Super Enhancer Regulatory Gene FYB1 Promotes the Progression of T Cell Acute Lymphoblastic Leukemia by Activating IGLL1

Abstract

Background: Arising from T progenitor cells, T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignant tumor, accounting for 15% of childhood ALL and 25% of adult ALL. Composing of putative enhancers in close genomic proximity, super enhancer (SE) is critical for cell identity and the pathogenesis of multiple cancers. Belonging to the cytosolute linker protein group, FYB1 is essential for TCR signaling and extensively studied in terms of tumor pathogenesis and metastasis. Dissecting the role of FYN binding protein 1 (FYB1) in T-ALL holds the potential to improve the treatment outcome and prognosis of T-ALL.

Methods: In this study, SEs were explored using public H3K27ac ChIP-seq data derived from T-ALL cell lines, AML cell lines and hematopoietic stem and progenitor cells (HSPCs). Downstream target of FYB1 gene was identified by RNA-seq. Effects of shRNA-mediated downregulation of FYB1 and immunoglobulin lambda-like polypeptide 1 (IGLL1) on self-renewal of T-ALL cells were evaluated in vitro and/or in vivo.

Results: As an SE-driven gene, overexpression of FYB1 was observed in T-ALL, according to the Cancer Cell Line Encyclopedia database. In vitro, knocking down FYB1 led to comprised growth and enhanced apoptosis of T-ALL cells. In vivo, downregulation of FYB1 significantly decreased the disease burden by suppressing tumor growth and improved survival rate. Knocking down FYB1 resulted in significantly decreased expression of IGLL1 that was also an SE-driven gene in T-ALL. As a downstream target of FYB1, IGLL1 exerted similar role as FYB1 in inhibiting growth of T-ALL cells.

Conclusion: Our results suggested that FYB1 gene played important role in regulating self-renewal of T-ALL cells by activating IGLL1, representing a promising therapeutic target for T-ALL patients.

Figure 1

ChIP-Seq H3K27ac datasets analysis of T-ALL cell lines, AML cell lines, and hematopoietic stem and progenitor cells (HSPCs). (a) PCA was performed for 7T-ALL cell lines, three AML cell lines, and three HSPCs based on the H3K27ac signals identified in each sample. Each circle represents a sample, and each color represents the type of sample. (b) Cluster analysis results of 7T-ALL cell lines, three AML cell lines, and three HSPCs based on the H3K27ac signals identified in each sample. (c) Super enhancer profiling in 7T-ALL cell lines. Enhancers were ranked by increasing H3K27ac signal. Number of super enhancers identified in each T-ALL cell line was shown. Examples of genes commonly associated with super enhancers in at least 6T-ALL cell lines were shown. the x-axis stands for Enhancers ranked by increasing H3K27ac signal, and the y-axis stands for H3K27ac signal at enhancers. (d–f) SEs were identified at the locus of CDK6 (d), CCND3 (e), and ETV6 (f). SE was highlighted by the shaded box. Peaks stand for H3K27ac signals.

Figure 2

FYB1 was super-enhancer associated and highly expressed in T-ALL cell lines. (a) The ChIP-Seq gene tracks represented the H3K27ac signal in 7T-ALL cell lines, three AML cell lines and three HSPCs at the FYB1 gene locus. The super enhancers were labeled by yellow boxes. (b) According to the CCLE (https://portals.broadinstitute.org/ccle), FYB1 was highly expressed in T-ALL. (c and d) The western blotting results showed that the protein level of FYB1 in T-ALL cells was significantly higher than that in non-T-ALL cells.

Figure 3

FYB1 knockdown inhibited the proliferation of T-ALL cells in vitro. (a and b) shRNA-mediated knockdown efficiency of FYB1 in the J-gamma1, 6 T-CEM and Jurkat cell lines was evaluated by qRT-PCR (a) and western blotting, respectively (b). (c) Downregulation of FYB1 significantly inhibited the proliferation of J-gamma1, 6T-CEM and Jurkat cell lines evaluated by imaging assay. (d) Downregulation of FYB1 significantly inhibited the proliferation of J-gamma1, 6T-CEM and Jurkat cell lines evaluated by cell proliferation assay. (e) Downregulation of FYB1 increased the apoptosis of J-gamma1 and 6T-CEM cells. (f) The cleavage of PARP, cleaved-caspase-3 and caspase-8 were enhanced due to downregulation of FYB1 in the J-gamma1, 6T-CEM and Jurkat cells. (g and h) Overexpression of FYB1 enhanced the proliferation of J-gamma1 and Jurkat cells.

Figure 4

FYB1 knockdown inhibited the growth of T-ALL cells in vivo. (a) Schematic diagram of in vivo experiments. (b, d) Jurkat cells expressing luciferase transfected with control or FYB1 knockdown were injected into Nod–Scid mice via tail vein injection; and the Night OWL in vivo imaging system was used to monitor the leukemia burden of each group on the 25th, 30th, and 35th day after injection. Quantified results of fluorescence were shown by the bar graph in panel D. (c) Fluorescence of livers and spleens from control and FYB1 knockdown groups was determined, and quantified results were shown by the bar graph. (e) Downregulation of FYB1 resulted in better survival rate compared to control group. P = 0.0026. (f) No significant difference of body weight was observed between control and FYB1 knockdown group. (g) Percentages of Jurkat cells in liver, peripheral blood (PB), bone marrow (BM), and spleen from control and FYB1 knockdown groups were determined by hCD45 flow cyteometry and quantified results were shown by the bar graph. (h) Representative HE staining of bone marrow and liver in control and FYB1 knockdown groups (zoom factor: 200). Scale bars: 100 μm.

Figure 5

FYB1 activated IGLL1 in the T-ALL cell line. (a) Differentially expressed genes between J-gamma1 cells with or without knocking down FYB1 gene were shown by the volcanic plot. (b) Top 20 downregulated and upregulated genes after the knockdown of FYB1 gene in J-gamma1 cell line. (c) A total of 17 common genes between the RNA-seq data (1,051 differentially expressed genes (DEGs)) and Chip-seq data (213 SE associated genes in T-ALL) were identified. (d) ChIP-seq data from T-ALL cell lines showed that the promoter and enhancer region of IGLL1 had a consistent H3K27ac signal (track 1–7).

Figure 6

IGLL1 knockdown inhibited T-ALL cell proliferation and promoted apoptosis. (a) According to the CCLE (https://portals.broadinstitute.org/ccle), IGLL1 expression in T-ALL cells was higher than that in non-T-ALL cell lines. (b) The knockdown efficiency of IGLL1 in J-gamma1 and 6T-CEM cells was detected by the western blotting, and the expression of PARP was increased aftefr IGLL1 knockdown. (c) The knockdown of IGLL1 significantly inhibited the proliferation of J-gamma1 and 6T-CEM cell lines by cell proliferation assay. (d) The knockdown of IGLL1 significantly inhibited the proliferation of J-gamma1 and 6T-CEM cell lines by imaging assay. (e) The flow cytometry showed that the apoptosis rate of J-gamma1 and 6T-CEM cell lines increased after IGLL1 knockdown. (f) The knockdown of the IGLL1 gene inhibited the growth of J-gamma1 and 6T-CEM cell lines by affecting the cell cycle.