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.

Abstract Tumor growth and relapse are often driven by cancer stem cells, but self-renewal mechanisms and genetic mutations that elevate their number are not fully understood. Here, we show that recurrent L122R mutation (Leucine to Argine change at amino acid 122) in the DNA-binding site of the Myogenic Differentiation 1 (MYOD1) transcription factor increases cancer stem cell frequency in aggressive Spindle cell/sclerosing rhabdomyosarcoma. MYOD1 L122R also makes tumors resistant to chemotherapy and radiation. Epigenetic analysis reveals that MYOD1 L122R binds MYC-like DNA recognition motifs to activate stem cell programs, while retaining some wild-type function to regulate muscle pathways that drive transformation. Mechanistically, MYOD1 L122R transcriptionally activates Receptor tyrosine kinase-like Orphan Receptor 2 (ROR2) to turn on the non-canonical Wingless/Integrated (WNT) planar cell polarity pathway to increase both cancer stemness and therapy resistance. Targeting ROR2 with antibody-drug conjugates kills MYOD1 L122R -mutated tumor cells, offering therapeutic opportunities. These findings provide insights into how MYOD1 L122R rewires rhabdomyosarcoma to a stem-like state and defines a unique class of oncogenic transcription factors found in aggressive cancers.

Abstract Rhabdomyosarcoma (RMS) is a high-risk and lethal pediatric sarcoma that resembles developing skeletal muscle. RMS tumors have low mutation burdens, but these scant mutations often alter genes involved in transcriptional control. Transcriptional dysregulation is critical to RMS pathogenesis, supported by studies in both RMS tumors carrying mutationally derived chimeric transcription factors (“fusion positive (FP)”), or those without (“fusion negative” (FN)). However, mechanisms to selectively target dysregulated transcription in RMS remain outstanding. Here, we develop a novel approach targeting RMS transcription comprising simultaneous targeting of two distinctly acting transcriptional co-activators. We discover a common cell identity-controlling pan-RMS core regulatory circuit (CRC) composed of oncogenic and lineage-specific myogenic master transcription factors (mTFs). These mTFs are regulated by super-enhancers, and they co-bind genome-wide to control the malignant transcriptome of both FP- and FN-RMS. Using a super-enhancer-based reporter screen, we identify the EP300/CBP inhibitor A485 as a potent inhibitor of the pan-RMS CRC, though efficacy of this compound was limited by toxicity. To enhance on-target specificity, we identify the protein EYA2 as a co-factor that binds directly to SIX1, a member of the pan-RMS CRC and exploit a recently developed second-generation EYA1/2 tyrosine phosphatase inhibitor, LG1-34, to inactivate its function. While A485 and LG1-34 independently reduce mTF transcription and drive RMS cell death, in combination, these agents function synergistically to reduce RMS growth in vitro and in vivo. These results demonstrate that combined targeting of enhancer maintenance and CRC cofactors is a powerful strategy to suppress the RMS transcriptome and enforce RMS cell death. Citation Format: Annika Gustafson, Stephanie Nance, Berkley Gryder, Noha A. Shendy, Lars Wick, Grace McKay-Corkum, K. Elaine Ritter, Stephen Connor Purdy, Arthur R. Wolin, Sheera R. Rosenbaum, Sabateeshan Mathavarajah, Nickerson A. Demelfi, Yueyang Wang, Yang Zhang, Mark W. W. Zimmerman, Anoop M. Kavirayani, John Hardin, Alexander LaVeck, Xiang Wang, Neekesh V. Dharia, Andrew Hong, Guillaume Kugener, Jesse S. Boehm, Jennifer Roth, Javed Khan, Francisca Vasquez, Kristin B. Artinger, Rui Zhao, David M. Langenau, Jun Qi, Kimberly Stegmaier, Heide L. Ford, Adam D. Durbin, Brian J. Abraham. Synergistic targeting of EP300/CBP and EYA co-activators collapses the rhabdomyosarcoma core regulatory circuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3500.

A small, unassuming, fleshy lobe at the base of the pectoral fin of fish has long been overlooked by scientists. In this issue of JEM, Castranova et al. (https://doi.org/10.1084/jem.20241435) describe this structure as a new secondary lymphoid organ. Translucent and externally located, the axillary lymphoid organ (ALO) shares striking structural similarities with mammalian secondary lymphoid organs. Due to its position and optical accessibility, the zebrafish ALO permitted noninvasive, high-resolution imaging of immune cell dynamics in live animals. These studies revealed likely interactions between T, B, and macrophage cells, arguing that the ALO may function in adaptive immune cell activation and provide a nexus for immune cell trafficking and communication.

The complex heterogeneity of Acute lymphoblastic leukemia (ALL) often predicts poor prognosis, high morbidity, and drug resistance. Yet, the molecular drivers that initiate aggressive ALL have yet to be fully elucidated, especially in the context of the >30 subtypes of human T- and B-ALL. Here, we used a large-scale F0 transgenic screen in zebrafish to identify synergistic combinations of 65 putative oncogenes at inducing both T- and B-cell leukemia. This approach identified a new proto-oncogene SET that collaborates with both notch1a icn and mutationally activated Interleukin 7 Receptor (IL7R) to initiate aggressive leukemia in vivo. SET is a multifunctional protein that can act as a transcriptional regulator, a histone chaperone, and an inhibitor of protein phosphatase 2A (PP2A). The high expression of SET along with mutational activation of NOTCH1 and IL7R was also found in human B- and T-ALL, supporting their oncogenic roles in leukemogenesis. Our discovery that SET is capable of independently synergizing with known drivers to initiate ALL presents a unique opportunity to explore the nature of oncogene driven ALL and define the mechanisms by which it drives transformation. We next used Non-negative Matrix Factorization to unbiasedly identify six gene programs shared across the zebrafish ALL subtypes and assessed if each was predictive of outcome in human disease. One program comprised a MYC-driven transcription signature that unexpectedly predicted good outcomes in human T-ALL patients. Additional conserved programs independently stratified patients into poor outcomes and when combined with the MYC signature profile were able to better discern disease outcomes in patients, including the aggressive ETP-like and TAL1 DP-like T-ALL. Taken together, we have uncovered new roles for SET in driving ALL initiation, uncovered unexpected high MYC expression as predictive of good outcome, and defined a new combination biomarker gene signature that predicts poor overall survival in aggressive subtypes of human T-ALL. Our zebrafish-based screening approach is a powerful tool for comparative genomic studies to identify new oncogenic drivers, vulnerability pathways, and new biomarkers of aggression in ALL. James R Allen, Luis Antonio Corchete Sanchez, Mohamed N Bakr, Miriam Fernández-Lajarín, Alexandra Hazelwood, Nathan Ford, Anna M Lucianò, Alexandra Veloso, Alexander D Weissman, Olivia A Strom, Esther Rheinbay, David M Langenau. Zebrafish modeling predicts clinical outcomes in human acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B035.

Fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood muscle cancer where relapse and refractory disease leads to poor outcomes. Our group has recently shown that FN-RMS is a cancer stem cell (CSC) disease and that these CD44+/CD90+/EGFR+ cells likely contribute to therapy-resistance, yet the precise mechanisms governing self-renewal and tumor regrowth after stress are not known. To better visualize and describe CSCs in the context of therapy response, we developed a fluorescent reporter using the EGFR promoter to drive expression of a photoconvertible-nuclear localized protein (H2B-Dendra) that specifically marks FN-RMS CSCs. Photoconversion and live cell imaging confirmed that the CSCs were largely quiescent under normal growth conditions. Yet, following stress, CSCs could re-enter the cell cycle and self-renew. Moreover, a single dose of chemotherapy was also able to elevate the overall number of CSCs by (1) inducing cell cycle re-entry and (2) by direct conversion of differentiated cells into CSCs. This latter phenomenon was completely unexpected and suggested that CSC hierarches are far less rigid than previously suggested. Indeed, time-lapse imaging verified direct reprograming of differentiated cells into CSCs. Mechanistically, we identified interferon-b (IFN-b) as the secreted factor emanating from damaged cells to directly reprogram cells into therapy-resistant CSCs. Indeed, the CGAS-STING-IFN pathway drove local paracrine-mediated reprogramming of CSCs and small molecule pathway inhibitors could sensitize FN-RMS to chemotherapy. Taken together, these findings reveal that FN-RMS utilizes paracrine expressed type I interferons to elevate the CSC population in response to therapy and suggests a far less rigid hierarchy of cell states in FN-RMS. Sabateeshan Mathavarajah, Tiffany C Eng, Anna Luciano, Elisa J Quantin, Devika D Kannambadi, Shuze Wang, Alexander D Weissman, Jihee Lee, Yueyang Wang, Yun Wei, Sara G Danielli, Michael A Marconi, David M Langenau. Therapy-associated paracrine signaling alters the cell states in Rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B007.

Olaparib and temozolomide (OT) combination therapy is in clinical trial evaluation for rhabdomyosarcoma (RMS). Unfortunately, OT resistance has been reported in other cancers. Using preclinical mouse xenograft experiments, we show that OT effectively suppresses RMS growth, yet over half of RMS tumors develop resistance associated with transcriptomic changes that occur in the absence of recurrent genomic mutation. Importantly, most resistant RMS models upregulate the PIK3CA/AKT pathway, activating NRF2 phosphorylation and subsequent transcriptional expression of multidrug resistance ABC transporters. PIK3CA inhibitor alpelisib re-sensitizes resistant cells to OT by suppressing expression of ABC transporters. The combination of OT + alpelisib also kills RMS cells which are resistant to standard-of-care combination chemotherapy and was effective in preclinical xenograft mouse models at curbing tumor growth. Our work defines a common resistance pathway in RMS and has credentialled PIK3CA/AKT inhibition as a preclinical strategy to kill therapy resistant RMS.

CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3K-AKT-mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood or adolescents. The spindle sclerosing (ss-RMS) subtype has the worst outcome of all RMS patients and often harbor recurrent mutation of Leucine 122 to Arginine within the DNA binding domain of the transcription factor MYOD1. To date, the molecular function of MYOD1L122Rremains largely unknown. Methods: Here, we create the first animal model of MYOD1L122R using transgenic zebrafish. We also used CRISPR-homology-directed-repair and dox-inducible systems to express MYOD1L122R in human RMS cells. These models, along with patient tumors and patient-derived xenografts, were assessed by RNA- and ChIP-sequencing, single-cell RNA and ATAC (assay for transposase-accessible chromatin) sequencing, and immunohistochemistry to identify direct targets and downstream pathways regulated by MYOD1L122R. We also discovered that MYOD1L122R activates non-canonical WNT signaling to elevate cancer stemness and to impart chemo- and radiation-resistance through molecular mechanism studies. Results: Utilizing transgenic zebrafish models, we uncovered that MYOD1L122R is not oncogenic by itself but requires RAS pathway activation to generate rhabdomyosarcoma, akin to known gene associations found in ss-RMS patients. Limiting dilution cell transplantation revealed that MYOD1L122R increased the numbers of tumor propagating cells in zebrafish tumors. We next showed that engineered MYOD1L122R human tumors also have increased numbers of TPCs and were refractory to standard of care VAC (vincristine, actinomycin, and cyclophosphamide) and gamma-irradiation. Indeed, scRNA- and scATAC-seq of patient and patient-derived xenografts confirmed the elevation of cancer stem cell programs within MYOD1L122R tumors. We next identified the shared and differential genomic regions bound by MYOD1L122R using whole-genome ChIP and RNA sequencing. Most notably, MYOD1L122R bound to new DNA binding motifs shared with the MYC oncoprotein and transcriptionally upregulated cancer stem cell programs through distal enhancers. Unexpectedly, MYOD1L122R also bound to shared genomic regions with normal MYOD1 at transcription start sites/promoters to regulate muscle specific gene expression. Mechanistic studies revealed that MYOD1L122R binds to regulatory elements of ROR2 (receptor tyrosine kinase like orphan receptor 2), activating its expression and turning on the non-canonical WNT11-ROR2-VANGL2 pathway to elevate cancer stemness and to drive chemo- and radiation- resistance. Conclusions: MYOD1L122R is an RMS modifying gene that elevates cancer stemness and promotes chemo- and radiation- resistance through epigenetic reprogramming and activation of non-canonical WNT signaling pathway. The discovery of ROR2 in MYOD1L122R RMS patients grants future evaluation of this protein as a therapeutic target in combination with current standard of care. Citation Format: Yun Wei, Luis Antonio Corchete Sanchez, Diego Antelo, Sabateeshan Mathavarajah, Shuze Wang, Alexander Daiki Weissman, Jihee Lee, Miguel N. Rivera, Esther Rheinbay, David M. Langenau. MYOD1L122R induces chemoresistance and elevates cancer stem cell programs through WNT11-ROR2-VANGL2 signaling in aggressive rhabdomyosarcoma [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 PR012.

Cancers exploit coinhibitory receptors on T cells to escape tumor immunity, and targeting such mechanisms has shown remarkable clinical benefit, but in a limited subset of patients. We hypothesized that cancer cells mimic noncanonical mechanisms of early development such as axon guidance pathways to evade T cell immunity. Using gain-of-function genetic screens, we profiled axon guidance proteins on human T cells and their cognate ligands and identified fibronectin leucine-rich transmembrane protein 3 (FLRT3) as a ligand that inhibits T cell activity. We demonstrated that FLRT3 inhibits T cells through UNC5B, an axon guidance receptor that is up-regulated on activated human T cells. FLRT3 expressed in human cancers favored tumor growth and inhibited CAR-T and BiTE + T cell killing and infiltration in humanized cancer models. An FLRT3 monoclonal antibody that blocked FLRT3-UNC5B interactions reversed these effects in an immune-dependent manner. This study supports the concept that axon guidance proteins mimic T cell checkpoints and can be targeted for cancer immunotherapy.

Innovative treatment options have greatly improved outcomes for acute lymphoblastic leukemia (ALL) patients over several decades, yet relapse and refractory disease remain a significant clinical challenge. The complex genomic heterogeneity of molecular subtypes contributes to poor prognosis, high morbidity, and recurrent drug resistance in relapse and refractory ALL patients. Identifying novel genetic interactions and molecular pathways capable of initiating ALL will help to develop new diagnostic criteria and to identify actionable drug targets. Here, we developed a novel in vivo screening method using transgenic zebrafish to unbiasedly identify collaborating oncogenic drivers that when co-expressed lead to T- and B-ALL. Our high-throughput approach allows for rapid screening of collaborating oncogenic drivers across hundreds of individual animals in a cost-effective manner. Specifically, we screened a transgene pool of 68 putative oncogenes identified from relapsed human ALL for synergies at inducing leukemia in a large-scale F0 transgenic screen. Using our approach, complex gene networks involved in cancer initiation in human patients can be rapidly interrogated and unraveled with in vivo resolution utilizing the zebrafish model. Low concentrations of transgene pools containing candidate cDNAs driven by a tissue specific immune cell promoter were co-injected into zebrafish along with a fluorescent reporter (rag2:mCherry). Animals that developed tumors express mCherry along with only 5 to 20 additional transgenes (median 15 genes), indicative of random concatemeric integration of transgenes into the genome. Using histopathologic analysis and bulk mRNA sequencing, we identified zebrafish with mCherry-labeled thymic T cell lymphomas, T-ALL, and B-ALL ((>140 animals). Analysis of transgene expression from bulk-RNA sequencing studies allowed us to nominate causative combinations of human transgenes that drive leukemia onset in the zebrafish. Most notably, we identified several unique leukemia and lymphoma transgenic zebrafish models, representing T-ALL, B-ALL, mixed-phenotype acute leukemia (MPAL), T- and B-cell lymphoma, and NK cell leukemia. Our analysis also elucidated novel synergies between the proto-oncogene SET and either activated NOTCH1 or IL7R mutations to initiate ALL. To verify nominated collaborating oncogenic drivers as causative at inducing cancer, we next performed microinjection validation studies with: 1) rag2:SET + rag2:intracellular notch1a + rag2:mCherry (n=7, mean latency 105±16 days) and 2) rag2:SET + rag2:mutationally-activated IL7R + rag2:mCherry (n=5, mean latency 120±25 days). Both combinations lead to robust ALL formation in zebrafish, validating the oncogenic role of these factors in leukemia initiation. Importantly, lymphoma and leukemia were not detected in animals injected with rag2:SET (n=19, >180 days followed), or rag2:intracellular notch1a (n=31, >180 days followed) or rag2:mutationally-activated IL7R (n=18; >180 days followed). SET is a multifunctional protein involved in transcriptional regulation and histone binding. While SET has been characterized in other cancers, we found that SET collaborates with known drivers to initiate T-ALL in vivo and is also highly expressed in a vast majority of human T-ALL. SET is a potent inhibitor of H3 acetylation, suggesting that epigenetic dysregulation has a critical role in ALL oncogenic transformation. Finally, to determine whether SET is also required for human ALL maintenance, we established doxycycline inducible shRNA human ALL lines (lentiviral pINDUCER10; PMID: 21307310). We found that induced knockdown of SET inhibited cell proliferation and viability in multiple T-ALL and B-ALL cell lines while also increasing apoptotic cell death and inhibiting cell cycle progression. Taken together, our work has identified a unique role for a SET in ALL initiation and maintenance. We have generated a robust transcriptome atlas of zebrafish lymphoid malignancies, representing novel transgenic zebrafish models of several acute lymphoblastic leukemia and lymphoma molecular subtypes. In addition, our unique screening approach can be further applied towards discovery of genetic factors capable of initiating pediatric leukemias, creating a molecular “roadmap” to leukemogenesis, and identify vulnerable pathways for clinical therapies.

Rhabdomyosarcoma (RMS) is a pediatric tumor that resembles undifferentiated muscle cells; yet the extent to which cell state heterogeneity is shared with human development has not been described. Using single-cell/nucleus RNA sequencing from patient tumors, patient-derived xenografts, primary in vitro cultures, and cell lines, we identify four dominant muscle-lineage cell states: progenitor, proliferative, differentiated, and ground cells. We stratify these RMS cells/nuclei along the continuum of human muscle development and show that they share expression patterns with fetal/embryonal myogenic precursors rather than postnatal satellite cells. Fusion-negative RMS (FN-RMS) have a discrete stem cell hierarchy that recapitulates fetal muscle development and contain therapy-resistant FN-RMS progenitors that share transcriptomic similarity with bipotent skeletal mesenchymal cells. Fusion-positive RMS have tumor-acquired cells states, including a neuronal cell state, that are not found in myogenic development. This work identifies previously underappreciated cell state heterogeneity including unique treatment-resistant and tumor-acquired cell states that differ across RMS subtypes.

Abstract Olaparib and temozolomide (OT) combination therapy is in clinical trial evaluation for adolescent rhabdomyosarcoma (RMS) muscle cancers. Unfortunately, resistance to OT has been reported in other cancers, with no counterstrategies available. Using preclinical mouse xenograft experiments, we show that OT is effective at curbing RMS growth, yet a subset of tumors develop resistance that is associated with transcriptomic changes that occur in the absence of recurrent genomic mutation. Importantly, a vast majority of resistant RMS models upregulate the PIK3CA/AKT pathway, which in turn activates NRF2 transcription factor phosphorylation and subsequent transcriptional expression of multidrug resistance ABC transport proteins that rapidly efflux drugs from cells. Using dynamic real-time imaging of tumor growth and assessing drug responses at single-cell resolution in human cell line models, we found that the PIK3CA inhibitor alpelisib re-sensitized resistant RMS cells to OT therapy by suppressing the expression of ABC transport proteins. Excitingly, RMS uses the same PIK3CA/AKT pathway activation to drive resistance to standard-of-care combination therapy vincristine, actinomycin D, and cyclophosphamide (VAC) and the combination of OT + alpelisib effectively killed VAC-resistant RMS. Alpelisib also re-sensitized resistant RMS to chemotherapy in preclinical xenograft mouse models resulting in reduced tumor burden and extended disease-free survival. Our work defines a common resistance pathway in RMS and has credentialled a new preclinical strategy to kill therapy-resistant RMS. Citation Format: Yueyang Wang, Qiqi Yang, Chuan Yan, David M. Langenau. The PIK3CA/AKT pathway drives therapy resistance in rhabdomyosarcoma [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 A072.