Abstract
Introduction:
RNA binding proteins (RBPs) are one of numerous ways of gene expression regulation. IGF2BPs (Insulin Like Growth Factor 2 Binding Proteins) are a group of oncofetal RBPs overexpressed in various epithelial malignancies and B-Cell Acute Lymphoblastic Leukemia (B-ALL). Several studies have reported that IGF2BPs act as readers of N-6 methyladenosine RNA modifications (m6A). m6A modifications are most commonly seen in the 3'-UTR of mRNAs and these are identified by IGF2BPs (m6A readers) leading to subsequent mRNA stabilization and translation.
M6A methylation on mRNA is carried out by METTL3 and METTL14 (m6A writers) and removed by demethylases ALKBH5 and FTO (m6A erasers). Dysregulated expression of all these genes has been reported in various cancers including Acute Myeloid Leukemia (AML).
There is no report on the expression of the m6A writers, erasers and readers in primary as well as relapsed B-ALL patient samples and their comprehensive correlation with patient prognosis and survival which formed the theme of our study.
Methods:
122 newly diagnosed (primary) and 55 relapsed B-ALL pediatric patient bone marrow samples were collected from the Cancer Hospital at AIIMS, New Delhi after proper ethics approval. RT-qPCR analysis was done to study gene expression. Multiple controls were used: CD19+ lymphocytes (10) and peripheral blood (20) from healthy individuals, 18 uninvolved bone marrow (BM) samples of patients with other malignancies. Analysis of public microarray and RNA-Seq data of B-ALL patients was done using cBioportal.
Results and Discussion:
Expression of the m6A writers, METTL3 and METTL14 as well as the reader IGF2BP3 was significantly higher in the primary and relapsed B-ALL patient samples (p<0.001) irrespective of translocation subtype compared to the controls. IGF2BP1 overexpression was specific to the ETV6-RUNX1 subtype as previously reported.
Interestingly, the expression of m6A eraser FTO was also higher in the B-ALL samples with ALKBH5 showing a significantly lower expression. FTO has previously been shown to be overexpressed in AML and it has demethylase independent functions also.
The ratio of m6A writers METTL3/14 to the erasers FTO/ALKBH5 was significantly higher in B-ALL patients. A colorimetric assay for m6A also revealed that m6A levels were significantly higher in primary and relapsed B-ALL. IGF2BP3 expression positively correlated with METTL3 and METTL14 expression in our B-ALL samples as well as from public analysis of the TARGET and St Jude B-ALL datasets. Interestingly, there was a significant positive correlation between METTL3 expression and FTO despite them having opposing functions.
Considering the significant dysregulation of these genes, we correlated demographic, prognostic and survival parameters with gene expression. Among the demographic parameters, there was a significant correlation of age with overall and event free survival (OS/EFS) with patients < 9 years of age having better survival (Hazard Ratio (HR) for OS 0.474, p=0.012). Translocation analysis correlated with existing data showing that ETV6-RUNX1 had the best prognosis followed by no known translocation group and the BCR-ABL1/MLL translocated groups having the worst prognosis. Interestingly there was no significant correlation with other prognostic parameters including WBC, platelet counts, hemoglobin levels and minimal residual disease (MRD).
Univariate analysis revealed that the expression of IGF2BP3 (HR 9.466 p=0.005 for OS) and FTO (HR 1.031 p=0.02 for OS) correlated with a poor OS/EFS. A multivariate analysis using backward elimination including all the prognostic parameters and gene expression revealed that the translocation status and IGF2BP3 expression (HR 25.4 p=0.01 for OS) were the most significant parameters which predict poor OS/EFS.
IGF2BP3 expression has been correlated with poor prognosis in other malignancies. Interestingly, despite an overexpression of m6A writers, it is the expression of the reader IGF2BP3 which strongly correlates with survival in our cohort. We can speculate that the readers may play a more important role in leukemogenesis. This is in line with our previous work which shows that IGF2BP3 overexpression leads to progenitor expansion in the mouse marrow and is essential for development of MLL-AF4 induced leukemogenesis. Our work also provides strong rationale for targeting this machinery for therapy in B-ALL.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
