Tolinapant (ASTX660) is an antagonist of the inhibitor of apoptosis proteins (IAPs) cIAP1 (BIRC2), cIAP2 (BIRC3), and XIAP. These IAPs function by inhibiting caspase activity, thereby suppressing apoptosis. Recently, tolinapant has been introduced in the treatment of hematological malignancies and has shown promising results in preclinical models of T-cell lymphoma (TCL), with beneficial immunomodulatory effects confirmed in a small cohort of TCL patients (Ferrari et al., Blood Adv, 5(20):4003–4016, 2021). In acute myeloid leukemia (AML), a study evaluating tolinapant as a single agent and in combination with cedazuridine (ASTX727) in patients with relapsed/refractory AML was prematurely terminated (NCT04155580). However, the pharmacological and molecular basis of tolinapant activity, as well as the role of its targets, remains largely unexplored in the context of AML.

In this study, we conducted a comprehensive analysis of the impact of tolinapant targets, BIRC2, BIRC3, and XIAP, on survival outcomes and their association with risk stratification in an AML cohort. Additionally, we evaluated the effects of tolinapant, both as monotherapy and in combination with venetoclax, in a genetically diverse panel of AML cell lines.

We analyzed data from non-APL AML patients in The Cancer Genome Atlas (TCGA) cohort who received intensive therapy (n = 121). Ex vivo assay data from the BeatAML cohort (n = 570), treated with a panel of antineoplastic agents (n = 165), were also included. Furthermore, data from the DepMap project comprising AML cell lines (n = 25) subjected to knockout of BIRC2, BIRC3, and XIAP were analyzed. The effects of tolinapant treatment (0–100 µM) on cell viability were evaluated by MTT assay in 14 AML cell lines. Combination treatments with tolinapant and venetoclax were tested in intrinsically drug-resistant AML lines, Kasumi-1 and OCI-AML3, using MTT assays (25 combinations), flow cytometry (Annexin V/PI), and Western blot (cleaved PARP1 and γH2AX).

Gene expression dichotomization was based on ROC curve analysis and C-index optimization. Survival was analyzed using Kaplan–Meier estimates and compared by log-rank tests. ANOVA or Kruskal–Wallis tests were used for continuous variables, and Spearman's correlation was applied for association analyses. IC50 values were calculated by non-linear regression. ZIP synergy scores for combination treatments were calculated using SynergyFinder software. A p-value < 0.05 was considered statistically significant.

High BIRC2 expression (p = 0.04), but not BIRC3 or XIAP, was associated with worse overall survival in AML patients. Elevated BIRC3 expression was observed in the intermediate-risk group. Cytogenetically, BIRC2 expression was higher in patients with del(5), t(9;11)+other, and del(7q), while BIRC3 expression was increased in del(7q)+other and del(5q)+other groups. Conversely, XIAP expression was reduced in patients with del(5).

In ex vivo analyses, BIRC2 expression correlated with resistance to 20 antineoplastic agents, BIRC3 with resistance to 28 and sensitivity to 7, and XIAP with resistance to 12 and sensitivity to 20 (all p < 0.05). DepMap data showed moderate dependency for BIRC2 (−0.334), and minimal for BIRC3 (0.02) and XIAP (0.017) in AML cell models.

Tolinapant showed modest cytotoxic effects across AML cell lines, with IC50 values measurable in 5 of 14 lines (range: 29.8–77.2 µM) and efficacy rates from 13.6% to 99.9%. Notably, the combination of tolinapant and venetoclax demonstrated synergistic effects in Kasumi-1 cells (ZIP score = 13.8) and additive effects in OCI-AML3 cells (ZIP score = 8.4), as confirmed by apoptosis assays (all p < 0.05). Molecular analyses supported these findings, showing increased PARP1 cleavage and γH2AX accumulation in combination treatments compared to monotherapy.

In conclusion, this study provides new insights into the expression patterns and functional relevance of tolinapant targets in AML. High BIRC2 expression was associated with poor overall survival and resistance to multiple antineoplastic agents, suggesting its potential as a prognostic biomarker and therapeutic target. Although tolinapant displayed limited single-agent activity, its combination with venetoclax yielded synergistic or additive cytotoxic effects in resistant models. These findings support further investigation of tolinapant-based combination therapies, particularly in biomarker-defined AML subsets. Supported by FAPESP, CAPES, and CNPq.

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2025
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