Extracellular arginine deprivation enhances venetoclax sensitivity in acute myeloidleukemia via mitochondrial fission impairment and apoptotic priming

Abstract Resistance to the BCL2 inhibitor venetoclax (VEN) remains a major therapeutic challenge in acute myeloid leukemia (AML), fostering the need for rational combination strategies. One such approach involves arginine deprivation using pegylated arginine deiminase (ADI-PEG20), which is currently under clinical investigation in combination with VEN and azacitidine (ASH 2023, Borthakur; NCT05001828). Although ADI-PEG20 has shown efficacy in AML cells deficient in argininosuccinate synthase 1 (ASS1), a key enzyme for endogenous arginine synthesis, our previous study demonstrated its ability to similarly enhance VEN and decitabine responses in ASS1-positive AML cells (ASH 2023, Yamatani). However, the molecular mechanisms underlying this synergy remain poorly understood. In this study, we investigated how extracellular arginine depletion by ADI-PEG20 modulates VEN sensitivity, with a focus on mitochondrial dynamics. Two VEN-resistant AML cell lines were used: P31/FUJ (ASS1-low, 0.02 relative to GAPDH) and MV4;11 with TP53 frameshift mutant (MV4;11 TP53fs, ASS1-high, 19.8). Cells were cultured in complete medium or medium preconditioned with ADI-PEG20 (100 ng/mL, 72 hours). LC-MS/MS analysis confirmed complete depletion of extracellular arginine in ADI-PEG20–treated medium (<LOD), whereas complete medium contained 400 nM arginine. ADI-PEG20 suppressed cell proliferation within 24 hours, with a more pronounced effect in P31/FUJ cells (46.7 ± 1.7%) compared to MV4;11 TP53fs (16.8 ± 2.1%). No such effect was observed with heat-inactivated ADI-PEG20, confirming that enzymatic activity is essential for this response. Notably, after 48 hours, intracellular arginine levels decreased by 14.9 ± 2.6% in ASS1-low P31/FUJ cells but increased by 151.7 ± 16.2% in ASS1-high MV4;11 TP53fs cells, indicating that growth inhibition in MV4;11 TP53fs cells is primarily driven by extracellular arginine depletion rather than by changes in intracellular arginine levels. ADI-PEG20 treatment significantly sensitized both cell lines to VEN; IC₅₀ values shifted from >3200 to 16.5 ± 1.5 nM in P31/FUJ, and from 1613 ± 157 to 242.5 ± 14.7 nM in MV4;11 TP53fs. This effect was reversed by L-arginine supplementation. The combination of ADI-PEG20 and VEN exerted synergistic anti-proliferative effects, with ZIP synergy scores of 28.7 in P31/FUJ and 16.2 in MV4;11 TP53fs, accompanied by increased apoptosis as confirmed by Annexin V staining. To elucidate the molecular basis of VEN sensitization under arginine-deprived conditions, we performed Western blot analysis of key signaling proteins. ADI-PEG20 treatment decreased β-catenin and increased ATF4 expression in both cell lines. These changes suggest that extracellular arginine deprivation is sensed via the BAG2–SAMD4B–β-catenin axis (Molecular Cell 2024, Chen, PMID: 40555234), which in turn activates the Integrated Stress Response (ISR). Moreover, phosphorylation of DRP1 at Ser616, a marker of mitochondrial fission, was decreased, and transmission electron microscopy revealed mitochondrial swelling and increased autophagosome formation following ADI-PEG20 treatment. These findings indicate an adaptive shift toward mitochondrial fusion and enhanced autophagy-mediated repair of damaged mitochondria in response to nutrient stress. Importantly, these mitochondrial alterations were further exacerbated by co-treatment with VEN. Under these conditions, mitochondrial damage appeared to exceed the threshold for repair, resulting in simultaneous BCL2-dependent apoptotic priming and mitophagy failure, ultimately culminating in apoptosis. In conclusion, our study reveals that extracellular arginine deprivation via ADI-PEG20 induces mitochondrial stress, impairs mitochondrial fission, and primes AML cells for VEN-induced apoptosis irrespective of ASS1 status.