Novel cumate-inducible models of MYC-driven neuroblastoma enable unconfounded mitochondrial synthetic lethality screens

Oncogenic MYC transcription factors profoundly alter cellular programs, imposing dependencies that can be therapeutically exploited in MYC-driven cancers such as high-risk neuroblastoma. However, dissecting such synthetic lethal vulnerabilities using controlled, tunable gene expression within a uniform genetic background remains challenging. Widely used tetracycline-regulated systems rely on inducers known to perturb mitochondrial function, introducing significant off-target effects that may confound interpretation. To overcome this limitation, we established novel cumate (p-isopropylbenzoate)-inducible neuroblastoma models that enable physiologically unbiased regulation of MYC(N) expression. Functional validation demonstrated that cumate itself does not induce off-target effects on neuroblastoma cell viability, mitochondrial membrane potential, morphology, proteostasis, or stress signaling, even at the highest recommended dose. The developed SHEP-CuO-MYC and -MYCN models show efficient, titratable, and reversible upregulation of c-MYC and N-MYC, respectively, recapitulating the expression levels observed in MYC(N)-amplified neuroblastoma. As a proof-of-concept, we applied these models to mechanistically validate the recently proposed mitoribosomal synthetic lethality, providing fully unbiased evidence that elevated c-MYC/N-MYC levels sensitize neuroblastoma cells to inhibitors of mitochondrial gene expression. Although impairing mitochondrial translation activated mitochondrial integrated stress response in both MYC-on and MYC-off states, it led to dramatic MYC downregulation coupled with enhanced caspase-dependent cell death in MYC-on cells. These findings reveal that MYC(N) overexpression confers a selective, proliferation-independent mitochondrial vulnerability that can be therapeutically targeted by repurposing well-tolerated mitochondrial ribosome-targeting antibiotics. Collectively, our models provide a robust platform for studying the MYC-mitochondria interplay and can be directly adapted for drug repurposing screens targeting mitochondrial dependencies in neuroblastoma and, potentially, other MYC-driven tumors. HIGHLIGHTSO_LICumate shows no inducer-associated mitochondrial or cytotoxic off-target effects C_LIO_LICumate-inducible MYC models enable mechanistic studies of mitochondrial synthetic lethality C_LIO_LIMYC overexpression drives neuroblastoma sensitivity to mitochondrial translation inhibition C_LIO_LICommon ribosomal antibiotics trigger caspase-dependent cell death in MYC-driven tumor cells C_LIO_LIContext-specific MYC downregulation links mitochondrial stress to MYC synthetic lethality C_LI