Guoqing Du

Purpose:BH3 mimetics targeting antiapoptotic BCL2 family proteins are promising therapeutics for T-cell acute lymphoblastic leukemia (T-ALL). However, their activity across genomic subtypes of this cancer and interactions with other antileukemic agents remain incompletely defined.Experimental Design:We evaluated the ex vivo sensitivity of BCL2/BCL-XL dual, BCL2-, BCL-XL-, and MCL1-selective inhibitors across 58 xenografts derived from T-ALL, representing diverse molecular subtypes. The BCL2/BCL-XL dual inhibitor AZD4320 was further assessed in combination with selected antileukemic agents. Drug responses were quantified by dose-dependent induction of apoptosis and integrated with genomic and functional analyses.Results:AZD4320 demonstrated subtype-specific cytotoxicity, with increased sensitivity in early T-cell precursor (ETP)-like T-ALL and resistance in TAL1 αβ–like T-ALL. Gene network analysis revealed subtype-dependent activation of distinct BCL2 family proteins, with AZD4320 response associated with BCL2 and MCL1 activity. Drug–drug interaction analysis using the Multi-dimensional Synergy of Combinations algorithm showed that AZD4320 synergized by potency—rather than maximal efficacy—with asparaginase and dasatinib, particularly broad interaction with asparaginase across subtypes. In vivo, AZD4320–asparaginase combination therapy conferred a survival benefit. Mechanistically, asparaginase-induced asparagine depletion promoted mitochondrial dysfunction, potentiating AZD4320-mediated cytotoxicity.Conclusions:These findings highlight the genomic context in shaping BH3 mimetic responses and point to the rational combination of this class of drugs with antileukemic agents such as asparaginase.

Supplementary Table 1. Concentrations used for ex vivo drug sensitivity assay Supplementary Table 2. Sample information

Supplementary Fig.1. Genomic alterations in paired T-ALL samples. Supplementary Fig. 2. Ex vivo sensitivity to dasatinib in T-ALL samples. Supplementary Fig. 3. The correlations between BCL2, BCL-XL (encoded by BCL2L1), and MCL1 expression levels and ex vivo sensitivity to the BH3 mimetics in the T-ALL cohort. Supplementary Fig. 4. BCL2 family member genes’ activities across T-ALL subtypes. Supplementary Fig. 5. The correlations of ex vivo sensitivity to other BH3 mimetics with the activities of their target genes Supplementary Fig. 6. Distributions of the average Bliss values for 40 T-ALL PDX samples treated with the combination of AZD4320 and dasatinib, asparaginase, nelarabine, or prednisolone. Supplementary Fig. 7. Synergistic potency (log10(α1)) of AZD4320 combined with asparaginase across T-ALL subtypes. Supplementary Fig. 8. MuSyC-derived metrics across T-ALL subtypes. Supplementary Fig. 9. In vivo efficacy of the combined BCL2/BCL-XL inhibition with dasatinib. Supplementary Fig. 10. AZD4320 and asparaginase synergize to induce cytotoxicity in Loucy. Supplementary Fig. 11. Mitochondrial bioenergetics in Loucy cells. Supplementary Fig. 12. Mitochondrial membrane potential in Loucy cells. Supplementary Fig. 13. TMRM staining of primary CD8+ T cells following AZD4320 and asparaginase treatment. Supplementary Fig. 14. The combination of AZD4320 and asparaginase induces caspase-dependent apoptotic cell death. Supplementary Fig. 15. Uncropped immunoblot images and corresponding gel images.

Purpose: BH3 mimetics targeting antiapoptotic BCL2 family proteins are promising therapeutics for T-cell acute lymphoblastic leukemia (T-ALL). However, their activity across genomic subtypes of this cancer and interactions with other antileukemic agents remain incompletely defined. Experimental Design: We evaluated the ex vivo sensitivity of BCL2/BCL-XL dual, BCL2-, BCL-XL-, and MCL1-selective inhibitors across 58 xenografts derived from T-ALL, representing diverse molecular subtypes. The BCL2/BCL-XL dual inhibitor AZD4320 was further assessed in combination with selected antileukemic agents. Drug responses were quantified by dose-dependent induction of apoptosis and integrated with genomic and functional analyses. Results: AZD4320 demonstrated subtype-specific cytotoxicity, with increased sensitivity in early T-cell precursor (ETP)-like T-ALL and resistance in TAL1 αβ–like T-ALL. Gene network analysis revealed subtype-dependent activation of distinct BCL2 family proteins, with AZD4320 response associated with BCL2 and MCL1 activity. Drug–drug interaction analysis using the Multi-dimensional Synergy of Combinations algorithm showed that AZD4320 synergized by potency—rather than maximal efficacy—with asparaginase and dasatinib, particularly broad interaction with asparaginase across subtypes. In vivo, AZD4320–asparaginase combination therapy conferred a survival benefit. Mechanistically, asparaginase-induced asparagine depletion promoted mitochondrial dysfunction, potentiating AZD4320-mediated cytotoxicity. Conclusions: These findings highlight the genomic context in shaping BH3 mimetic responses and point to the rational combination of this class of drugs with antileukemic agents such as asparaginase.