Integrated stress response activation induced by usnic acid alleviates BCL-2 inhibitor ABT-199 resistance in acute myeloid leukemia

Abstract

The cotreatment of ABT-199 and usnic acid induced ISR activation through HRI with increased expression levels of the transcription factors ATF4, CHOP, and the pro-apoptotic protein NOXA, and the downregulation of anti-apoptotic protein MCL-1 through proteasomal degradation. The increase in the NOXA levels coupled with the reduction in MCL-1 levels caused by combined therapy, is most likely correlated with the recovery of sensitivity recovery in ABT-199-resistant cells. • Acquired resistance to BCL-2 inhibitor (ABT-199) in AML remains a critical clinical challenge. • Usnic acid at low toxicity dose synergistically anti-AML with ABT-199 both in in vitro and in vivo. • MCL-1 protein strongly degraded with the co-treatment of usnic acid and ABT-199. • The deduced MCL-1 may triggered by the activation of integrated stress response induced by usnic acid. • HRI kinase activation plays a critical role in the process of ISR induced MCL-1 degradation. ABT-199 (venetoclax) is a BCL-2 suppressor with pronounced effects on acute myeloid leukemia (AML). However, its usefulness as a monotherapy or in combination with hypomethylating medicines like azacitidine is debatable due to acquired resistance. Usnic acid, a dibenzofuran extracted from lichen Usnea diffracta Vain, exhibits anticancer properties and may counteract multidrug resistance in leukemia cells. This study investigated whether usnic acid at low-cytotoxicity level could enhance sensitivity of AML cells with acquired resistance to ABT-199 by targeting the integrated stress response pathways. To investigate the combined effects on AML cells, we used a cell viability test, flow cytometry to quantify apoptosis, cell cycle analysis, and mitochondrial membrane potential measurement. RNA-seq and immunoblot were used to determine the potential mechanisms of ABT-199 + usnic acid combination. Usnic acid, at a low cytotoxicity level, successfully restored ABT-199 sensitivity in AML cell lines that had developed ABT-199 resistance and increased ABT-199′s antileukemic activity in a xenograft model. Mechanistically, the combination of usnic acid and ABT-199 cooperated to boost the expression of the integrated stress response (ISR)-associated genes ATF4, CHOP, and NOXA through the heme-regulated inhibitor kinase (HRI), while also promoting the degradation of the anti-apoptotic protein MCL-1. ISRIB, a compound that blocks the ISR, was able to reverse the growth suppression and cell death, the increase in expression of genes related with the ISR, and the inhibition of MCL-1 protein caused by combination therapy. Additionally, the downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome. In summary, usnic acid improves chemosensitivity to ABT-199 by triggering the integrated stress response, leading to decreased levels of MCL-1 protein, suggesting a potential treatment for AML cases resistant to Bcl-2 inhibitors.

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