Unraveling the Function of PAX3-FOXO1’s Activation Domain in Rhabdomyosarcoma
Rhabdomyosarcoma is a childhood cancer that often resurfaces after chemotherapy, making it a challenging disease to tackle. The main culprit is a fusion protein called PAX3-FOXO1 (P3F), which causes the cancer by meddling with our DNA. Imagine this protein as a small machine that tweaks the opening, closing and 3D folding of the DNA instructions in our cells, leading to the growth of cancer. One part of the machine binds DNA, but little is known about the other half of the machine. Our recent research has discovered some new features in this non-DNA binding half, in particular a small 'hook' in an otherwise very floppy part of the P3F protein. We believe that this 'hook' acts like a magnet, pulling in other proteins and molecular tools necessary for P3F to modify DNA.
Two proteins, CBP and p300, are particularly attracted to this 'hook'. These proteins are known for their roles in changing the structure of DNA, which can switch genes on or off. This process might be how the cancer starts. Our latest findings suggest that when we block CBP and p300, we can stop the P3F protein from causing these harmful changes.
Project Goals:
Armed with this knowledge, our project aims to dig deeper into the function of the 'hook' in P3F and its interaction with CBP and p300. Our first goal is to understand how this 'hook' drives the changes in DNA, leading to cancer. We will use advanced techniques to identify the other proteins that interact with the 'hook', and to measure the effects of disrupting the 'hook' on P3F's function. We will also study how it informs the 3D folding of DNA, an essential part of P3F's function.
Our second goal is to investigate the role of CBP and p300 in more detail. We have developed new molecular tools that can degrade these proteins, giving us a unique opportunity to understand their role in the cancer process. We aim to understand which of these proteins are essential for P3F to activate its target genes.
Through these studies, we hope to uncover the mechanisms through which P3F causes cancer. If successful, we could potentially develop drugs that target CBP and p300, effectively stopping P3F and preventing this type of cancer. This could provide a new therapeutic strategy for patients suffering from rhabdomyosarcoma.
2025 Update:
Alveolar rhabdomyosarcoma (FP-RMS) is a deadly childhood cancer driven by the fusion protein PAX3-FOXO1 (P3F), which hijacks normal gene regulation to promote tumor growth. While P3F binds to critical regulatory DNA regions, the function of its activation domain (AD) remains a mystery. Our previous work uncovered a small but essential structural feature—an “AD hook”—that enables P3F to recruit co-activators and drive cancerous gene expression.
Building on these discoveries, we have now developed a novel, highly potent CBP/p300 inhibitor that selectively shuts down P3F-driven gene programs in FP-RMS. This new molecule demonstrates improved potency, reduced toxicity, and strong repression of P3F target genes, as confirmed by RNA sequencing. Encouragingly, this inhibitor outperforms all other epigenetic drugs we have tested for FP-RMS. We are scaling up production for preclinical efficacy studies. Further, our research reveals that histone H2B lysine acetylation is more dependent on CBP/p300 than the traditionally studied H3K27ac mark, providing a new perspective on how P3F alters chromatin. We are also conducting cutting-edge transcriptional and chromatin profiling (ChRO-seq and ChIP-seq) to understand how this inhibitor disrupts P3F function at a mechanistic level. These studies will not only enhance our understanding of P3F-driven gene regulation but also pave the way for a much-needed targeted therapy for FP-RMS. By leveraging chemical biology and epigenetic insights, we aim to transform treatment options for children with this aggressive cancer.
