Deciphering the bone marrow inflammatory cytokine network in AML prognosis and immune evasion: A focus on the IL-1A-CCL3/4 axis as a novel therapeutic target Free

Background: Acute Myeloid Leukemia (AML) is characterized by a profoundly dysregulated bone marrow microenvironment (BME) that fosters disease progression and immune escape. While inflammatory cytokines are pivotal in shaping immune responses, a comprehensive understanding of their complex interplay, prognostic significance, and mechanistic roles in AML, particularly across different FAB subtypes, remains a critical unmet need.

Aims: This study aimed to: 1) comprehensively profile inflammatory cytokines in AML patient BME and identify those with prognostic significance; 2) delineate the cellular sources and inter-cytokine regulatory mechanisms within the AML BME using cutting-edge single-cell transcriptomics; and 3) investigate the functional impact of key inflammatory axes on immune cell function and explore their potential as novel therapeutic targets.

Methods: We analyzed bone marrow supernatant from46 AML patients and 20 healthy controls for 20 inflammatory cytokines. Clinical correlations, including 5-year overall survival (OS), genetic risk stratification, and FAB subtype, were assessed using ROC analysis, univariate and multivariate Cox regression. For mechanistic insights, single-cell RNA sequencing (scRNA-seq) was performed on bone marrow samples from AML patients and healthy individuals. We employed an innovative CNV, tumor score, classification score methods for robust tumor cell clustering. Subsequent analysis involved mapping cytokine expression to specific cell types, performing ligand-receptor pairing, immune cell communication analysis, and signaling pathway enrichment. Inflammatory cytokine interaction networks were constructed using Cytoscape and STRING. Functional studies utilized in vitro co-culture systems with AML cell lines and primary patient cells, assessing T cell chemotaxis and cytotoxicity, followed by rescue experiments with cytokine modulation.

Results: Our initial profiling identified 7 significantly dysregulated inflammatory cytokines in AML patient BME. Among these, 5 were significantly associated with 5-year OS, and 2, including IL-1A, emerged as independent prognostic factors for adverse OS. Cytokine expression also varied significantly across FAB subtypes, with M1 and M2 exhibiting distinct inflammatory profiles compared to M5 AML, suggesting an influence of tumor cell origin. ScRNA-seq revealed CCL3 and CCL4 were primarily expressed by healthy T cells, whose numbers and functional capacity were notably reduced in AML patients, with increased naive T cells and decreased CCL3/4 expression. Ligand-receptor and cell communication analyses confirmed reduced CCL3/4 impaired immune cell recruitment and tumor-killing. Network analysis highlighted IL-1A as a central regulator that significantly influenced CCL3/4 expression on T cells. Elevated BME IL-1A was found to suppress T cell CCL3/4 expression, directly contributing to impaired T cell chemotaxis and cytotoxicity. In vitro functional assays demonstrated IL-1A inhibition (using antagonists) restored T cell CCL3/4 expression and anti-leukemic activity. Importantly, exogenous CCL3/4 supplementation synergized with IL-1A inhibition, leading to robust recovery of T cell function and enhanced tumor suppression.

Conclusion: Our study provides a comprehensive systems-level inflammatory cytokine atlas in AML, identifying IL-1A as a critical independent prognostic factor and a central player in AML immune evasion. We innovatively demonstrate that IL-1A orchestrates the suppression of the CCL3/4-T cell chemotaxis axis, leading to impaired T cell function and compromised anti-tumor immunity. This finding, particularly pronounced in specific FAB subtypes like M5 AML, highlights a novel mechanism by which the AML microenvironment promotes disease progression. Our preclinical data suggest that targeting IL-1A, either alone or in combination with CCL3/4 supplementation, represents a promising immunomodulatory strategy to restore T cell function and improve therapeutic outcomes in AML. These insights open new avenues for refining prognostic models and developing precision immunotherapies for AML patients.