Targeting FGFR4, RAS, and PAX3-FOXO1 gene in Rhabdomyosarcoma

Division Of Basic Sciences - Nci

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Abstract

Introduction Rhabdomyosarcoma (RMS) remains the most common pediatric soft tissue sarcoma, yet clinical outcomes for high-risk and relapsed patients are dismal, with survival rates below 30%. Novel, targeted therapies are urgently needed. Our multidisciplinary, high-throughput approach aims to identify and translate actionable therapeutic targets into clinical interventions, focusing on FGFR4, RAS pathway mutations, and the PAX3-FOXO1 fusion oncogene. Comprehensive Assay Platform for Target Discovery Our project integrates genomics, epigenetics, functional CRISPR screening, and phenotypic assays such as the Incucyte and iCELLigence platforms to uncover key genetic and epigenetic regulators in RMS. Drug screening employs the Mechanism Interrogation Plate (MIPE) from NCATS, evaluating over 2,000 compounds with known mechanisms. These include FDA-approved agents and investigational drugs in clinical development. Targeting PAX3-FOXO1: A Central Oncogenic Driver in FP-RMS PAX3-FOXO1 is the hallmark driver of fusion-positive RMS (FP-RMS), locking RMS cells in a proliferative, myoblast-like state through extensive epigenetic reprogramming. Using an engineered ALK super-enhancer GFP-Luc reporter cell line, we screen for agents that inhibit PAX3-FOXO1 transcriptional activity. In parallel, a CRISPR knock-in model fuses a HiBiT peptide to endogenous PAX3-FOXO1, enabling real-time tracking of protein levels. We have identified that BRD4, a key co-activator of super-enhancers, is crucial for PAX3-FOXO1 function. BRD4 inhibitors JQ1 and CPI-0610 suppress FP-RMS xenograft growth and reduce PAX3-FOXO1 protein levels, highlighting a critical dependency exploitable by small molecule inhibitors. Further, we identified KDM3B as an essential co-regulator of PAX3-FOXO1 activity through a high-throughput screen of 62,643 compounds. P3FI-63 and its optimized analog P3FI-90 were found to selectively inhibit KDM3B, suppressing PAX3-FOXO1-driven transcription and tumor growth in vitro and in vivo. FGFR4: A Pan-RMS Surface Target FGFR4 is a tyrosine kinase receptor overexpressed in virtually all RMS subtypes, driven either by PAX3-FOXO1 in FP-RMS or activating mutations in FN-RMS (15%). Our studies show FGFR4 is minimally expressed in healthy tissues, making it an ideal immunotherapy target. A second-generation FGFR4 CAR T cell construct, based on a high-affinity 3A11 monoclonal antibody, demonstrates potent cytotoxicity against RMS cells and is currently progressing to a Phase I clinical trial at the NCI (NCT06865664). These CARs eradicate tumors in both metastatic and orthotopic RMS models without toxicity to normal tissues. Dual-Antigen CAR T Cell Therapies: FGFR4 and CD276 To overcome tumor heterogeneity and antigen escape, we engineered bicistronic CARs (BiCisCARs) co-targeting FGFR4 and CD276. These CARs combine two co-stimulatory domains (CD28 and 4-1BB), resulting in synergistic cytokine production, enhanced persistence, and reduced exhaustion. The BiCisCARs significantly outperform single-target CARs in multiple RMS xenograft models. ADC and Bispecific Antibody Strategies In parallel to CAR T cell approaches, we are developing FGFR4-targeted antibody-drug conjugates (ADCs) using humanized 3A11 antibodies conjugated with exatecan or MMAE payloads. These ADCs demonstrate selective cytotoxicity in vitro and potent efficacy in vivo in both FP- and FN-RMS xenograft models. Bispecific antibodies (BsAbs) using humanized 3A11 and anti-CD3 (huOKT3) fragments are also in development. These BsAbs activate host T cells to kill FGFR4-expressing RMS cells and exhibit robust activity across various xenograft models. Translational Pipeline and Future Clinical Trials Lead targets validated in vitro and in vivo are prioritized for clinical translation. FGFR4 CAR T cells have received IND approval (IND 28682), and the NCI clinical trial NCT06865664 is expected to begin patient enrollment shortly. Additional trials are in planning stages for BiCisCARs and FGFR4-targeted ADCs. Conclusion Our multimodal, integrated research strategy is delivering a pipeline of targeted therapies for high-risk RMS. Through functional genomics, drug screening, and immunotherapy engineering, we are advancing first-in-class treatments targeting PAX3-FOXO1, FGFR4, and RAS, and translating these into clinical trials that offer new hope for children and adolescents facing this aggressive cancer.