Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Although most AML patients achieve complete remission after standard chemotherapy, refractory and relapsed disease remain a significant challenge. Over the past decade, immunotherapy has been widely applied in cancer treatment, particularly chimeric antigen receptor T-cell (CAR-T) therapy, which has shown remarkable success in treating hematological malignancies, especially B-cell malignancies. However, the efficacy of CAR-T therapy in AML has been relatively limited, with CAR-T cell exhaustion being a major limiting factor. Exhaustion of CAR-T cells is considered a key factor affecting their efficacy, and mitigating exhaustion to maintain the effector function and persistence of CAR-T cells remains a core challenge.

T cells play a protective role in the body by eliminating recognized antigens. However, under certain conditions, T cell function can be suppressed by exhaustion, which may arise from persistent antigen exposure, the surrounding microenvironment, and immunosuppressive cells. Exhausted T cells exhibit increased expression of inhibitory receptors such as PD-1, TIM-3, LAG-3, and CTLA4, as well as metabolic dysregulation, functional impairment, and changes in key transcription factor genes. Similarly, CAR-T cell exhaustion is driven by sustained tumor antigen stimulation and an immunosuppressive tumor microenvironment, leading to transcriptional and epigenetic changes. CAR-T cell exhaustion not only affects their function but is also associated with patient prognosis.

T cell exhaustion is a multistage, progressive process with significant heterogeneity. T cells at different stages of exhaustion exhibit distinct epigenetic and transcriptional characteristics. Studies have shown that exhausted T cells can be divided into several stages: progenitor, intermediate, terminal, and proliferating exhaustion. Progenitor exhausted T cells, which express higher levels of naïve and memory T cell signature genes, can be reprogrammed and continue to proliferate even after PD-1/PD-L1 blockade. In contrast, terminal exhausted T cells, marked by higher expression of inhibitory receptors such as PD-1 and TIM-3, cannot be reprogrammed and lose proliferative capacity after PD-1/PD-L1 blockade.

The BET protein family, including BRD2, BRD3, BRD4, and BRDT, plays a crucial role in maintaining cellular homeostasis. Among these, BRD4 is the most extensively studied. Dysfunction of BET proteins, particularly BRD4, can lead to gene mutations and abnormal cell proliferation, resulting in malignant cell growth and carcinogenesis. In recent years, anti-tumor drugs targeting BET proteins have shown significant effects in preclinical studies and clinical trials. JQ1, a first-generation synthetic BET inhibitor, has demonstrated significant anti-tumor activity in mouse models and early clinical trials for hematologic malignancies. In addition to directly inhibiting tumor cells, recent research has found that BET inhibitors can also inhibit malignancy progression by enhancing anti-tumor immune responses. JQ1 can maintain the properties of central memory T cells (TCM) and enhance the functional persistence of CD8+ T cells by directly inhibiting BATF, a regulatory factor associated with the phenotypic differentiation of effector memory cells in CD8+ T cells.

Source of the Paper

This paper was co-authored by Songnan Sui, Mengjun Zhong, and others, published in the 2024 issue of Biomarker Research. The authors are affiliated with the First Affiliated Hospital of Jinan University, the Institute of Hematology at Jinan University School of Medicine, and other institutions. The study used single-cell RNA sequencing (scRNA-seq) to explore the mechanism by which the BRD4 inhibitor JQ1 reduces CAR-T cell exhaustion.

Research Process

1. Construction of the CAR-T Cell Exhaustion Model:
   Researchers first constructed a CAR-T cell exhaustion model. Specifically, CD123 CAR-T cells were co-cultured with MV411 cells expressing high levels of CD123 for 72 hours. The expression of inhibitory receptors (such as PD-1, TIM-3, and LAG3) on the CAR-T cell surface was then detected by flow cytometry to confirm the exhaustion state. The exhausted CAR-T cells were divided into two groups and treated with DMSO (control group) or 0.2 µM JQ1 for 72 hours. The treated CAR-T cells were sorted by flow cytometry and subjected to single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq).

2. Single-Cell RNA Sequencing and T Cell Receptor Sequencing:
   scRNA-seq and scTCR-seq libraries were constructed using the 10x Genomics Chromium Single Cell 5’ kit, and sequencing was performed on the Illumina NovaSeq 6000 platform. The sequencing data were mapped to the human GRCh38 reference genome using the CellRanger v6.1.2 pipeline, generating a cell-by-gene expression matrix. Quality control, filtering, and clustering analysis were performed using Seurat v4.3.0.

3. Data Analysis:
   Researchers used Seurat v4.3.0 to normalize, reduce dimensionality, and cluster the scRNA-seq data. Batch effects were mitigated, and samples were merged using the Harmony v0.1.1 package. Exhaustion and proliferation scores were calculated based on scRNA-seq data using the AddModuleScore function in Seurat v4.3.0. Gene set functional enrichment analysis was performed using the ClusterProfiler v4.2.2 package, including GO and KEGG enrichment analysis. Gene regulatory network analysis was conducted using pySCENIC v0.12.1, constructing gene regulatory networks (GRNs) and evaluating the activity of each regulon.

Main Results

1. JQ1 Reduces CAR-T Cell Exhaustion:
   Compared to the control group, the proportion of exhausted CD8+ CAR-T cells and the exhaustion score were significantly reduced in the JQ1-treated group. Additionally, JQ1 treatment increased the proportion of naïve, memory, and progenitor exhausted CD8+ CAR-T cells while decreasing the proportion of terminally exhausted CD8+ CAR-T cells. JQ1 treatment also enhanced the proliferation, differentiation, and activation capacities of CAR-T cells.

2. JQ1 Enhances the Function of Naïve and Memory CD8+ CAR-T Cells:
   In JQ1-treated naïve and memory CD8+ CAR-T cells, genes related to T cell proliferation, activation, and differentiation were significantly upregulated. Transcriptional regulatory network analysis revealed that the activity of transcription factors such as EGR1 and BCLAF1 increased in JQ1-treated naïve CD8+ CAR-T cells, while the activity of POU2F2 and ZBTB38 decreased.

3. JQ1 Maintains the Early Exhaustion State of CD8+ CAR-T Cells:
   JQ1 treatment increased the proportion of progenitor exhausted CD8+ CAR-T cells and prevented their differentiation into terminally exhausted cells. Functional enrichment analysis showed that genes related to T cell differentiation, activation, and proliferation were upregulated in JQ1-treated progenitor exhausted CD8+ CAR-T cells.

4. JQ1 Reduces CAR-T Cell Exhaustion by Modulating BATF and EGR1:
   In JQ1-treated naïve, memory, and progenitor exhausted CD8+ CAR-T cells, the activity and expression of BATF were downregulated, while the activity and expression of EGR1 were upregulated. The target genes of these transcription factors were associated with T cell proliferation, activation, and differentiation.

Conclusion

This study demonstrates that the BRD4 inhibitor JQ1 can reduce CAR-T cell exhaustion and block terminal differentiation by downregulating BATF activity and expression while upregulating EGR1 activity and expression. This provides a novel strategy for improving the effectiveness of CAR-T cell therapy.

Research Highlights

1. Key Finding: JQ1 significantly reduces CAR-T cell exhaustion and enhances their function by modulating the activity of BATF and EGR1.
2. Methodological Innovation: The study is the first to use single-cell RNA sequencing to detail the mechanism by which JQ1 regulates CAR-T cell exhaustion.
3. Clinical Relevance: The findings offer new insights into the application of CAR-T cell therapy in AML and other hematological malignancies, with significant clinical implications.

Additional Valuable Information

The study also found that higher expression of EGR1 and its target genes in AML patients was associated with better prognosis, while lower expression of BATF and its target genes was also linked to better prognosis. These findings further support the mechanism by which JQ1 improves CAR-T cell function through the regulation of BATF and EGR1.