Background Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity, and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell death that is caused by oxidative stress through excess levels of iron-dependent peroxidation of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain to be elucidated. Methods We used quantitative single-cell imaging of fluorescent metabolic probes, transcriptomics, proteomics, and lipidomics to perform a longitudinal analysis of the adaptive response to androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate cancer (PCa). Results We discovered that cessation of cell proliferation and a robust reduction in bioenergetic processes were associated with multidrug tolerance and a strong accumulation of lipids. The gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid remodeling of all major phospholipid classes at the expense of storage lipids, leading to increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR and fatty acid desaturation, lipase activities, or growth medium supplementation with antioxidants or PUFAs altered GPX4 dependence. Conclusions Our work provides mechanistic insight into processes of lipid metabolism that underpin the acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard of care therapies in PCa. It demonstrates novel strategies to suppress the therapy-tolerant state that may have potential to delay and combat resistance to androgen receptor-targeted therapies, a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an adaptive phenotype shared by several types of cancer in response to different therapies, our work might have universal implications for our understanding of metabolic events that underpin resistance to cancer therapies.

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29Background 30 Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity and 31 multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, 32 therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell 33 death that is caused by oxidative stress through excess levels of iron-dependent peroxidation 34 of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced 35 ferroptosis hypersensitivity remain to be elucidated. 37Methods 38 We used quantitative single cell imaging of fluorescent metabolic probes, transcriptomics, 39 proteomics and lipidomics to perform a longitudinal analysis of the adaptive response to 40 androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate 41 cancer (PCa). 43Results 44 We discovered that cessation of cell proliferation and a robust reduction in bioenergetic 45 processes were associated with multidrug tolerance and a strong accumulation of lipids. The 46 gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective 47 (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), 48 whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid 49 remodeling of all major phospholipid classes at the expense of storage lipids, leading to 50 increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA 51 levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to 52 glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR 53 and fatty acid desaturation, lipase activities or growth medium supplementation with 54 antioxidants or PUFAs altered GPX4 dependence. Despite multidrug tolerance, PCa cells 55 displayed an enhanced sensitivity to inhibition of lysosomal processing of exogenous lipids, 56 highlighting an increased dependence on lipid uptake in the therapy-tolerant state. 57 58 Conclusions 59 Our work provides mechanistic insight into processes of lipid metabolism that underpin the 60 acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard 61 of care therapies in PCa. It demonstrated novel strategies to suppress the therapy-tolerant state 62 that may have potential to delay and combat resistance to androgen receptor-targeted therapies, 63 a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an 64 adaptive phenotype shared by several types of cancer in response to different therapies, our 65 work might have universal implications for our understanding of metabolic events that 66 underpin resistance to cancer therapies. 67 68 69 Background 74 Despite significant advancements in detection and treatment over the past decades, prostate 75 cancer (PCa) remains the second most commonly diagnosed cancer among men and the third 76 leading cause of cancer mortality in men worldwide [...