TKI-Tolerant Persisters Emerge from a PKCα-Dependent and Highly Plastic Subpopulation of Stem-Like Cells in NSCLC

Reversible drug-tolerant persister states are emerging as key drivers of limited therapeutic durability, offering a complementary non-genetic perspective distinct from traditional models of acquired resistance. This is of particular interest in lung adenocarcinoma where EGFR tyrosine kinase inhibitors (TKIs) elicit dramatic responses, yet residual surviving cells persist and ultimately seed relapse. To define mechanisms that enable survival during this earliest residual-disease phase, we focused on the drug-tolerant persister population that remains after EGFR TKI exposure and can later give rise to outgrowth. Initial observations of elevated transcript levels of PRKCA , which encodes PKCα, in established TKI-resistant models, together with markedly delayed tumor relapse following PKCα suppression in vivo, nominated PKCα as a candidate regulator of the persister-to-relapse transition. Genetic ablation of PRKCA or its inhibition with enzastaurin reduced residual survival and outgrowth after TKI exposure, indicating that PKCα functions as an early dependency of drug-tolerant persisters rather than as a general mediator of acquired resistance. Mechanistically, PKCα was required for persister-associated EMT, migratory capacity, and robust induction of ALDH1A1, the latter constraining oxidative stress and enhancing persister survival. Functionally, PKCα was specifically necessary for survival of a rare, pre-existing CD44 ^High stem-like subpopulation that exhibited marked plasticity and ultimately seeded persistence. Together, these data identify a PKCα-dependent EMT/stemness/ROS pathway as a critical survival program in EGFR TKI-tolerant persister cells and support therapeutic strategies aimed at eliminating residual disease to prolong clinical responses.