094 - Dual Inhibition of PAX3-FOXO1 in Rhabdomyosarcoma

Publication Number: 94.4909

Rishika Mehta, The University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States; Amit J.. Sabnis, UCSF Benioff Children's Hospital San Francisco, San Francisco, CA, United States

Background: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood, and FOXO1 fusions including PAX3-FOXO1 are a major risk factor for metastasis, relapse, and death. The PAX3-FOXO1 oncoprotein transforms cells by re-organizing chromatin and driving the transcription of cancer-associated genes, including itself. To date, directly inhibiting PAX3-FOXO1 function has not been possible.

Objective: In preliminary work, our laboratory discovered that mTORC1 inhibitors capable of suppressing cap-dependent translation halts the ribosomal synthesis of PAX3-FOXO1. This project investigated the effects of combining this translational inhibition with BET inhibitors to halt the transcription of PAX3-FOXO1. Our objectives included assessing the impact of mTORC1 inhibition on PAX3-FOXO1 mRNA and protein expression, evaluating the influence of drug sequencing on PAX3-FOXO1 levels, and developing a strategy for preclinical trials combining RMC-6272 (an mTORC1 inhibitor) and BMS-986158 (a BET inhibitor).

Design/Methods: We conducted experiments in two patient-derived PAX3-FOXO1 positive cell lines, Rh 30 and Rh4, and used both qPCR and Western Blots to quantify our results. First, we analyzed the effects of RMC-6272 on PAX3-FOXO1 mRNA and protein levels over different time points. Based on the results of the first experiment, we next focused on optimizing the sequence of administering RMC-6272 and BMS-986158 to suppress PAX3-FOXO1 as combined dosing was not tolerable in murine patient-derived xenograft (PDX) models.

Results: While RMC-6272 initially reduced PAX3-FOXO1 protein levels, a rebound effect was observed. This was matched by an upregulation of mRNA levels, suggesting transcriptional mechanisms and providing rationale to study mTORC1 and BET inhibitors in combination. The second experiment of mTORC1 and BET inhibitors together revealed that the combination of RMC-6272 followed by BMS-986158 resulted in the greatest reduction in PAX3-FOXO1 levels aside from combined treatment.

Conclusion(s): Our findings highlight that while mTORC1 inhibition can suppress PAX3-FOXO1 translation, it also provokes increased transcription of PAX3-FOXO1 mRNA. The greatest suppression of PAX3-FOXO1 with the lowest toxicity and highest therapeutic effect is achieved by sequential administration of RMC-6272 followed by BMS-986158. This schema will be explored in PDX models as a prelude to clinical testing, with the ultimate goal of directly targeting PAX3-FOXO1 to improve cure rates for children with fusion positive rhabdomyosarcoma.