Inhibition of the m6A reader IGF2BP2 as a strategy against T-cell acute lymphoblastic leukemia

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant leukemia with extremely limited treatment for relapsed patients. N6-methyladenosine (m⁶A) reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) participates in the initiation and growth of cancers by communicating with various targets. Here, we found IGF2BP2 was highly expressed in T-ALL. Gain and loss of IGF2BP2 demonstrated IGF2BP2 was essential for T-ALL cell proliferation in vitro and loss of IGF2BP2 prolonged animal survival in a human T-ALL xenograft model. Mechanistically, IGF2BP2 directly bound to T-ALL oncogene NOTCH1 via an m⁶A dependent manner. Furthermore, we identified a small-molecule IGF2BP2 inhibitor JX5 and treatment of T-ALL with JX5 showed similar functions as knockdown of IGF2BP2. These findings not only shed light on the role of IGF2BP2 in T-ALL, but also provide an alternative γ‑Secretase inhibitors (GSI) therapy to treat T-ALL.

Fig. 1. IGF2BP2 is highly expressed in T-ALL.

A IGF2BP2 expression levels were analyzed among 14 human lymphoblastic leukemia cell lines in CCLE database (https://portals.broadinstitute.org/ccle). B IGF2BP2 expression levels in normal T cells (n = 17) and primary T-ALL patients cells (n = 13) from GEO database, GSE48558. C IGF2BP2 expression levels in T-ALL. IGF2BP2 mRNA expression levels (left) were analyzed in normal human bone marrow cells (n = 6) and primary T-ALL patients cells (n = 9) by RT-qPCR assays. IGF2BP2 protein expression levels (right) were assessed in normal human bone marrow cells (n = 3) and primary T-ALL patients cells (n = 3) by western blot. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. IGF2BP2 promotes cell survival and impairs chemotherapy sensitivity in T-ALL.

A Proliferation of Jurkat cells (siIGF2BP2 or siNC) and Molt4 cells (siIGF2BP2 or siNC) was assessed by CCK8 assays, and proliferation rates at 0, 12, 24, 48, 72 and 96 h were normalized to the absorbance at 0 h. B Apoptosis analysis of Jurkat cells (siIGF2BP2 or siNC) after 24 h treatment with Ara-C, VCR and venetoclax were measured by flow cytometry. Percentages were representative of cell apoptosis from three replicate experiments. C Proliferation of Jurkat cells (oeIGF2BP2 or NC) and Molt4 cells (oeIGF2BP2 or NC) were assessed by CCK8 assays, and proliferation rates at 0, 12, 24, 48, 72 and 96 h were normalized to the absorbance at 0 h. D Apoptosis analysis of Jurkat cells (oeIGF2BP2 or NC) after 24 h treatment with Ara-C, VCR and venetoclax were measured by flow cytometry. Percentages were representative of cell apoptosis from three replicate experiments. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Transcriptome-wide iRIP-sequence assays identifies potential targets of IGF2BP2 in T-ALL.

A The Top 10 RNA reads within different gene types bound by IGF2BP2 from two replicate IGF2BP2 binding iRIP-sequence data. Protein_coding, contains an open reading frame (ORF); for detailed explanations of other gene types, refer to following website, https://www.gencodegenes.org/pages/biotypes.html. B Metagene profiles of enrichment of IGF2BP2-binding sites and their Input control analyzed by two replicate iRIP-sequence. CDS, coding sequence. C The distribution of IGF2BP2-binding peaks within different gene regions from two replicate IGF2BP2 binding iRIP-sequence data. D Venn diagram was plotted to show the intersected genes from IGF2BP2 binding iRIP-sequence, RNA-sequence of IGF2BP2 downregulation associated gene and IGF2BP2 upregulation associated gene. 2309 common genes were screened out. E The enrichment of NOTCH1 was analyzed by IGF2BP2 RIP-qPCR in Jurkat and Molt4 cells. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4. IGF2BP2 enhances NOTCH1 mRNA stability via an m⁶A-dependent manner.

A Distribution of m⁶A peaks and IGF2BP2-iRIP peaks across NOTCH1 mRNA transcript. The overlap region is highlighted in yellow. m⁶A-seq was repeated once, whereas RIP-sequence was performed twice. B M⁶A enrichment of NOTCH1 mRNA in Jurkat cells by m⁶A-RIP-qPCR. Results are presented relative to those obtained with immunoglobulin G (IgG). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), m⁶A negative control; MYC peak, m⁶A positive control. C IGF2BP2 expression was measured by western blot in Jurkat cells after RNA pull down assay using single-stranded NOTCH1 RNA with methylated or unmethylated adenosine. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5. IGF2BP2 knockdown significantly decreases leukemia burden in the human T-ALL xenograft.

A Kaplan–Meier survival curves of Jurkat xenografts in each group as indicated (n = 7 for each group). Survival of KD or LCV2 mice (orange and green lines) are compared with p value provided as shown (log-rank test). B Representative spleen and bone images of mice carrying Jurkat cells with or without IGF2BP2 expression. C Human CD45⁺ cells from bone marrow, spleen and peripheral blood were analyzed by flow cytometry. Data from five individual mice were plotted and shown on the right. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6. Small-molecule inhibitor of IGF2BP2 as a strategy against T-ALL.

A Small-molecule inhibitor JX5. Chemical structure JX5 (left side). 3D (left) and surface (right) view models show the binding pocket and binding of IGF2BP2 and JX5. Molecular docking of JX5 with IGF2BP2 (PDB: 6ROL) were performed (right side). B Cell growth inhibition of Jurkat cells (oeIGF2BP2 or NC, shIGF2BP2 or shNC) were assessed by CCK8 assays after 48 h treatment with serial dilutions of JX5. C Proliferation of Jurkat cells (oeIGF2BP2 or NC, shIGF2BP2 or shNC) treated with 25uM JX5 were assessed by CCK8 assays, and proliferation rates at 0, 12, 24, 48, 72 and 96 h were normalized to the absorbance at 0 h. Data are mean ± SD values. *P < 0.05; **P < 0.01; ***P < 0.001.