Abstract 1093: Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is commonly divided into subtypes based on histopathological appearance. The oncogenic fusion proteins PAX3: : FOXO1 or PAX7: : FOXO1 are present in the alveolar subtype of RMS (ARMS). These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. These fusion proteins have been shown to drive malignant transformation in ARMS, and patients with fusion-positive ARMS have a worse prognosis than patients with fusion-negative ARMS and embryonal RMS. We recently reported that piperacetazine, a phenothiazine derivative and first-generation antipsychotic, is capable of binding to and inhibiting the transcriptional activity of PAX3: : FOXO1, reducing the expression of PAX3: : FOXO1 target genes, and inhibiting the ability of fusion-positive RMS cells to grow in soft agar. We hypothesize that other phenothiazine derivatives may have stronger binding affinity for PAX3: : FOXO1 and greater ability to reduce tumor growth in vivo. We employed a structure-activity relationship campaign to develop new phenothiazine derivatives that may be more potent inhibitors of PAX3: : FOXO1 than piperacetazine. We synthesized and tested 10 new compounds, in addition to 9 existing FDA-approved phenothiazine derivatives, for their ability to bind to PAX3: : FOXO1 and inhibit its transcriptional activity and target gene expression as well as inhibiting the growth of fusion-positive RMS cells in 3D culture. Seven derivatives inhibited PAX3: : FOXO1 reporter activity better than piperacetazine, nine of them performed worse, and three of them showed comparable activity to piperacetazine. We demonstrated that piperacetazine does not alter PAX3: : FOXO1 levels, subcellular localization, binding to a PAX3: : FOXO1 target DNA sequence, or phosphorylation at Ser256 in the FOXO1 portion of the fusion protein. We therefore hypothesize that piperacetazine may be disrupting interactions between PAX3: : FOXO1 and its partner proteins. We are employing a proximity-based biotinylation method to identify proteins that are competed away from PAX3: : FOXO1 by piperacetazine. Our data suggest that it is possible to target PAX3: : FOXO1 protein using small molecules that can directly bind to it and inhibit its activity in fusion-positive RMS cells. Citation Format: Taryn Shaw, Kay Nakazawa, Purushottam Tiwari, Eryn Nelson, Jeffrey Formen, Christian Wolf, Jeffrey Toretsky, Aykut Üren. Small molecule inhibitors of the PAX3: : FOXO1 fusion protein in rhabdomyosarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1093.