Schoofs, A.; Chen, J.; Abou El Asrar, R.; Ryckaert, E.; Delarbre, L.; Azfar, M.; Lu, N. G.-H.; Sukhai, A.; De Jaeger, M.; Vrijsen, S.; Chakrabarty, S.; Bejster, J.; Meeus, E.; Fayt, Y.; Vercauteren, A.; Ausloos, E.; Van den Haute, C.; Gijsbers, R.; Agostinis, P.; Verhelst, S.; Murai, N.; Eggermont, J.; van Veen, S.; Vangheluwe, P.

Cellular polyamine depletion is a promising anticancer strategy, but compensatory polyamine uptake limits efficacy when synthesis is blocked by DFMO (difluoromethylornithine), a clinically approved inhibitor of ornithine decarboxylase. The transporter and feedback mechanism driving this adaptive response have remained unclear. Despite their similar biochemical properties, we identify ATP13A3, rather than ATP13A2, as the principal DFMO-responsive polyamine importer, suggesting that these isoforms regulate distinct polyamine fluxes. Mechanistically, the polyamine sensor antizyme not only restrains polyamine biosynthesis but also selectively inhibits ATP13A3-mediated uptake, a brake that is relieved upon DFMO treatment. This regulatory circuit exposes distinct polyamine-acquisition states across cancers, defining synthesis- and/or uptake-biased subtypes that can shift during disease progression. Melanoma metastasis and vemurafenib resistance evolve toward increased ATP13A3-dependent uptake. The polyamine uptake branch controlled by ATP13A3-antizyme regulation can be pharmacologically blocked by AMXT 1501, which directly inhibits ATP13A3. Together, our findings explain DFMO adaptation through ATP13A3-antizyme control and establish ATP13A3 as a targetable node for polyamine depletion strategies in multiple cancers, supporting ongoing clinical evaluation of combined DFMO/AMXT 1501 therapy.