TP53 activation by the natural quassinoid brusatol enhances venetoclax efficacy and overcomes resistance in myeloid leukemias

Abstract Introduction Based on the encouraging results from the VIALE-A and VIALE-C trials, venetoclax in combination with azacitidine or cytarabine has become a standard treatment option for elderly patients or those ineligible for intensive chemotherapy. Nevertheless, virtually all patients eventually relapse, primarily due to intrinsic and acquired resistance, necessitating the development of alternative combination strategies. Brusatol, a natural quassinoid, has been shown to possess a broad spectrum of biological activities, with increasing attention to its potential applications in cancer treatment. It demonstrates strong anti-tumor activities against various types of malignancies while also overcoming resistance and potentiating the efficacy of other anticancer agents. However, the precise mechanism by which brusatol induces cell death remains unclear, and most current evidence stems from research on solid tumors. This study aimed to assess whether, and if so how, brusatol enhances venetoclax efficacy and its potential as a novel combination partner for overcoming resistance in AML. Methods Five genetically and biologically distinct human AML cell lines were used in this study, including MOLM-14, HL60, KG-1a, THP-1, and U937. A venetoclax-resistant MOLM-14 variant (VEN-R) was generated by continuous exposure to increasing concentrations of venetoclax. For the establishment of cell line-derived xenograft (CDX) models, NSG mice received 250 cGy of total body irradiation, followed by intravenous injection of 0.2 million AML cells. For RNA-seq analysis, MOLM-14 cells were treated with DMSO, 2.5 nM venetoclax, 5.0 nM brusatol, or combination for 24 hours. Results Brusatol rapidly induced apoptosis in all AML cell lines in a dose-dependent manner. MOLM-14 cells were the most susceptible, whereas THP-1 and U937 were comparatively less sensitive, yet still showed IC50 values below 100 nM. Similarly, venetoclax exhibited strong anti-leukemia effects in AML cell lines, but was less effective against U937. Notably, brusatol synergized with venetoclax and overcame its resistance, as shown by lowering the apoptotic threshold for venetoclax and inhibiting colony formation across all AML cell lines, including venetoclax-refractory U937 and VEN-R MOLM-14 cells. Synergism analysis using HSA and Bliss independence models confirmed the synergy of the combination, with scores of 28.6 and 23.9 in MOLM-14, and 31.8 and 27.3 in U937, respectively, well above the synergy threshold of 10. Three independent CDX models using MOLM-14, U937, and VEN-R MOLM-14 cells showed significant tumor reduction and prolonged the survival of all mice treated with the combination (median survival: 24 vs. 51 days, 16 vs. 21 days, 25 vs. 34 days, respectively, all p < 0.05), while mice treated with single agents demonstrated only limited therapeutic efficacy. RNA-seq and subsequent pathway analysis using GSEA revealed that brusatol treatment led to upregulation of p53- and apoptosis-related genes, such as BBC3, TRAILR2, and MDM2. It also upregulated CDKN1A, while downregulating CCND1, AURKB, and CDC25C, suggesting negative regulation of cell cycle progression. Immunoblotting confirmed increased expression of p53 and its targets p21 and PUMA following brusatol treatment. Consistent with this, cell cycle analysis revealed that a suboptimal dose of brusatol induced G0/G1 arrest, while higher concentrations increased sub-G1 apoptosis. Genetic knockdown of TP53 largely restored the cell viability, further supporting that TP53 activation is a primary mechanism underlying the anti-leukemic effects of brusatol. Interestingly, SREBP2 downstream genes, such as HMGCS1, DHCR7, and CYP51A1, which encode enzymes critical for sterol biosynthesis, were specifically suppressed by the combination. As observed with sterol synthesis inhibitors like statins, the combination increased ROS accumulation and reduced mitochondrial membrane potential and OXPHOS activity compared with single agents, suggesting that the combination impairs mitochondrial membrane integrity, thereby promoting apoptosis independently of TP53 gene status. Conclusions Our study demonstrates that brusatol synergizes with venetoclax to induce apoptosis through activation of the TP53-axis, including CDKN1A, one of the key genes involved in venetoclax resistance in AML. This combination overcomes venetoclax resistance in both in vitro and in vivo models, providing a strong rationale for its further clinical investigation.