Ultra-low-dose same-target combinations control drug resistance across stages

The emergence of drug resistance continues to limit the long term effectiveness of cancer therapies [1], as tumour cells adapt to therapeutic selective pressure through diverse genetic and non-genetic routes [2,3]. Despite advances in more potent and next generation inhibitors and mechanism based combination therapies [4,5], durable disease control remains difficult to achieve. Here we tested an alternative strategy: combining multiple inhibitors directed against the same oncogenic driver, each at ultra low doses with minimal individual activity, to delay resistance emergence. In epidermal growth factor receptor (EGFR) driven cancer models, such ultra low dose same target combinations (ULDCs) distributed individually weak perturbations across multiple inhibitors directed against the same target and achieved substantial growth inhibition in vitro and antitumour activity in vivo despite weak effects of the corresponding single agents. Under similar levels of growth inhibition (~50%), single-agent EGFR inhibitors generated resistant outgrowth, whereas a five drug ULDC delayed resistance emergence. Increasing the number of inhibitors further delayed resistance emergence, with no detectable resistant outgrowth observed for combinations containing 8 to 14 inhibitors within the experimental timeframe. The five drug ULDC also retained measurable growth inhibitory activity against gefitinib resistant PC-9/GR cells, and resistant cells arising under five drug ULDC treatment exhibited markedly greater reversibility following drug withdrawal than those arising under single agent treatment. Although pharmacological additivity or synergy may contribute to the collective growth inhibitory effects of ULDCs, the divergent long term resistance outcomes observed under similar levels of growth inhibition indicate that resistance behaviour is not determined by inhibitory strength alone. Instead, our findings support a model in which increasing same target inhibitor diversity progressively constrains the adaptive trajectories available during resistance evolution, while therapeutic perturbation architecture may also influence the stability of resistant states that emerge. More broadly, these findings support the concept of same target collective suppression, in which multiple individually weak perturbations directed against the same target can collectively produce substantial antitumour activity and constrain resistance evolution across multiple stages. We propose that increasing same target inhibitor diversity may restrict accessible adaptive trajectories, while therapeutic perturbation architecture may also influence the stability of resistant states.