Mark E. Hatley

Summary: A large proportion of pediatric sarcomas are driven by neomorphic transcription factors that are challenging therapeutic targets. In this issue, two independent studies identify CDK8 as a shared therapeutic vulnerability in pediatric fusion oncoprotein–driven sarcomas and reveal that CDK8 inhibitors disrupt oncogenic transcriptional programs through an unexpected gain-of-function mechanism involving Mediator complex trapping on chromatin. See related article by Zhang et al., p. 2344 See related article by McGinnis et al., p. 2366

Alveolar rhabdomyosarcoma (ARMS) is largely characterized by the PAX3::FOXO1 (P3F) fusion oncoprotein, yet the mechanisms by which P3F initiates tumorigenesis and cell identity remain incompletely understood. Here, we describe a doxycycline-inducible, degradable PAX3::FOXO1 iPSC-derived ARMS model (ddP3F) to interrogate the temporal requirements of P3F for ARMS cell identity and proliferation. We show that, while P3F is essential for initiating ARMS transformation from precursor cells, continued expression of P3F is not required for maintaining proliferation, viability, or RMS-associated transcriptional programs. Integration of CUT&RUN, bulk RNA-seq, and 10X multiome profiling during endothelial-directed differentiation revealed that P3F rapidly pioneers a novel enhancer landscape within two days of expression, activating transcriptional circuits anchored by both specific myogenic and neural transcription factors (TFs), including MYOD1, PRDM12, and POU4F1 (BRN3A). Our single-cell analysis shows that these TFs are specifically co-expressed with P3F from the earliest stages of transformation. Knockout of MYOD1 or POU4F1 impairs ARMS identity acquisition without preventing transformation, whereas PRDM12 loss enhances P3F expression programs and myogenic differentiation. Together, these findings underscore a previously unappreciated interplay between myogenic and neural transcriptional programs during ARMS initiation. Furthermore, these data demonstrate that P3F establishes a stable oncogenic state that persists even after removal, thus providing a framework for evaluating P3F-dependent and P3F-independent therapeutic vulnerabilities. STATEMENT OF SIGNIFICANCE: This study reveals that PAX3::FOXO1 initiates rhabdomyosarcoma by establishing myogenic and neural transcriptional programs, yet becomes dispensable for tumor maintenance, uncovering stable oncogenic states and highlighting opportunities for targeting early transforming events.

Pediatric sarcomas are a heterogeneous group of rare mesodermal malignancies. These cancers, which affect children from infancy through adolescence and young adulthood, are in general challenging to treat with currently available therapies. Biologically, many are characterized by quiet genomes, fusion oncoproteins, immune “cold” microenvironments, and vast epigenetic deregulation that contributes to diverse and complex mechanistic drivers. Multifaceted advancements in research strategies, including high-throughput screening, new model systems, surfaceome profiling, and study of oncogenic fusion condensates have led to new opportunities for understanding the biology of pediatric sarcomas. To continue to make progress for these difficult to treat cancers, it will be critical to continue to improve access to bioinformatic data, approach patient care using innovative clinical trial frameworks, and foster interdisciplinary partnerships among medicinal chemists, scientists, clinicians, advocates, and industry partners.

The third in a series of Paediatric Therapeutic Development Workshops focused on rhabdomyosarcoma. Rhabdomyosarcoma is the most common soft tissue sarcoma in children, with 90% survival for those with the lowest risk disease, but just 20-30% in children with metastatic disease. An urgent unmet need exists to develop targeted therapeutics for high-risk disease and to reduce the toxicity of treatment. The results of trials of CAR T-cells and ADCs against FGFR4 are awaited with great interest, and developing a FGFR4 degrader is a priority. Directly targeting PAX3::FOXO1 and PAX7::FOXO1 fusion proteins is a high priority. An in vivo study of a MYOD1 degrader approach is required prior to clinical development. Degraders of P300/CBP should be evaluated preclinically with a view to clinical investigation. ROR2 is an interesting target for the L122R mutant MYOD1 rhabdomyosarcoma. Development of a TEAD degrader is a high priority, and this should be evaluated in combination with a Notch inhibitor. Considering targets with existing clinical agents, antibody-drug conjugates targeting cell-surface antigen B7-H3/CD276 are showing preclinical promise in other paediatric cancers and are also deemed a high priority for evaluation in rhabdomyosarcoma. Based on currently available evidence, MEK inhibitors should be evaluated, potentially with BRAF or PI3K inhibitors, in combination with chemotherapy in the maintenance setting. Understanding the mechanism of action underpinning drug combinations, gaining access to therapeutics and optimising clinical trial design will be essential to enable combinatorial testing in patients.

Despite comprehensive and multi-modal therapy, outcomes for children and adolescents with rhabdomyosarcoma (RMS) have plateaued over the past four decades. This is not for a lack of progress in the basic and translational studies of RMS. Indeed, advances in animal models and/or patient tissue sample acquisition and analysis have improved our understanding of RMS biology. Large-scale sequencing efforts have generated transcriptomic, genomic, and epigenomic datasets that highlight the heterogeneity of RMS and have the potential to improve prognostication and the application of precision medicine in patients with RMS. However, few of these discoveries have been clinically translated, and limitations to the accessibility, uniformity, and application of these new models and datasets hinder their utility. Here, we discuss how advances in understanding RMS biology, optimization of preclinical models, and strategies for translating basic science discoveries to the clinic can potentially improve outcomes for patients with RMS.

Rhabdomyosarcoma (RMS) is a pediatric embryonal solid tumor and the most common pediatric soft tissue sarcoma. The histology and transcriptome of RMS resemble skeletal muscle progenitor cells that have failed to terminally differentiate. Thus, RMS is typically thought to arise from corrupted skeletal muscle progenitor cells during development. However, RMS can occur in body regions devoid of skeletal muscle, suggesting the potential for nonmyogenic cells of origin. Here, we discuss the interplay between RMS driver mutations and cell(s) of origin with an emphasis on driving location specificity. Additionally, we discuss the mechanisms governing RMS transformation events and tumor heterogeneity through the lens of transcriptional networks and epigenetic control. Finally, we reimagine Waddington's developmental landscape to include a plane of transformation connecting distinct lineage landscapes to more accurately reflect the phenomena observed in pediatric cancers.

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.