiri, Edison Tse, Salvador Garcia, Ben Norman, Jafar Moininazeri, AnneMarie Pferdekamper, Evan Holmes, Debbie Liao, Rachelle Elsdon, Joshua Lange, Anthony Pinkerton, Julius Apuy, Sudhir Chowdhry, Christian Hassig, Shailaja Kasibhatla. Oral CHK1 inhibitor BBI-355 allows flexibility of dose and schedule with demonstration of monotherapy and combinational antitumor activity in extrachromosomal DNA (ecDNA) driven preclinical models [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 613.

Abstract 6: Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy

Abstract Sarcomas are a heterogenous group of mesenchymal cell malignancies that are difficult to treat and often respond poorly to current immunotherapies. Approximately 30% of sarcomas are associated with oncogenic driver fusion proteins. Desmoplastic small round cell tumor (DSRCT) is a prototypical fusion-driven sarcoma defined by a pathognomonic EWSR1-WT1 fusion event. The resultant EWSR1-WT1 fusion protein contains a shared junctional amino acid sequence that is clonally conserved and significantly divergent from normal human proteins. Consequently, the EWSR1-WT1 fusion might yield a highly immunogenic shared, or public, neoantigen (NeoAg) that may serve as a target for novel immunotherapeutic approaches.Using a functional HLA-immunoprecipitation/mass spectrometry (HLA-IP/MS) screen, we identified a 9-amino acid peptide sequence derived from the junction of the most common EWSR1-WT1 fusion protein that is presented in the context of the prevalent HLA-A*03 and -A*11 alleles. We confirmed that HLA-A*03+ DSRCT cells physiologically present the same peptide sequence. Using fluorophore-conjugated HLA-multimers loaded with the HLA-IP/MS-derived fusion NeoAg, we identified a rare population of circulating NeoAg-specific CD8+ T cells in HLA+ DSRCT patients. Through antigen-directed clonal expansion, we discovered n=3 HLA-A*03-restricted and n=1 HLA-A*11-restricted fusion NeoAg-specific T cell clones and retrieved the paired TCRαβ gene sequences of their T cell receptors (TCRs) using 10x single-cell sequencing. Polyclonal CD8+ T cells transduced with candidate TCRαβ gene sequences robustly upregulated TNFα after coculture with HLA+/Fusion+ target cells but not HLA−/Fusion+ or HLA+/Fusion− control cells. Further, T cells expressing candidate TCRs, but not viral-protein-specific TCRs, lysed HLA+ DSRCT cells in vitro and controlled established DSRCT tumors in vivo. Finally, we discovered that one TCR was capable of cross HLA allele fusion NeoAg recognition and could lyse both HLA-A*03+ and -A*11+ DSRCT cells. These findings imply that a single TCR therapeutic could cover >36% of North American DSRCT patients.Collectively, our data establish for the first time that DSRCT cells present a junction-spanning fusion-derived neoantigen in the context of two prevalent HLA alleles that may be therapeutically targeted using TCR gene therapy. These data lay the foundation for first-in-human clinical translation of a T-cell based therapy targeting EWSR1-WT1. More broadly, these findings establish proof-of-principle that recurrent fusion proteins are an actionable source of immunogenic public NeoAgs, providing a framework for developing targeted immunotherapies for other fusion-driven malignancies. Citation Format: Lauren Banks, Hannah Arkin, Smita Chandran, Emily Slotkin, Madelyn Espinosa-Cotton, Neerav Shukla, Luc Morris, Marc Ladanyi, Andrew Kung, Martin Klatt, Christopher Klebanoff. Defining and therapeutically targeting a fusion-derived public neoantigen in desmoplastic small round cell tumor using T cell receptor gene therapy [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 6.

Abstract 3365: Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort

Abstract Background Focal high-level oncogene amplifications (FH-amp) (e.g., MYC, EGFR) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular non-chromosomal DNA. FH-amp on ecDNA promote intra-tumoral heterogeneity, resistance to therapies, and poor prognosis. Recently, a Phase 1 clinical trial exploring ecDNA-directed treatments has begun. However, to date, bioinformatic detection of ecDNA has relied on whole-genome sequencing data, and no standard method exists to detect ecDNA on targeted NGS. We thus sought to explore the feasibility of detecting ecDNA in a large cohort of patients with solid tumors sequenced with the targeted NGS panel, MSK-IMPACT, and describe the pan-cancer prevalence of oncogenes amplified on ecDNA. Methods We utilized a novel bioinformatic ecDNA-diagnostic algorithm, ECHO, to determine the presence of oncogene FH-amp on ecDNA (including HSR) vs. chromosomal in clinical-grade NGS data. 72,651 tumor samples profiled by MSK-IMPACT were queried for high level oncogene FH-amp (AR, CCNE1, CDK4/6, EGFR, FGFR1-4, KRAS, MDM2, MDM4, MET, MYC, MYCL, and MYCN) above a fold-change of three, and subsequently annotated with ECHO. Patients with concomitant oncogenic driver mutations or fusions (e.g., EGFR L858R, EML4-ALK) were excluded based on the hypothesis that they might be mutually exclusive with oncogene FH-amp. Results FH-amp of any above listed oncogene were found in 3065 (4.2%) samples. Cancer types with frequent FH-amp without concomitant driver alterations were liposarcoma (LPS) (61% FH-amp; 43% were ecDNA+), glioblastoma (GBM) (22%; 72%), gastroesophageal adenocarcinoma (GE AD) (10%; 57%) squamous cell lung cancer (7%; 18%), and breast cancer (BRCA) (7%; 23%). Among tumors with oncogene FH-amp, GBM and GE AD had high frequencies of ecDNA+ FH-amp of EGFR (39% and 15% of all EGFR ecDNA+). LPS had the highest frequency of ecDNA+ FH-amp of CDK4 (64%), followed by GBM (15%). The majority of ecDNA+ FGFR1 and FGFR2 FH-amp were in BRCA (75% and 47%). EGFR in GBM and EGFR or FGFR2 in GE AD were ecDNA+ in more than 90% of patients, while CDK4 in LPS and FGFR1 in BRCA were ecDNA+ in 45% and 39% respectively.Evaluation the relationship between ecDNA+ by ECHO and amp FC range showed higher ecDNA+ in samples with higher level amp (FC > 5; 58% ecDNA+) compared to lower level amp (FC 4~5; 12%, FC 3~4; 6% ecDNA+). Conclusions A novel ecDNA clinical diagnostic tool, ECHO, was successfully applied to a large clinical cohort of patients enriched for actionable oncogene amplifications sequenced with MSK-IMPACT. The results demonstrate that detection of ecDNA using clinical-grade NGS assays is feasible and that key oncogene FH-amp on ecDNA (e.g., EGFR, FGFR2) are common in select tumor types. ECHO may be developed as a clinical trial device to prospectively identify patients with oncogene ecDNA+ FH-amp for clinical testing of ecDNA-directed therapies. Citation Format: Matteo Repetto, Klaus Wagner, Shalabh Suman, Peter Krein, Pavitra Rao, Maria Arcila, Christian Hassig, Mark Donoghue, Alexander Drilon, Michael Berger. Reliable detection of potentially therapeutically actionable ecDNA using clinical-grade NGS in a large pan-cancer cohort [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 3365.

Abstract 6836: Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape

Abstract Myxoid/Round Cell Liposarcoma (MRCL) is an understudied soft tissue sarcoma that typically affects young adults and accounts for 30% of all liposarcomas. MRCL is characterized by oncogenic DDIT3 fusions, with FUS::DDIT3 being present in over 90% of cases. However, the cellular origins and oncogenic mechanisms of MRCL remain incompletely defined. Here we report oncogene transcription, and tumor-immune interactions in a cohort of 5 patient tumors with matched normal tissue for which we sequenced the transcriptomes of approximately 45,000 single cells using 10X Genomics, and full-length transcripts in approximately 20,000 single cells via Oxford Nanopore in 2 of these patients. Our results reveal two distinct cancer cell populations: Early Cancer Cells (ECC) and Advanced Cancer Cells (ACC). These cells appear to derive from adipose stem progenitor cells (ASPC) and fall along an ASPC→ECC→ACC pseudo-time axis. Along this axis, a distinct pattern emerges where oncogenes tend to be upregulated and tumor suppressor genes (TSG) tend to be downregulated and these appear to be mutually exclusive. Comparing ECC to ASPC we see increased expression of HIPPO and PI3K pathway oncogenes, FGFR2 and IGF1R RTKs, and downregulation of the TSG CDKN2A and CDKN1A. ACC show upregulation of hedgehog, ErbB, WNT, HIPPO, PI3K, and MAPK signaling pathways compared to ECC. Full-length transcript sequencing at the single-cell level shows a consistent expression of the FUS::DDIT3 fusion transcript in both ECC and ACC populations, confirming that these populations are cancer cells. Furthermore, MRCL displays several immune escape mechanisms, including poor immune infiltration and a marked decline of HLA Class I expression along the pseudo-time axis from ECC to ACC, suggesting a complex interplay between oncogenic signaling and immune evasion. These results suggest that MRCL likely originates from ASPC and expression of FUS:DDIT3 oncoprotein induces early HIPPO, PI3K, FGFR2, and insulin signaling pathway expression in ECC with further hedgehog, ErbB, MAPK, and WNT pathway activation in ACC. This progressive activation in oncogenic signaling is associated with immune evasion. This is the first single-cell RNA seq analysis to detect the presence of the FUS::DDIT3 fusion in ECC and ACC significantly advancing our understanding of the mechanisms of MRCL oncogenesis, immune escape, and identifies potential novel therapeutic targets. Citation Format: Rodrigo Gularte-Mérida, Evan Seffar, George Li, Young-Mi Kim, Narasimhan P. Agaram, Nicholas Socci, Francisco Sanchez-Vega, Nikolaus Schultz, Samuel Singer. Single-cell RNA sequencing of FUS::DDIT3 myxoid/round cell liposarcoma delineates mechanisms of oncogenic progression and immune escape [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 6836.

Abstract 2147: Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort

Abstract In prostate cancer, Black men face higher incidence rates, more advanced diagnoses and worse outcomes than White men. These disparities partially stem from environmental, socioeconomic, and healthcare access factors. Recent data highlight racial differences in prostate cancer molecular profiles, with tumors from Black men showing increased SPOP and decreased PIK3CA mutations and amplified androgen receptor signaling and inflammatory pathway activations. Thus, somatic mutation patterns may also contribute to these disparities. We sought to identify associations of mutational profiles in prostate adenocarcinoma (PRAD) with genetic ancestry instead of race and ethnicity categories, analyzing a de-identified cohort of 5,959 patients who underwent the Tempus xT 648-gene NGS tumor profiling test. We used 654 ancestry-informative markers to estimate genetic ancestry by calculating similarity to reference populations for five regions: Africa (AFR), Americas (AMR), East Asia (EAS), Europe (EUR), and South Asia (SAS). Associations between genetic ancestry proportions and somatic variants were assessed for 67 PRAD-related genes using logistic regression models. Analyses included copy number alterations (CNAs), oncogenic gene fusions, small splice and non-synonymous mutations (NS), OncoKB L1/2 & R1, and small missense driver somatic mutations predicted with boostDM. Likelihood ratio tests were employed to compare models with and without ancestry terms. The Benjamini-Hochberg method was utilized to adjust p-values and control the FDR at 5%. We confirmed reported associations in PRAD between AFR ancestry and SPOP NS mutations (OR=1.03 per doubling of AFR ancestry proportion, p=0.03) and decreased AFR with PIK3CA OncoKB and driver mutations (OR=0.94, p=0.02), PTEN CNAs (OR=0.95, p=0.0001), and TMPRSS2-ERG fusions (OR=0.95, p<0.0001). However, we also observed negative associations between AFR and AR CNAs (OR=0.95, p=0.004), EAS and PTEN CNAs (OR=0.96, p=0.01), and EAS and TMPRSS2-ERG fusions (OR=0.97, p=0.02). Additionally, a negative association was found between EAS and TP53 driver NS mutations (OR=0.96, p=0.04), while positive associations with driver mutations in FOXA1 (OR=1.06, p=0.03) and NS mutations in the PI3K/AKT/mTOR pathway genes (OR=1.04, p=0.01) were noted. Lastly, we identified novel positive associations between AMR and driver mutations in PIK3C3A (OR=1.12, p=0.0003) as well as small NS mutations or CNAs in the PI3K/AKT/mTOR pathway genes (OR=1.03, p=0.04). By analyzing a large, diverse real-world cohort and leveraging NGS-inferred genetic ancestry, our study confirms known associations between somatic alterations in PRAD cancer genes and race and ethnicity, while unveiling novel associations in understudied populations of potential significance for understanding disparities in disease outcomes. Citation Format: Brooke Rhead, Yannick Pouliot, Francisco M. De La Vega, David W. Craig, John Carpten. Genetic ancestry associations with prostate adenocarcinoma mutational profiles: New Insights from a diverse 5,959-patient real-world cohort [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 2147.

Abstract 1093: Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma

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.

Abstract 5777: Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs)

Abstract The purpose of this study was to determine if EWS/FLI1 (E/F), the oncogenic fusion protein that drives Ewing Sarcoma (ES), can be rewired to enhance expression of BCL6-controlled genes using Transcriptional Chemical Inducers of Proximity (TCIPs), a novel bivalent small molecule platform. TCIP molecules recruit a transcriptional regulator, such as BRD4, to the transcriptional repressor BCL6. Upon binding, a ternary complex is formed on chromatin at BCL6-bound loci, causing transcriptional upregulation of BCL6 target genes. Since E/F does not have a bona fide ligand, we have used an exogenously expressed N terminal FKBPF36V-EWS/FLI1 (N-FK-E/F) fusion system to study how modulation of E/F by TCIPs impacts the transcriptome of ES cells. To eliminate background signal from endogenous E/F, we used CRISPR/Cas9 to knockout (KO) endogenous E/F. Our exogenously expressed N-FK-E/F has mutated PAM motifs and is resistant to KO. In our proof-of-concept study we used TCIP1 consisting of ortho-AP1867 (oAP), a synthetic ligand for FKBPF36V, linked to the BCL6 inhibitor BI3812 (BI) to rewire N-FK-E/F. We have also tested a negative control TCIP (TCIP NEG) that links oAP to a BI derivative that abolishes BCL6 binding. TCIP NEG has similar physiochemical properties to our active TCIP1; however, it cannot bind BCL6, serving as a negative control for ternary complex formation. TCIP1 rapidly and dose-dependently increases the RNA and protein expression of several BCL6 target genes, like p21, in multiple ES models that stably express N-FK-E/F. TCIP NEG does not increase the expression of BCL6 target genes in ES cells. TCIP1 induces ternary complex formation in ES cell lysates, and ternary complex formation is necessary for activity in ES cells. TCIP1 is specific and does not increase BCL6 target gene levels to the same extent in ES cells expressing a FKBP-GFP construct or in parental ES cells lacking N-FK-E/F. RNA sequencing of EWS502 N-FK-E/F expressing cells shows that TCIP1 treatment induces a larger increase in expression of BCL6 target gene RNA than BCL6 inhibition. Moreover, CUT&RUN experiments in EWS502 N-FK-E/F expressing cells suggest TCIP1 leads to an accumulation of N-FK-E/F at the promoters of BCL6 target genes. Motif analysis showed that TCIP1 increases the likelihood of N-FK-E/F binding to BCL6 consensus sequences as wells as increases the likelihood of BCL6 binding to E/F recognition sequences. In future studies we will conduct RNA sequencing, CUT&RUN, and other ‘omics’ approaches (e.g. DisP-seq) to compare TCIP1 to TCIP NEG in multiple cell lines at multiple concentrations and time points. Our data suggests that N-FK-E/F can be rewired to induce the expression of BCL6 target genes in ES. Since ES is driven solely by E/F, TCIPs may be promising next generation therapeutics. Our study provides a proof-of-concept that will be the foundation for the development of future TCIP molecules that recruit endogenous E/F once suitable ligands are developed. Citation Format: Michael J. Bond, Ryan P. Golden, Roman C. Sarott, Giulia DiGiovanni, Basel A. Karim, Briana Howard, Kenneth Ross, Nathanael S. Gray, Kimberly Stegmaier. Rewiring EWS/FLI1 with transcriptional chemical inducers of proximity (TCIPs) [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 5777.

Abstract 131: Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is a childhood soft tissue sarcoma composed of skeletal myoblast-like cells that lack the capacity to differentiate despite their high expression of terminal differentiation markers like myogenin. There are two main types of RMS: fusion-positive RMS (FP-RMS), which is associated with a chromosomal translocation resulting in an oncogenic fusion protein (PAX3-FOXO1), and the more common form, fusion-negative RMS (FN-RMS) which lacks this translocation and is instead thought to be driven by numerous other oncogenes. Our lab used a Bayesian-based computational algorithm to analyze NGS sequencing data from 290 RMS samples and identified 25 potential genetic drivers of RMS. We aim to use induced pluripotent stem cells as a new model to study how these drivers, including the PAX3-FOXO1 fusion gene, influence RMS formation and progression. To this aim, we have successfully made a doxycycline inducible lentiviral vector containing the full-length sequence of human PAX3-FOXO1 which we used to ectopically express PAX3-FOXO1 in two well-characterized iPSC lines. We subcutaneously injected these iPSCs into NOD/SCID mice and treated them with doxycycline to form teratomas, non-malignant tumors containing tissue from all three germ layers. We hypothesized that PAX3-FOXO1 induction in these differentiating teratomas, may have transforming capabilities and may deregulate transcriptional programs to form FP-RMS-like tumors. We have found that PAX3-FOXO1 overexpressing teratomas grew faster and histologically look more “immature” than their control counterparts but do not seem to give rise to rhabdo-like tumors. We want to further understand what other critical factors are necessary to drive RMS formation by focusing on one of our drivers of interest EZH2, a histone methyltransferase implicated as either an oncogene or a tumor suppressor in certain forms of cancer. We have data suggesting that EZH2-dependent histone methylation activity may act as a proto-oncogene with PAX3-FOXO1 to silence the expression of key tumor suppressor genes CDKN2A and CDKN2B in RMS. To this aim, we reasoned that iPSCs differentiated into myogenic progenitors, one of the potential cells of origin of RMS, will be a novel way to study the role of oncogenes like PAX3-FOXO1 and EZH2 in RMS. We have derived myogenic progenitors from iPSCs (iPSC-MPCs) that express key myogenic factors like PAX7 and MYOD and differentiate and fuse to form mature myofibers. We plan to next explore how ectopic expression of EZH2 and/or PAX3-FOXO1 alters gene expression and histone methylation and whether these alterations can drive RMS formation in these iPSC-MPCs. Using this model system of iPSC-MPCs to drive expression of these oncogenes has the potential to offer new and exciting ways to identify the most important genes in driving cancer formation hopefully providing insight into better targets for treatment of RMS. Citation Format: Celeste Romero, Yanbin Zheng, Lin Xu, Stephen Skapek. Utilizing human induced pluripotent stem cells (hiPSCs) as a model to study the genetic drivers of 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 131.

Abstract 647: FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types

Abstract There is a compelling need for the development of more effective and less toxic therapies for patients with Ewing Sarcoma (ES) and Clear Cell Sarcoma (CCS). ES and CCS are characterized by chromosomal translocations which result in two distinct fusion proteins: EWS-ATF1 (t(12;22)(q13;q12)) in CCS and EWS-FLI1 (t(11;22)(q24;q12)) in ES. There is a strong premise that targeting the fusion proteins or their key downstream signaling pathways will be disease modifying and improve outcomes for patients. Preliminary high throughput screening studies using an FDA approved small molecule compound library identified filanesib, a kinesin spindle protein (KIF11) inhibitor, as having potent anti-proliferative activity in ES and CCS cells over other cancer subtypes. The mechanism linking the fusion proteins with KIF11 and the tumorigenic role of KIF11 in ES and CCS has yet to be described. Previous studies have shown that the EWS-ATF1 fusion protein forms a heterodimer with transcription factors to target the promoters of its downstream genes. In breast cancer, the knockdown of KIF11 has been shown to significantly reduce the phosphorylation of these transcription factors. Additionally, KIF11 has been shown to directly interact with p300/CBP-associated factor (PCAF), a histone acetyltransferase (HAT). HATs are necessary for EWS-FLI1-induced chromatin remodeling. We hypothesize that the fusion proteins, EWS-FLI1 and EWS-ATF1, form a complex with KIF11 that regulates the activity of co-transcription factors during chromatin remodeling. The target of filanesib, KIF11, has essential roles in maintaining cell cycle progression through mitosis and its inhibition results in mitotic arrest and subsequent cell death. We used ImageStream to assess the effects of filanesib on the cell cycle. ImageStream allows both a quantitative and qualitative analysis of the cell cycle by measuring the population of cells that are positive for a given marker as well as imaging each cell by microscopy. Results demonstrate that filanesib promotes an increase in cells arrested in prophase with dramatically altered spindle formation. We are currently addressing the role of KIF11 in regulating the expression levels of the fusion proteins’ associated transcriptional coactivators and downstream target genes. By completing these studies, we aim to, i) outline the mechanism underpinning the relationship between KIF11 and the oncogenic fusion proteins in ES and CCS cells, and ii) gain an understanding of why ES and CCS cells are particularly vulnerable to KIF11 inhibition. This work has been supported in part by the Flow Cytometry and the Molecular Genomics Cores at the H. Lee Moffitt Cancer Center & Research Institute, a comprehensive cancer center designated by the National Cancer Institute and funded in part by Moffitt’s Cancer Center Support Grant (P30-CA076292) Citation Format: Hannah L. Walker-Mimms, Nicole Londono, Yi Liao, Neelkamal Chaudhary, Dessiree Pena Gomez, Yonghong Zhang, Mingxiang Teng, Uwe Rix, Derek R. Duckett. FDA approved library screen revealed Ewing and clear cell sarcomas have increased sensitivity to filanesib over other cancer types [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 647.