Integrated Stress Response Activation Induced By Usnic Acid Alleviates BCL-2 Inhibitor ABT-199 Resistance in Acute Myeloid Leukemia

Drug resistance is an inevitable issue with targeted therapeutics. A targeted BCL-2 inhibitor, Venetoclax, is approved for newly diagnosed AML cases that can't undergo rigorous chemotherapy when combined with hypomethylating agents. However, most responders eventually develop acquired resistance and experience subsequent tumor progression. Therefore, there is an urgent need for new regimens with reduced toxicity and higher efficacy. Usnic acid, a dibenzofuran compound originating from Usnea diffracta Vain, has potential anticancer effects in various cancers, such as liver, breast, non-small cell lung and colon cancers. Studies utilizing usnic acid against cultured malignant cells have reported its capability of inducing cell cycle arrest, autophagy, and apoptosis. Additionally, usnic acid effectively overcomes the multidrug resistance of K562/Adr cells by enhancing ROS-dependent apoptosis and arresting the cell cycle at G1/G0, indicating its potential as a candidate to synergize with antileukemic drugs. Here, we investigated whether usnic acid at low-cytotoxicity level enhances sensitivity to ABT-199 in cells with acquired ABT-199 resistance and in xenograft model. In this study, MOLM-13 and THP-1 cells were employed to establish ABT-199-resistant AML cell lines, named MOLM-13 R and THP-1 R, with distinct genomic backgrounds (FLT3-ITD mutant in MOLM-13 vs. TP53 mutant in THP-1), which are more likely to develop adaptive resistance to venetoclax-containing combinations. Our results indicate that usnic acid combined with ABT-199 effectively restored ABT-199 sensitivity to drug-resistant cells. The drug resistance reversal fold for 1.5 μM usnic acid in MOLM-13 R cells was 9.7, versus 9.5 for 4 μM usnic acid in THP-1 R cells. The combination therapy also demonstrated prominent anti-leukemic effects in the xenograft model. The combined administration reduced the average proportion of human CD45+ leukemic cells from 43.17% to 11.50%. RNA-seq showed that response of EIF2AK1 (HRI) to heme deficiency was markedly enriched in the combination group by reactome enrichment analysis. HRI, an eIF2α kinase, can induce eIF2-mediated integrated stress response. To examine the effects on downstream ISR signaling, real-time PCR assay and western blot were conducted in MOLM-13 R and THP-1 R cells administered the study drugs for 24 h. The combination of usnic acid and ABT-199 boosted the expression of the integrated stress response (ISR)-associated genes (ATF4, CHOP, and NOXA), CHOP protein levels and eIF2α phosphorylation at serine 51. ISRIB, a chemical inhibitor of the ISR, could rescue combination therapy-induced apoptosis, upregulation of ISR-associated genes. These findings demonstrate that ABT-199 and usnic acid jointly induce the ISR, which subsequently triggers cell death. Furthermore, our findings showed that treatment with ABT-199 combined with usnic acid markedly increased NOXA protein amounts, and reduced anti-apoptotic factor MCL-1 protein levels. The downregulation of MCL-1 was linked to an increase in MCL-1 phosphorylation at serine 159 and subsequent destruction by the proteasome. MOLM-13 R cells overexpressing MCL-1 exhibited dramatically weakened apoptosis induced by usnic acid and ABT-199 co-administration, highlighting the significance of MCL-1 in the impact of ABT-199-usnic acid. In addition, ISRIB was able to reverse the inhibition of MCL-1 protein, and impair the degradation process in the combination group. Overall, addition of usnic acid to ABT-199 results in an enhancement of its inhibitory effect on resistant AML in cell culture and animal experiments, because of enhanced MCL-1 downregulation resulting from ISR hyperactivation. These findings provide compelling evidence that combined treatment with usnic acid and ABT-199 constitutes a novel approach for AML cases that are more likely to develop resistance to ABT-199 and should be further investigated in clinical settings.