Abstract
Acute myeloid leukemia (AML) remains one of the most lethal hematologic malignancies, particularly in elderly patients and those harboring TP53 mutations. Standard-of-care chemotherapies offer limited long-term survival benefits, especially in TP53-mutated patients with chemotherapy-resistant phenotypes. Recent insights into the tumor microenvironment (TME) suggest that immune evasion mechanisms mediated by myeloid cells play a critical role in leukemia progression and therapeutic failure. Therefore, our central hypothesis is to overcome myeloid cell-mediated immunosuppression for mounting anti-tumor response in acute myeloid leukemia.
Bioinformatics and flow cytometry analysis were done on bone marrow and PBMC of AML patients to investigate VIP profiling in TP53-mutated vs non-mutated cases, respectively. Human and murine AML cell lines were deployed to study impact of TP53 on VIP production and phagocytosis vulnerability of VIP-producing cells, respectively. In vivo efficacy of novel long-acting VIP receptor antagonist, ANT308-IgG4 Fc fusion was studied in the murine AML model.
Our flow cytometry analysis showed a higher level of VIP expression in PBMC of ~36% AML patients relative to healthy human PBMC. Survival analysis using cBioportal datasets revealed direct association of high VIP expression with poor survival in AML patients. TP53 loss-of-function mutation followed the same pattern of mortality as VIPhi AML. Differential gene expression analysis showed an enrichment of VIP and their receptor on TP53-mutated patients, but not PD-1, other immune checkpoint or anti-apoptosis proteins. Corroborating secretome analysis of human leukemia cells demonstrated a higher VIP secretion from TP53 LOF-mutated leukemia cells (HL-60 harboring homozygous deletion in TP53; Kasumi-1 with homozygous TP53-mutation, and heterozygous TP53-mutation present in CCRF-CEM) relative to control (TP53-wildtype present in THP-1). In murine VIP-secreting WEHI3 leukemic cells treatment with the VIP receptor antagonist, ANT308 enhanced phagocytosis of leukemia cells by both M1-/M2-like macrophages but did not alter macrophage phagocytosis of VIP non-secreting C1498 cells , suggesting VIP may function as a phagocytosis checkpoint in TP53-mutated AML. Additionally, flow cytometry demonstrated increased VIP expression in non-leukemic myeloid cells in AML patients. Testing the effects of VIP signaling in host cells, we also observed a significant survival benefit in VIP knock-out mice as well as VPAC1 or VPAC2-knockout mice bearing C1498 leukemia relative to VIP-wild type mice. In vivo treatment of wild-type mice harboring C1498 with ANT308 or the long-acting Fc fusion form eradicated established C1498 leukemia in up to 65% of leukemic mice.Conclusions: The VIP/VPAC axis functions as a previously unrecognized phagocytosis checkpoint in AML. TP53-mutation evades anti-tumor immune response via VIP. These findings support the evaluation of ANT308-IgG4 Fc3 fusion, alone or in rational combinations, for high-risk AML.
