Earlier reports showed that hyperplasia of sympathoadrenal cell precursors during embryogenesis in Nf1-deficient mice is independent of Nf1’s role in down-modulating RAS-MAPK signaling. We demonstrate in zebrafish that nf1 loss leads to aberrant activation of RAS signaling in MYCN-induced neuroblastomas that arise in these precursors, and that the GTPase-activating protein (GAP)-related domain (GRD) is sufficient to suppress the acceleration of neuroblastoma in nf1-deficient fish, but not the hypertrophy of sympathoadrenal cells in nf1 mutant embryos. Thus, even though neuroblastoma is a classical “developmental tumor”, NF1 relies on a very different mechanism to suppress malignant transformation than it does to modulate normal neural crest cell growth. We also show marked synergy in tumor cell killing between MEK inhibitors (trametinib) and retinoids (isotretinoin) in primary nf1a-/- zebrafish neuroblastomas. Thus, our model system has considerable translational potential for investigating new strategies to improve the treatment of very high-risk neuroblastomas with aberrant RAS-MAPK activation.

Transcriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms. This protocol combines rapid degradation of endogenous transcription factors with nascent transcript analysis to define the earliest, and likely direct, regulatory targets of transcription factors.

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma.

Background: Gene expression networks are perturbed in every human malignancy. One common way that this occurs is through chromosomal translocations such as the the t(2;13)(q35;q14), which creates the oncogenic transcription factor, PAX3-FOXO1, in alveolar rhabdomyosarcoma (aRMS). Identification of the direct transcriptional targets and mechanisms of control by translocated transcription factors is critical to our understanding of disease etiology and the development of targeted therapeutic approaches. However, defining the genes directly controlled by DNA binding proteins and the mechanisms of regulation at those direct target genes is challenging, because traditional genetic approaches to inactivate transcription factors cannot distinguish direct from indirect changes in gene expression. Methods: A CRISPR-based approach was used to engineer the endogenous PAX3-FOXO1 locus to incorporate the FKBP12F36V degron tag. This allowed degradation of PAX3-FOXO1 within 2 hr of treatment with the proteolysis targeting chimera, dTAG-47. We performed short time courses to analyze nascent transcription (precision nuclear run-on sequencing; PRO-seq), chromatin accessibility (ATAC-seq), and genome-wide mapping of transcriptional complexes (CUT&RUN). This identified a small cohort of high-confidence PAX3-FOXO1 gene targets and associated PAX3-FOXO1-regulated enhancer elements. In addition, we used CRISPR editing to add APEX2 and 3XFLAG tags to PAX3-FOXO1 to enable deep proteomics to identify PAX3-FOXO1-associated transcriptional complexes in aRMS cell lines. Results: Degradation of PAX3-FOXO1-FKBP12F36V led to growth inhibition, hallmarks of myogenic differentiation, and reduced growth in soft agar. PAX3-FOXO1 binding was detected at over 44,000 sites throughout the genome using CUT&RUN, and loss of chromatin bound PAX3-FOXO1 was observed within 2-4hr of PAX3-FOXO1 degradation. PRO-seq analysis defined a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Unexpectedly, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. Deep proteomic analysis showed that PAX3-FOXO1 was in close proximity to many transcriptional regulatory complexes, but appeared to coordinate the activity of these complexes at only a few hundred sites. Moreover, the continued presence of PAX3-FOXO1 at these elements was required to maintain chromatin accessibility. Conclusion: This work demonstrates the utility of using rapid degradation of endogenous transcription factors to define their direct transcription targets and the mechanisms by which these factors control gene expression. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network, and emphasizes the utility of PAX3-FOXO1 as a therapeutic target in aRMS. Citation Format: Susu Zhang, Jing Wang, Qi Liu, Hayes McDonald, Monica Bomber, Hillary Layden, Jacob Ellis, Scott Borinstein, Scott Hiebert, Kristy Stengel. Pax3-foxo1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2362.