Delineating the Role of Interplay between m6A Machinery Genes and IGF2BP Group of RNA-Binding Proteins in B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Abstract

Introduction: N-6-methyladenosine (m6A) is the most common, dynamic and reversible RNA modification with implications in various cancers including leukemia. Deregulation of m6A writer METTL3 has been shown to promote disease progression in various cancers, including Acute Myeloid Leukemia(AML). Overexpression of METTL3 led to increase in cell growth and inhibition of apoptosis, thereby promoting leukemia progression. Interestingly, m6A demethylases (erasers) ALKBH5 and FTO have also seen to play a critical role in progression of AML by mediating cancer stem cell renewal. The IGF2BP family of RNA binding, oncofetal proteins have recently been identified as m6A readers and have also been shown to be deregulated in B-ALL. In this work, we have studied the expression of m6A machinery (writers, erasers and readers) in primary (naïve and relapsed) B-ALL patient samples. The percentage of methylated RNA (m6A%) was also evaluated in B-ALL patient samples.Materials and Methods: 91 newly diagnosed (naïve) and 47 relapsed B-ALL pediatric patient bone marrow samples were collected from BRAIRCH, AIIMS, New Delhi. Gene expression of m6A writer (METTL3), readers (IGF2BP1/3) and erasers (ALKBH5, FTO) was studied by RT-qPCR. Peripheral blood (PB) of 20 healthy individuals and 18 uninvolved bone marrow (BM) samples of patients with other malignancies were used as controls. m6A% was also measured in B-ALL patients (naïve n=47, relapsed n=43,) and controls (PB n=20, BM n=16, CD34+ cells from normal donors n=5) by an anti-m6A based colorimetric assay.Results: The ratio of m6A writer METTL3 to m6A eraser ALKBH5 was significantly higher in the naïve and relapsed B-ALL patients as compared to all controls. Interestingly, the ratio of the m6A writer METTL3 to m6A eraser FTO was also significantly high in naïve BM patient sample than controls. The expression of m6A readers IGF2BP1/3 that stabilize the methylated target mRNA, was also studied. IGF2BP1/3 m6A reader was significantly higher in naïve and relapsed patient samples. Increased expression of the writers and readers implied an increase in the m6A levels in B-ALL patients. The m6A% assay showed that the percentage of m6A was significantly higher in naïve and relapsed BM patient samples than both controls corroborating the RT-qPCR data.Discussion: METTL3 m6A methyl transferase has been identified a key factor in mediating the pathogenesis of AML. In our data, we have shown overexpression of METTL3 in B-ALL patient BM samples compared to controls. We have also seen an overexpression of m6A demethylase FTO in B-ALL patient samples. In order to identify the major factor among m6A writers and erasers that might play a role in pathogenesis of B-ALL, we calculated the ratio of m6A writer to m6A eraser. We have observed that ratio of METTL3 to ALKBH5 and METTL3 to FTO was significantly higher in B-ALL patient samples than both the controls. This signifies that overexpression of METTL3 subsequently leading to dysregulated methylation of its targets might influence the development and onset of relapse in B-ALL.It is well known that m6A bound target mRNAs are read by m6A readers like IGF2BPs that stabilize these m6A bound mRNAs leading to overexpression and thereby cancer progression. We have also studied expression of IGF2BP1/3 in B-ALL and seen significant overexpression of both IGF2BP1 and IGF2BP3 in B-ALL samples. These findings indicate a combined dysregulation of m6A writers, erasers and readers in B-ALL. This corroborates with the findings seen in AML, which also shows overexpression of METTL3, ALKBH5 and FTO.Our gene expression studies together point towards an increased percentage of m6A methylated RNA in B-ALL. We have evaluated the percentage of m6A in B-ALL patient samples to confirm our gene expression findings. We observed presence of significantly higher percentage of m6A in B-ALL patient samples (naïve and relapse) than both the controls. m6A% was significantly higher in naïve B-ALL patient samples compared to CD34+ HSCs also.Our findings reveal overall high m6A% in B-ALL, attributed to overexpression of m6A writer METTL3 and m6A readers IGF2BP1/3. This RNA methylation and stabilization might be dysregulated and concentrated in oncogenic genes leading to leukemogenesis. Our results provide a rationale for targeting of these m6A machinery genes dysregulation of which can be instrumental in pathogenesis of B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.