# AICAr Inhibition of cardiomyocyte autophagy promotes p62-dependent NRF2 expression and protection against doxorubicin toxicity

## TL;DR

AICAr protects heart cells from doxorubicin toxicity by boosting NRF2 through p62, but not through AMPK or autophagy.

## Contribution

AICAr's AMPK-independent protection against doxorubicin is revealed to depend on p62 and NRF2, not autophagy.

## Key findings

- AICAr inhibits autophagy in heart cells via ADK, not AMPK.
- AICAr increases p62 and NRF2, reducing doxorubicin-induced cell death.
- Breast cancer cells with low ADK are not protected by AICAr and are more sensitive to doxorubicin.

## Abstract

Doxorubicin is an effective cancer chemotherapeutic, but its use is complicated by cardiotoxic side-effects. 5-amino-4-imidazolecarboxamide ribonucleoside (AICAr) is a widely used pharmacological activator of adenosine monophosphate-activated kinase (AMPK), but also exerts AMPK-independent actions that may have unrealized therapeutic potential. Here, we identified a novel mechanism by which pretreatment with AICAr protects neonatal rat cardiomyocytes against doxorubicin toxicity. Despite increasing AMPKThr172 and ULK1Ser555 phosphorylation, AICAr suppressed cardiomyocyte LC3 lipidation and caused accumulation of the autophagy receptor, p62 SQST1, through an adenosine kinase (ADK)-dependent, AMPK-independent mechanism. The accumulation of p62 was associated with increased expression and transcriptional activity of NRF2, as well as decreased doxorubicin-induced reactive oxygen species and cell death. Notably, AICAr-induced increase of NRF2, antioxidant gene expression, and doxorubicin resistance were blocked by RNAi depletion of p62, indicating that the protective effects of AICAr rely upon the secondary effects of autophagy inhibition, rather than autophagy inhibition per se. While doxorubicin treatment alone did not affect cardiomyocyte LC3 lipidation, it did significantly decrease p62 levels and diminish NRF2 nuclear localization. Pretreatment with AICAr to provide surplus p62 and nuclear NRF2 diminished the impact of these doxorubicin effects. Importantly, MCF7 breast cancer cells, which poorly express ADK, were not protected by AICAr pretreatment and instead were sensitized to doxorubicin-induced cell death. These findings raise the possibility that differences in ADK expression between cardiomyocytes and breast cancer cells might be exploited by pretreatment with AICAr or similar ADK-dependent drugs to provide dual benefits in doxorubicin therapy.

The online version contains supplementary material available at 10.1007/s00018-025-05905-4.