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Background <p>Arsenic trioxide (ATO) therapy is highly successful in majority of acute promyelocytic leukemia (APL) patients with PML-RARA fusion oncoprotein. However, after initial response to therapy, 8–10% experienced relapse and became resistant to ATO. ATO-resistant APL cells show elevated BCL2 expression and increased reliance on oxidative phosphorylation (OXPHOS) for survival. Venetoclax (Ven), a BCL2 inhibitor, induces apoptosis by suppressing OXPHOS and increasing mitochondrial reactive oxygen species (ROS).</p> Method <p>Cytotoxic effects of venetoclax were assessed in APL cell lines and primary blasts using CTG assays. Mitochondrial function, biogenesis, and apoptosis were evaluated through Seahorse extracellular flux analysis, flow cytometry–based assays, transmission electron microscopy, and immunoblotting. For mechanistic studies, we carried out genetic knockdown of BCL2 and Beclin-1 and performed co-immunoprecipitation. Proteomic profiling of ATO-resistant cells treated with venetoclax or ATO was conducted using high-resolution LC-MS. Leukemic burden, apoptosis, and mitochondrial ROS were studied in NOD/SCID orthotopic xenografts. An FVB/N-PML-RARA syngeneic model was used to establish in vivo ATO resistance and evaluate OXPHOS, apoptosis, mitochondrial ROS and tumor burden.</p> Results <p>Antiproliferative activity of venetoclax in primary APL blasts (<i>n</i> = 25) was in nanomolar concentration (mean = 39 nM; range = 0.015-280 nM). Venetoclax in ATO-resistant cells decreases mitochondrial respiration, increases mitochondrial ROS, and disrupts mitochondrial membrane potential. These events led to cytochrome C release to activate mitochondrial apoptosis. Proteomic profiling using high-resolution Liquid Chromatography-Mass Spectrometry identified differentially expressed proteins, revealing dysregulated pathways associated with apoptosis and autophagy. Venetoclax downregulated anti-apoptotic proteins and degraded PML-RARA. Mechanistically, venetoclax disrupted BCL2/Beclin-1 interaction, releasing Beclin-1 to initiate autophagy by modulating the levels of p62 and LC3-I to LC3-II conversion. Furthermore, in orthotopic xenografts, venetoclax treatment results in the reduction of hCD45+ cells in bone marrow to 4.8% (range: 2.5–5.5%) compared to 9.4% (range: 8.6–10.5%) in vehicle control (<i>p</i> &lt; 0.0001). Ven-ATO further reduced this to 3.8% (range: 1.6–4.8%; <i>p</i> &lt; 0.0001), whereas ATO decreased hCD45+ cells to 8.0% (range: 5.5–12.3%; <i>p</i> = 0.16). Additionally, the therapeutic efficacy of venetoclax was confirmed using in vivo ATO-resistant FVB/N-PML-RARA model.</p> Conclusions <p>These findings suggest that venetoclax induced apoptosis and autophagy in ATO-resistant cells by increasing mitochondrial stress and disrupting BCL2/Beclin-1 interaction.</p> Graphical abstract <p></p>

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Venetoclax induces mitochondrial apoptosis and autophagy to overcome arsenic trioxide resistance in acute promyelocytic leukemia

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  • 发表日期：

    2026年1月12日

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  • Deepshikha Dutta,
  • Akash Maity,
  • Saurabh Kumar Gupta,
  • Poonam Gera,
  • Hasmukh Jain,
  • Bhausaheb Bagal,
  • Gaurav Chatterjee,
  • Sweta Rajpal,
  • Lingaraj Nayak,
  • Sachin Punatar,
  • Sabyasachi Bandyopadhyay,
  • Avik Chakraborty,
  • Ezhilarasi Chendamarai,
  • Nithya Balasundaram,
  • Vikram Gota,
  • Navin Khattry,
  • Manju Sengar,
  • Vikram Mathews,
  • Syed K. Hasan

摘要

Background

Arsenic trioxide (ATO) therapy is highly successful in majority of acute promyelocytic leukemia (APL) patients with PML-RARA fusion oncoprotein. However, after initial response to therapy, 8–10% experienced relapse and became resistant to ATO. ATO-resistant APL cells show elevated BCL2 expression and increased reliance on oxidative phosphorylation (OXPHOS) for survival. Venetoclax (Ven), a BCL2 inhibitor, induces apoptosis by suppressing OXPHOS and increasing mitochondrial reactive oxygen species (ROS).

Method

Cytotoxic effects of venetoclax were assessed in APL cell lines and primary blasts using CTG assays. Mitochondrial function, biogenesis, and apoptosis were evaluated through Seahorse extracellular flux analysis, flow cytometry–based assays, transmission electron microscopy, and immunoblotting. For mechanistic studies, we carried out genetic knockdown of BCL2 and Beclin-1 and performed co-immunoprecipitation. Proteomic profiling of ATO-resistant cells treated with venetoclax or ATO was conducted using high-resolution LC-MS. Leukemic burden, apoptosis, and mitochondrial ROS were studied in NOD/SCID orthotopic xenografts. An FVB/N-PML-RARA syngeneic model was used to establish in vivo ATO resistance and evaluate OXPHOS, apoptosis, mitochondrial ROS and tumor burden.

Results

Antiproliferative activity of venetoclax in primary APL blasts (n = 25) was in nanomolar concentration (mean = 39 nM; range = 0.015-280 nM). Venetoclax in ATO-resistant cells decreases mitochondrial respiration, increases mitochondrial ROS, and disrupts mitochondrial membrane potential. These events led to cytochrome C release to activate mitochondrial apoptosis. Proteomic profiling using high-resolution Liquid Chromatography-Mass Spectrometry identified differentially expressed proteins, revealing dysregulated pathways associated with apoptosis and autophagy. Venetoclax downregulated anti-apoptotic proteins and degraded PML-RARA. Mechanistically, venetoclax disrupted BCL2/Beclin-1 interaction, releasing Beclin-1 to initiate autophagy by modulating the levels of p62 and LC3-I to LC3-II conversion. Furthermore, in orthotopic xenografts, venetoclax treatment results in the reduction of hCD45+ cells in bone marrow to 4.8% (range: 2.5–5.5%) compared to 9.4% (range: 8.6–10.5%) in vehicle control (p < 0.0001). Ven-ATO further reduced this to 3.8% (range: 1.6–4.8%; p < 0.0001), whereas ATO decreased hCD45+ cells to 8.0% (range: 5.5–12.3%; p = 0.16). Additionally, the therapeutic efficacy of venetoclax was confirmed using in vivo ATO-resistant FVB/N-PML-RARA model.

Conclusions

These findings suggest that venetoclax induced apoptosis and autophagy in ATO-resistant cells by increasing mitochondrial stress and disrupting BCL2/Beclin-1 interaction.

Graphical abstract