Abstract B020: PI3K-Dependent Tunnelling Nanotubes Drive Cooperative Chemotherapy Resistance in Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Current standard-of-care treatment combines vincristine, actinomycin D, and cyclophosphamide (VAC) chemotherapy with surgery and radiation. However, approximately 30% of patients develop relapse disease, which carries a dismal five-year survival rate of only 17%. Thus, new therapeutic strategies to overcome chemotherapy resistance are urgently needed. We recently identified activation of the PIK3CA/AKT signaling pathway as a key driver of chemotherapy resistance in RMS by transcriptionally upregulating ATP-binding cassette (ABC) transporters that mediate drug efflux. Using cell mixing experiments with fluorescently labelled treatment responsive and resistant cells, we found that co-culture led to the unexpected retention of parental responsive cells after therapy. High-resolution real-time imaging using fluorescently labeled drug revealed the creation of tunnelling nanotubes (TNTs) originating from resistant cells that extend and contact adjacent cells to directly facilitate drug efflux. In addition, resistant cells transferred damaged mitochondria to sensitive cells through TNTs, increasing ROS levels in sensitive cells and promoting cell survival by upregulating the ABC transport pathway. Treatment with the FDA-approved PI3K inhibitor Alpelisib inhibited TNT formation and significantly reduced both TNT-mediated drug efflux and mitochondrial transfer, confirming roles for the PI3K pathway in regulating the creation and function of TNTs. Taken together, our findings reveal a novel PIK3CA/AKT-dependent mechanism by which drug-resistant and drug-sensitive RMS cells cooperatively interact through the creation of TNTs to (i) actively remove chemotherapeutic agents from sensitive cells and (ii) transfer damaged mitochondria from resistant cells to induce ROS-mediated activation of ABC transporters within adjacent cells. These findings identify TNTs as a promising therapeutic target and support the clinical translation of PI3K inhibition in combination with existing chemotherapy to overcome drug resistance in rhabdomyosarcoma.