BACH2-mediated CD28 and CD40LG axes contribute to pathogenesis and progression of T-cell lymphoblastic leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by its high heterogeneity and unfavorable clinical features. Despite improved insights in genetic and epigenetic landscapes of T-ALL, the molecular mechanisms that drive malignant T-cell development remain unclear. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in B-cell malignancies, but little is known about its function and regulatory network in T-ALL. Here we found extremely low levels of BACH2 in T-ALL clinical samples and cell lines compared to normal T cells. Overexpression of BACH2 in T-ALL cells not only induced cell growth retardation but also inhibited cancer progression and infiltration in xenografts. Further RNA sequencing (RNA-seq) analysis revealed significant alterations in regulation of defense and immune responses in T-ALL cells upon BACH2 overexpression. Strikingly, CD28 and CD40LG, two essential stimulatory molecules on T cells, were for the first time identified as novel downstream targets repressed by BACH2 in T-ALL cells. Interestingly, both CD28 and CD40LG were indispensable for T-ALL survival, since largely or completely silencing CD28 and CD40LG led to rapid cell death, whereas partial knockdown of them resulted in cell-cycle arrest and enhanced apoptosis. More importantly, BACH2-mediated CD28 and CD40LG signals contributed to cell migration and dissemination of T-ALL cells to the bone marrow, thus adding a new layer to the BACH2-mediated tumor immunoregulation in T-cell malignancies.

Fig. 1. Decreased BACH2 levels in T-ALL patients and tumor-suppressor-like role of BACH2 in T-ALL cells and xenografts.

A BACH2 expression values in T-ALL patient samples (n = 9) vs. normal thymic T cells (n = 4) from GSE63602 (left), as well as in T-ALL patient samples (n = 4) vs. normal peripheral CD3⁺ T cells (n = 4) from GSE26530 (right). B Relative BACH2 mRNA levels in three T-ALL patient samples (#1-#3) vs. normal peripheral CD3⁺ T cells and different T-cell subsets including DN, SP (CD4⁺ T and CD8⁺ T) and DP T cells. The fold change in expression relative to the BACH2 levels of CD3⁺ T cells is shown as the mean ± SD. Each of the T-cell subsets is compared to three T-ALL samples, respectively, with the p values indicated. C The overexpression efficiency of BACH2 (BACH2^OE) in Jurkat (left) and MOLT-4 (right) T-ALL cells were validated by immunoblots with a nonsilencing shRNA plasmid (BACH2^Con) as a negative control. β-actin was used as a loading control. Viable cells were counted in manipulated Jurkat and MOLT-4 cells (lower). D Representative cell-cycle distribution of manipulated Jurkat and MOLT-4 cells (left). The % population of cells in each phase is shown as the mean ± SD from three independent experiments (right). E Representative cell apoptosis in manipulated Jurkat and MOLT-4 cells staining with Annexin V/7-AAD (left). The % population of early and late apoptotic cells in each group is shown as the mean ± SD from three independent experiments (right). NS not significant; *p < 0.05; **p < 0.01 (vs. control group). F Manipulated Jurkat cells (2 × 10⁶ cells/mouse) were intravenously (i.v.) injected into NOD/SCID mice via tail vein (n = 8). Mice #1 to #4 were injected with BACH2^Con cells, whereas mice #5 to #8 were injected with BACH2^OE cells. Xenografts were sacrificed when they presented leukemic phenotypes. Spleens were isolated and photographed against a ruler in centimeters. G GFP⁺ cells from spleens (left) and bone marrows (right) in each group were analyzed using flow cytometry. Data are presented as the mean ± SEM.

Fig. 2. RNA-seq analysis of manipulated T-ALL cells.

A Visualization of BACH2 transcriptional levels (red peaks) with IGV from two replicates in each group. GRCh38 genome was used as a human reference genome (upper). Volcano plot was generated with 137 Up-DEGs and 96 Down-DEGs in BACH2^OE Jurkat cells vs. control cells (lower). The Log₂(FC), adjusted p values were calculated using DESeq2 default settings, and significant genes were considered the ones with adjusted p values < 0.05. B Heatmap of the top 20 Up- and Down-DEGs from two replicates in each group. The level of gene expression is indicated by the color intensity, with red representing high expression and blue representing low expression. C Top 10 of GO terms enriched in BP category based on the enrichment analysis of the obtained DEGs. D DEGs that were enriched in the top 5 GO-BP terms. E Top 15 hub genes in BACH2-involving gene regulatory network using weighted gene co-expression network analysis. The black arrows pointed to the downstream target genes and the lines indicated the gene-gene interactions. Red, green, and blue represent the Up-DEGs (n = 5), Down-DEGs (n = 4), and nonsignificant genes (n = 6), respectively. F Top 12 hub nodes in BACH2-involving PPI network, with red representing the Up-DEGs (n = 8) and blue representing the Down-DEGs (n = 4).

Fig. 3. Identification of the downstream transcriptional targets of BACH2 in T-ALL cells.

A Venn diagram showing the overlap of annotated peaks containing promoter region identified from CUT&Tag-seq for BACH2 and the DEGs from RNA-seq. B GO enrichment analysis of the overlapping genes (n = 129) including the top 5 terms in BP category and the top 5 terms in cellular component (CC) category. C Log₂(FC) of the selected 22 DEGs from RNA-seq. Each value from the RNA-seq in BACH2^OE Jurkat cells was relative to the control cells. Chr, chromosome. Relative mRNA levels of Down-DEGs (n = 9) D and Up-DEGs (n = 13) E in manipulated Jurkat and MOLT-4 cells. Each value was normalized to ACTB and is presented as the mean ± SD from three independent experiments. *p < 0.05; **p < 0.01 (vs. control group). Log₂(FC) of each gene in BACH2^OE cells relative to the control cells is indicated under the bar chart, with the upper row representing Jurkat and the lower row representing MOLT-4.

Fig. 4. CD28 and CD40LG are novel downstream targets repressed by BACH2 in T-ALL cells.

A Visualization of BACH2 peaks at CD28 (left) and CD40LG (right) promoters with orange reads representing antibodies against control immunoglobulin G (IgG) and blue reads representing antibodies against human BACH2. Transcriptional levels of the CD28 and CD40LG genes from RNA-seq are indicated with red reads representing upregulation while green reads representing downregulation. GRCh38 genome was used as a human reference genome. Truncated promoter construct containing three putative BACH2-binding sites (MARE1-3) of the CD28 gene (B) or the CD40LG gene (C) is indicated (left). 293T cells were transfected with truncated promoter plasmids, BACH2 expression plasmids, or control plasmids (pcDNA3.1). An empty pGL3-basic plasmid was used as a negative control. The Renilla luciferase reporter pRL-SV40 was used as an internal control for normalization. Luciferase activity was measured 48 h after transfection. Data are presented as the relative luciferase activity compared with the cells transfected with control plasmids (right). Data are shown as the mean ± SD from three independent experiments. D Representative expression levels of CD28 in manipulated Jurkat and MOLT-4 cells using flow cytometry (upper). The relative intensity of CD28 positive (CD28⁺) cells were normalized to control cells and shown as the mean ± SD from three independent experiments (lower). E Representative expression levels of CD40LG in manipulated Jurkat and MOLT-4 cells using flow cytometry (upper). The % population of CD40LG positive (CD40LG⁺) cells were normalized to control cells and shown as the mean ± SD from three independent experiments (lower). *p < 0.05; **p < 0.01 (vs. control group).

Fig. 5. Knockdown of CD28 in T-ALL cells inhibits cell growth by inducing cell-cycle arrest and remarkable apoptosis.

A Representative expression levels of CD28 in manipulated Jurkat and MOLT-4 cells using flow cytometry (upper). The relative intensity of CD28⁺ cells were normalized to control cells and shown as the mean ± SD from three independent experiments (lower). B Viable cells were counted in manipulated Jurkat and MOLT-4 cells. C Representative cell-cycle distribution of manipulated Jurkat and MOLT-4 cells (left). The % population of cells in each phase is shown as the mean ± SD from three independent experiments (right). D Representative cell apoptosis and necrosis in manipulated Jurkat and MOLT-4 cells staining with Annexin V/7-AAD (left). The % population of apoptotic and necrotic cells in each group is shown as the mean ± SD from three independent experiments (right). NS not significant; *p < 0.05; **p < 0.01 (vs. control group).

Fig. 6. Knockdown of CD40LG in T-ALL cells inhibits cell growth by inducing cell-cycle arrest and apoptosis.

A Representative expression levels of CD40LG in manipulated Jurkat and MOLT-4 cells using flow cytometry. B Viable cells were counted in manipulated Jurkat and MOLT-4 cells. C Representative cell-cycle distribution of manipulated Jurkat and MOLT-4 cells (left). The % population of cells in each phase is shown as the mean ± SD from three independent experiments (right). D Representative cell apoptosis and necrosis in manipulated Jurkat and MOLT-4 cells staining with Annexin V/7-AAD (left). The % population of apoptotic and necrotic cells in each group is shown as the mean ± SD from three independent experiments (right). NS not significant; *p < 0.05; **p < 0.01 (vs. control group).

Fig. 7. BACH2-mediated CD28 and CD40LG axes contribute to dissemination of T-ALL cells to the BM.

A Cells were stained with PKH26 prior to seeding onto the pre-established monolayer of HS-5 BMSCs. PKH26 dye intensity in manipulated Jurkat and MOLT-4 cells were normalized to control cells. Data are shown as the mean ± SD from three independent experiments. B ELISA analyses of IL-6 and IL-8 using conditioned media upon coculturing CD28^KD or CD40LG^KD T-ALL cells with HS-5 cells. The colorimetric values were normalized to the control group. C Experimental design for transwell migration assays. Cells were pre-stained with PKH26 followed by the treatment with PBS (control), hCD28-mAb (0.5 μg/mL), rhCD40 (0.5 μg/mL) or both (hCD28-mAb+rhCD40). PKH26 dye intensity of migrated cells in the lower chamber were measured. D PKH26 dye intensity of migrated cells was normalized to the control group and shown as the mean ± SD from three independent experiments. E Experimental design for testing the effects of activating CD28 and/or CD40LG signals on T-ALL xenografts. F Spleens were isolated and photographed against a ruler in centimeters. G GFP⁺ cells from spleens (upper) and bone marrows (lower) in each group were analyzed using flow cytometry. Data are presented as the mean ± SEM. NS not significant; *p < 0.05; **p < 0.01 (vs. control group).