Targeted therapies have transformed the treatment landscape of hematological malignancies; however, meaningful progress for older/unfit patients with acute myeloid leukemia (AML) remained limited until the advent of the BCL-2 inhibitor venetoclax (VEN) combined with azacitidine (AZA)—the VA regimen. Despite its efficacy, only 43.4% of newly diagnosed (ND) older/unfit AML patients achieved composite complete remission (CRc) after one cycle of VA (DiNardo et al., NEJM 2020). In our phase II trial (ChiCTR2300078290), augmentation of VA with granulocyte colony-stimulating factor (G-CSF) (VAG regimen) significantly improved CRc rates to >80% (in review). While prior studies demonstrate that VEN synergizes with AZA to impair leukemia stem cell (LSC) metabolism and activate T cells, the mechanisms underlying G-CSF-mediated synergy remain unknown.

To interrogate the anti‐leukemic mechanisms of the VAG regimen, we replicated the VA/VAG protocols used in AML patients in two unirradiated murine models:C1498 cells engrafted into C57BL/6 (immunocompetent) and NSG (immunodeficient) mice; MLL-AF9 cells transplanted into aged C57BL/6 (immunocompetent) mice. In the immunodeficient NSG model, VAG treatment did not significantly prolong survival compared with VA treatment. By contrast, in the immunocompetent model, VAG treatment group conferred a clear survival advantage (P < 0.05) especially in aged mice AML model (P < 0.01) with a significant reduction of leukemia burden in VAG treatment group versus VA treatment (P < 0.05). These findings support the hypothesis that G-CSF addition to the VA regimen reactivates the AML microenvironment to mediate its anti-leukemic effect.

To further elucidate these mechanisms, we performed longitudinal multi-omic profiling—including single-cell RNA sequencing (scRNA-seq), whole-exome sequencing (WES), and flow cytometric immunophenotyping—on 12 newly diagnosed older/unfit AML patients undergoing VA or VAG treatment. Samples were collected at five timepoints from both peripheral blood (PB) and bone marrow (BM). Following identification of leukemic cells, we first assessed dynamic changes in immune cell composition in PB. Notably, patients receiving the VAG regimen exhibited a more diverse and abundant immune landscape early in treatment, with significant increases in T cells, monocytes, and neutrophil cells. Focusing on early timepoints (day 3 and 7), we identified two CD8+ effector T cell subsets (CD8T_GNLY and CD8T_GZMK) and natural killer T (NKT) cells that expanded robustly in the VAG-treated cohort. Conversely, these effector populations gradually declined in VA-treated patients. Transcriptional analysis revealed that effector T cells enriched in VAG-treated patients expressed elevated levels of cytotoxicity- and activation-related genes including JUN, JUNB, JUND, CD69, and CXCR4. Importantly, after one cycle of induction, BM effector T cells in VAG-treated patients retained an activated transcriptional signature, with enrichment of key regulators such as JUN and STAT3. In contrast, the immunosuppressive CD4+ regulatory T cell subset (CD4T_Treg_FOXP3) was preferentially enriched in VA-treated patients.

Collectively, these findings suggest that the VAG regimen alleviates immune suppression and promotes the activation and retention of cytotoxic CD8+ T cells within the AML niche. The VAG regimen enhances leukemic clearance in vivo and improves immune cell abundance and activation, particularly in older AML patients. G-CSF may synergize with targeted therapy to restore anti-leukemic immunity and represents as a promising strategy for immunocompromised AML patients.

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2025
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