Overexpression of GAD1 in Pediatric Acute Myeloid Leukemia Cells Impairs Cytotoxic T Lymphocyte Response to Tumor Cells

Background:

In acute myeloid leukemia (AML), as the efficacy of traditional chemotherapy combined with hematopoietic stem cell transplantation has plateaued over the last decades, new approaches are urgently needed. Understanding factors that shape the immune landscape is essential for immunotherapy development. This study aims to elucidate the immunological landscape across pediatric AML subtypes and its impact on patient survival, with a focus on identifying new therapeutic targets.

Methods:

AML patients enrolled in this study were diagnosed and treated between 2009-2018 at Shanghai Children’s Medical Center, excluding those with acute promyelocytic leukemia. We utilized CIBERSORT and TRUST4 algorithms to analyze transcriptome data for immune cell proportion and T-cell receptor (TCR) repertoire, complemented by flow cytometry, to comprehensively examine the tumor microenvironment. Through differential gene expression analysis, we identified crucial factors of T-cell infiltration. The potential of these factors as therapeutic targets was validated using in vitro experiments and AML animal models.

Results:

Totally, 287 pediatric AML patients were included in the study. We reanalyzed the flow cytometry data of patients at the time of diagnosis and determined the total lymphocytes and subpopulation percentages in bone marrow. Our results revealed that patients who achieved CR demonstrated higher lymphocytes, particularly elevated T lymphocytes, regardless of tumor burden. Survival analysis indicated that a diminished CD8+ T-cell proportion and lower TCR clonality at diagnosis in bone marrow were closely linked to worse outcomes. Through differential expression (DE) analysis, we identified upregulated genes associated with T-cell activation and lymphocyte differentiation in patients with higher clonality, who exhibited superior survival rates. However, the set of DE genes were enriched for GO biological processes related to synaptic membrane, γ-aminobutyric acid (GABA) -ergic synapse and GABA receptor complex in those with low clonality and adverse survival. Glutamic acid decarboxylase-1 (GAD1), the enzyme that catalyzes the production of GABA from glutamate, was highly expressed in AML cases with CBFA2T3::GLIS2, FUS::ERG, and NUP98::KDM5A rearrangements, which show poor prognosis. The levels of GABA were increased in the culture supernatant of cell lines with high expression of GAD1. Additionally, elevated GABA levels were observed in the bone marrow and spleen microenvironment of NUP98::KDM5A mouse model. Tumor-derived GAD1 resulted in elevated levels of γ-aminobutyric acid in the microenvironment, which in turn inhibited the proliferation, activation, and cytotoxic functions of T cells. In vivo inhibiting GAD1 activity with 3-MPA reduced tumor proliferation and improved survival in a mouse model of NUP98::KDM5A AML.

Conclusions:

Our study highlights the significant role of T-cell immunity in achieving remission and securing long-term survival in AML. Notably, we have identified GAD1 as a critical modulator of T-cell efficacy, providing a potential new target for the treatment of AML.